A Twisted Tale By Way of Background
Phenoxodiol owes its discovery to a misguided belief in the early 1990s (and unfortunately still present in some quarters today) that dietary isoflavones are bad for us….that they will cause female babies to develop reproductive abnormalities, that they are behind the falling sperm counts in modern man, and are responsible for the modern phenomena of a rising incidence in breast cancer and a lowering of the age of puberty in girls. The only surprise is that global warning wasn’t included in that list.
The toxicologists in the FDA were the most alarmist…the world as we knew it was about to end unless isoflavones were removed from the human diet, and in particular from the diet of babies being fed soya-based infant formula. At an FDA-sponsored conference in Arkansas in 1990, FDA toxicologists presented results of studies on guinea pigs and rats that showed isoflavones causing all sorts of female dysfunctions, and bayed for the blood of anybody who dared to question this view of the world.
What remarkably seemed to escape the attention of those alarmists is that dietary isoflavones have been part of the human diet since the dawn of time and that the fact that most Western diets have virtually no isoflavones is a modern aberration, not a truism. Some communities (well, actually, the bulk of the world’s population) today retain the sort of high dietary isoflavone intake that has typified much of human development ….Asian communities, India, North African and Mediterranean communities, vegetarians, Seventh-Day Adventists. And not only do these communities not have the sort of sexual dysfunction that the alarmists were propounding, but they actually enjoy lower incidences of diseases such as breast cancer, prostate cancer, bowel cancer etc. And if high isoflavone intake was causing such havoc with male genitalia, then the men of China and India seemed to be performing pretty well.
The root cause of this nonsense was a condition in Australian sheep known as Clover Disease. First observed in the 1940s, sheep of both sexes developed alarming problems with fertility and their genitalia. Ewes stopped ovulating and pregnant animals aborted. Sperm production in rams fell away, and castrated male sheep underwent breast development to the point of milk production. From a limited beginning, the problem soon spread to involve a substantial proportion of the national flock, something of major concern to a country that was supplying some 90% of the world’s wool.
The problem fairly smartly was traced to the presence in the pasture of large levels of the 4 estrogenic isoflavones, genistein, biochanin, daidzein and formononetin, a direct result of the introduction into those pastures of subterranean clover grasses. The clovers had been introduced in the late-1940s into Australian pastures as part of a national agronomy program to improve nitrogen levels in the soil. The Australian continent, the driest in the world, and without native leguminous grasses such as clovers, has significantly lower nitrogen levels in the soil compared to Europe and North America. But rather than introducing the red and white clovers found so prolifically in those regions, Australian farmers sowed pastures with subterranean clovers, so-called because they are a prostrate plant that sows its seeds below the surface of the soil. Being legumes, all clovers contain isoflavones, but sub-clovers just happen to have the highest isoflavone content of any plant in the world, with isoflavones making up to 5% of the dry weight of the plant.
This meant that for the poor old sheep grazing on a pasture rich in sub-clovers, they were capable of ingesting several gm each day of estrogenic isoflavones. And despite these isoflavones only being about 1/10,000th as estrogenic as the sheep’s own estradiol, the sheer quantity of isoflavone being ingested meant that the sheep were being exposed to a massive estrogen overdose. It was equivalent to a woman swallowing several monthly packets of the oral contraceptive pill every day of her life. Little wonder then that female sheep became infertile and castrated male sheep underwent gender relocation.
A lot of research was conducted by Australian agricultural scientists in the 1950s and 1960s into the nature of the problem, in particular looking at the fate of the isoflavones once they were eaten by the sheep. What they found was that the isoflavones got caught up in the extensive fermentation activity in the sheep’s rumen, undergoing a range of chemical modifications by the rumenal bacteria before being absorbed by the sheep. Two of the isoflavones, genistein and biochanin, were converted by the bacteria into biologically inactive substances, and so these two isoflavones were thought to play no role in the condition. The other two isoflavones, daidzein and formononetin, were almost completely converted into the compound, equol, which was the predominant isoflavonoid found in the blood and urine of sheep grazing on sub-clover-enriched pasture. The significance of this chemical conversion was that equol was 10x more estrogenic than the plant isoflavones, making it about 1/1000th as estrogenic as estradiol. So equol became the villain of the story.
The problem for Australian sheep eventually was solved by developing strains of sub-clover that had low levels of isoflavones, bringing the daily isoflavone intake of Australian sheep into line with those overseas. Dietary isoflavones then quietly exited the scientific agenda.
The story probably would have lain silent at that point had it not been for a study published in 1984 by the English scientist, Dr. Ken Setchell, showing that equol was present in the urine of women eating soya products. The study didn’t draw any specific conclusions about this observation, although the subject of Clover Disease was raised in the study’s discussion with the question being posed of whether the presence of equol might be responsible for unexplained infertility in women. Suddenly dietary isoflavones were back on the scientific agenda. The spectre of sheep with equol in their urine displaying gross abnormalities of their genitalia was too much for the toxicologists. And this is where the FDA came in with concerns that soya = equol = estrogen overdose = female babies with abnormalities of their female genitalia. Followed shortly afterwards by the notion that dietary isoflavones = equol = increased risk of breast cancer.
It seemed to me at the time that this line of thinking was on a par with proposing that vitamin A should be avoided because you can go blind from eating too much liver (hypervitaminosis A), or that fats should be removed completely from the diet because obesity is a health risk. Yes, equol was estrogenic, but the levels reported by Setchell were hundreds of times less than those found in affected sheep. And the whole discussion about the dangers of soya conveniently ignored the fact that all legumes, not just soya, contained isoflavones, meaning that two-thirds of the world’s population that relied on legumes for a large part of their daily protein needs were in deep trouble according to this line of thinking.
More significantly, however, this line of thinking stood in stark contrast to the fact that communities with the highest intake of dietary isoflavones had the lowest incidences of cancers of the reproductive system – cancers of the prostate, breast, uterus and ovaries – the so-called Western diseases. Far from causing problems of too much estrogen, a diet high in estrogenic isoflavones appeared to be providing protection from the ravages of our own steroidal hormones.
In an attempt to better understand the role of isoflavones in human health, a study was undertaken in 1990 at The University of Sydney to look at the fate of dietary isoflavones in the human body. That study showed that humans handled isoflavones in a substantially different way to sheep, the only other species in which such research had been done. The difference essentially came down to two factors – (a) the much lower level of fermentation in the human gut compared to the sheep gut, and (b) the fact that genistein was converted by bacteria into biologically active and not inactive compounds.
Normal digestive processes in the human stomach and small intestine where the bulk of our food is digested) appeared to release only a small proportion (about 10-20%) of the isoflavones from food. The isoflavones (daidzein, genistein, formononetin, biochanin) released at that point were absorbed into the bloodstream and taken to the liver where formononetin was converted to daidzein, and biochanin converted to genistein. The bulk of the dietary isoflavones, however, remained in the gut along with the dietary fibre and moved further down the digestive tract where they were subjected to bacterial fermentation within the large bowel. That fermentation was similar to what occurred in the sheep’s rumen, modifying the plant isoflavones and converting them into new isoflavonoid structures (referred to as isoflavonoid metabolites) that were absorbed into the body and eventually excreted in the urine.
Equol was certainly a major component in this group of metabolites, but 12 others also were identified. Collectively, these 13 new compounds accounted for 80-90% of the isoflavonoid compounds present in the body after eating food such as soya. By their sheer overwhelming presence, it was looking as though whatever health benefits were being ascribed to dietary isoflavones could be laid at the feet of these new compounds. Genistein and daidzein were present as well, but at levels that suggested that their contribution to any biological outcome was subordinate to that of the metabolites.
And in the same way that structurally altering daidzein to make equol resulted in a 10-fold increase in its estrogenic potency, so the question needed to be asked if the same thing happened to all other biological functions at that time being assigned to plant isoflavones, such as anti-cancer activity. Genistein showed modest anti-cancer activity, but did any of the metabolites show any greater activity?
This question could only be answered by conducting laboratory studies on all 13 isoflavonoid compounds. And to do that meant that they would have to be made synthetically. The alternative of trying to isolate enough compounds from hundreds of litres of human urine was not something that excited me. So the synthetic chemists were brought in.
The task began by looking at the 13 new isoflavonoid metabolites identified in human urine, and trying to determine how the gut bacteria might have gone about making them. Those pathways are shown in the diagram below. The chemical pathway coming from genistein, yielding metabolites such as dihydrogenistein, seemed relatively simple, more than likely involving just simple snipping off of certain groups or their replacement with new groups. But for those metabolites coming from daidzein, the chemical pathway appeared to be far more complex, almost certainly requiring a number of separate chemical steps, based on the bacteria manufacturing ‘intermediate’ compounds. The main intermediate compound proposed for those compounds coming from daidzein was a theoretical structure given the name of dehydroequol. Dehydroequol was entirely theoretical and was never detected in the body, so its existence (assuming that it does exist) must be restricted to the bacteria in the gut, and probably only then for very short periods of time. Dehydroequol later was renamed phenoxodiol.
Following the establishment of Novogen, all 13 confirmed metabolites plus the one putative metabolite (dehydroequol) were made synthetically and subjected to a battery of tests to determine their biological function. All 14 compounds proved to have some biological activity; most displayed varying levels of anti-inflammatory, anti-oxidant and cardiovascular activities; only one showed any anti-cancer activity, and that was dehydroequol.
Mechanism of anti-cancer action
The anti-cancer mechanism of action of phenoxodiol has been the subject of considerable study, mainly because it represents a unique and previously unrecognised mode of drug action.
There are 3 distinct steps in the process, summarised in the following diagram. Each of the three steps involves the plasma membrane, the membrane providing the interaction between the cell and its environment.
1. Proton pump
The primary target of phenoxodiol is the mechanism by which the cell eliminates waste hydrogen from its interior, the so-called proton pump.
The proton pump is an integral part of a cell’s survival mechanism. It is the means by which it generates most of its energy needs. It is part of a cell’s reduction-oxidation activity because it involves the movement of hydrogen (reducing) and oxygen (oxidizing) atoms both around the cell and between the cell and its external environment.
All cells have an extensive membranous structure comprising both the plasma membrane and surrounding all internal structures such as mitochondria. The proton pump operates in all of these membranous structures, and in the membranes surrounding internal structures, it is a vital source of energy generation. The pump essentially involves taking hydrogen ions (H+) (or protons) from the cytoplasm of the cell where they are generated as by-products of metabolic processes, and actively moving them into the membrane space where they become concentrated. The result is a considerable concentration gradient across the cell membrane, and it is this proton gradient that generates the energy required by the cell’s mitochondria to manufacture the compound, ATP, which provides 95% of the body’s energy needs.
The following is a general description of the proton pump process.
It starts with the capture of a hydrogen ions (H+) by the compound, nicotinamide adenosine dinucleotide (NAD), a compound found abundantly within cells and bodily fluids. This result in the formation of NADH.
NADH in turn is coupled to the protein, NADH reductase, which moves as a complex to the membrane where it passes the hydrogen ion onto the compound ubiquinone (also known as coenzyme Q10). In the process of doing this, 4 other protons also are pumped into the membrane space. Ubiquinone is transformed in coenzyme Q10H2.
In the case of the plasma membrane, the proton pump is less concerned with energy generation than it is with the expulsion of excess H+ ions from the cell. To achieve this, the proton pump involves an additional enzyme known as NAHD oxidase. The role of this enzyme is to remove the hydrogen ions from ubiquinone and pass them to NAD in the space outside of the cell. For the average cell, this process serves to remove a potentially toxic compound from the cell where its build-up could be fatal; for the cells lining the stomach it serves the additional role of secreting acid into the stomach as part of the digestive process.
So where does this fit into the phenoxodiol story?
Studies conducted by a group at Purdue University in Indiana, USA, and headed by Professor James Morre, are pointing to the fact that the NADH oxidase protein undergoes structural changes in certain disease states. The best studied of those disease states is cancer. Cancer cells express a structurally different form of NADH oxidase that Morre’s team have termed tumour-associated NADH oxidase, abbreviated to tNOX. tNOX is found only on cancer cells and appears to occur on all cancer cells across all forms of cancer. It is both structurally and functionally different to the form of NADH oxidase found on the cell surface of normal cells, referred to as constitutive NADH oxidase (abbreviated to CNOX).
Phenoxodiol inhibits tNOX, but has no effect on CNOX.
The following figure shows the effect of adding phenoxodiol to two types of cells. The first of these (MCF-10A cells) are normal human breast cells. These cells only express CNOX and as the figure shows, the addition of phenoxodiol produces no change in the activity of the proton pump in these cells, with their regular cycling of about 24 minutes.
The second type of cell in this study, BT-20, are the same MCF-1oA breast cells transfected with tNOX, so that the cells are now expressing both CNOX and tNOX. The addition of phenoxodiol to these cells results in an immediate shut-down of the proton pump driven by tNOX, while that driven by CNOX remains unaffected. While cancer cells also express some CNOX activity, it is completely subservient to the more prevalent tNOX activity, so that there is complete shut-down of the proton pump in cancer cells once tNOX is switched off. The absence of tNOX on normal cells accounts for the reason why the effect of phenoxodiol is limited to cancer cells.
This shut-down of the plasma membrane proton pump has two immediate consequences. The first is that CoQ10H2 stops losing the hydrogen ions that it is carrying removed at the cell’s surface, leading to a build-up of CoQ10H2 within the plasma membrane. The following figure shows this as measured by the immediate cessation of oxidation (loss of hydrogen ions) by ubuiqinol following the addition of phenoxodiol.
The second consequence of course is that hydrogen ions build up within the cell. Without an active proton pump, the cell has no means of eradicating hydrogen ions being produced as by-products of metabolism. This in itself would be toxic, disrupting all metabolic processes in the short-term, and proving lethal ultimately.
But in terms of how phenoxodiol drives the cancer cell towards death, it appears that it is the first outcome (rising CoQ10H2 levels) that is most relevant to the final outcome. The build-up of CoQ10H2 within the plasma membrane has a critical effect on the next part of the sequence of events, the sphingomyelin pathway.
2. Sphingomyelin pathway
A key component of cell membranes are compounds known as sphingolipids. The German biochemist, Johann Thurdichum, so-named them in 1874 after the sphinx of Greek mythology who devoured all who could not answer her riddles, because to him it appeared that these chemicals went a long way to answering the riddle that was the brain. Sphingolipids are fatty substances that form much of the myelin sheath surrounding and insulating brain cells and nerve fibres, and thereby facilitating the passage of nerve impulses.
They also play another crucial role in that they provide the structural framework of cell membranes, and in particular the plasma membrane, providing a support structure for all the cell’s various protein receptors and other functions such as the proton pump.
The membrane is made up of two inverted layers of sphingolipids known as sphingomyelin molecules, much like two pieces of bread in a sandwich. Each sphingomyelin molecule has a head (polar head) and tail structure, with the polar heads presenting to the internal and external edges of the membrane as shown in the figure. The various protein receptors then are embedded within these two lipid layers.
The sphingomyelin molecules, however, provide much more than a passive cytoskeleton. They play an active role in signal transduction and cell survival, and it is these pro-survival roles that are disrupted as a consequence of the failure of the proton pump.
An important characteristic of the membrane sphingolipid structure is that it is in a constant state of turn-over. Individual sphingomyelin molecules are being broken down to smaller compounds and then reassembled again from those same compounds. The first break-down product is another sphingolipid, ceramide, which in turn is broken down to sphingosine, which in turn is phosphorylated to sphingosine-1-phosphate. Each of these conversion steps is reversible via the action of different enzymes. This dynamic state of play is known as the sphingomyelin cycle.
At any one time the plasma membrane contains variable amounts of sphingosine, ceramide, and sphingosine-1-phosphate. The biological significance of this situation is that ceramide and sphingosine-1-phosphate (Sph-1-P) are biologivally active messengers known as secondary messengers, so-called because they respond to primary messages entering the cell via the various protein receptors, adding to the independent effects of those primary messengers.
Ceramide is essentially a pro-death messenger, while sphingosine is pro-survival. Ceramide blocks cell division and is thought to induce apoptosis via a direct disruptive effect on mitochondria (intrinsic apoptotic pathway). Sphingosine, on the other hand activates a number of different pro-survival pathways (principally the Akt and ERK pathways), a key outcome being the production of proteins that block apoptosis (so blocking the extrinsic apoptotic pathway). The pro-survival pathways being triggered by sphingosine also initiate a number of other important cellular functions including the generation of new blood vessels (angiogenesis), the expression of adhesion molecules, and cell motility.
Ceramide is biologically actrive in its own right, while sphingosine, in order to become active, needs to have a phosphate group attached to it (termed phosphorylation). This is accomplished by the action of the enzyme, sphingosine kinase, yielding sphingosine-1-phosphate (Sph-1-P).
In normal, healthy cells, an appropriate balance is maintained between levels of ceramide and sphingosine in the plasma membrane. In this way, a balance is struck between pro-survival and pro-death messages, with the pro-survival messages dominating. Events that tip the scales in favour of ceramide production include any stress on the cell such as inflammation and irradiation. The one event known to tip the scales the other way in favor of production of Sph-1-P is cancer.
Cancer cells express abnormally high levels of Sph-1-P, thought to be the result of increased activity of the enzyme sphingosine kinase, driving the sphingomyelin cycle towards the production of sphingosine. Over-expression of Sph-1-P is a key event in carcinogenesis, preventing apoptosis, and increasing the ability of the cell to migrate and to generate the development of new blood vessels, both important underwriting factors of cancer metastasis.
Phenoxodiol is able to affect this critically important ceramide:sphingosine balance by virtue of the fact that CoQ10H2 levels directly regulate the sphingomyelin pathway. Increasing CoQ10H2 levels have two effects. The first is that the activity of the enzyme, sphingosine kinase, is directly inhibited. The second is that the activity of the enzyme, sphingomyelinase, is increased, and this enzyme is responsible for the breakdown of sphingomyelin to ceramide. The result of this is that ceramide levels within the cancer cell rise, while Sph-1-P levels fall dramatically.
This opens the door for the involvement of the processes next in Step 3.
3. Apoptosis and death receptors
Apoptosis means programmed cell death. It is distinguished from necrosis, where gross damage to a cell will result in it expanding before exploding, so releasing intact cell contents into surrounding tissues where they can cause inflammation. Apoptosis is a far more controlled form of death, where the cell essentially undergoes internal auto-digestion, generally being removed by the body’s primary defence cells (macrophages) before it completely implodes.
There are two pathways by which a cell can undergo apoptosis. The first is called the intrinsic pathway which, as the name suggests, comes from within. This pathway is activated when the cell suffers gross injury to its DNA by way of radiotherapy, chemotherapy or heat stress. The second is called the extrinsic pathway and involves the inability of the cell to reject death signals being received by the cell. Both pathways are immobilized in cancer cells, and both pathways are restored by the action of phenoxodiol on the sphingomyelin pathway.
With both pathways, the end result is the release into the cytoplasm of enzymes known as caspases (cysteine-aspartic acid proteases). These are proteolytic enzymes held in sacs within the cytoplasm that when released from the sacs, auto-digest a cell’s protein structure. To ensure that caspases are not triggered either inadvertently or by very minor events, the cell produces a wide range of chemical factors that must be overcome before the various pro-apoptotic signals can activate the caspases. Cancer cells invariably over-express these protective factors, making it that much harder for the cell, no matter how severely damaged, to either self-destruct (intrinsic pathway) or be instructed by the body to die (external pathway).
The following figure from the American Association for Cancer Research nicely summarises the various pathways and their chemical cascades that lead to apoptosis.
Without getting bogged down in the detail of what is a very complex story, there are a couple of key points to note.
The intrinsic apoptotic pathway is dormant under normal circumstances. It is only when the cell suffers major injury, particularly to its DNA, that the cell attempts to activate it. The extrinsic apoptotic pathway, on the other hand, is continuously on active status. Chemical signals known as death signals are being received by all of our cells on a second-by-second basis. They are received by protein receptors located on the cell’s surface, which once activated, trigger a cascade of chemical events designed to activate the caspases both directly and via the mitochondria. To stay alive, all cells must maintain the production of IAPs (Inhibitor of Apoptosis Proteins) whose task it is to act as decoys for the pro-apoptotic cascades being triggered by the death receptors.
Those anti-apoptotic factors (eg. C-FLIP and XIAP), and the point at which they block, are denoted in the diagram by black bars. Cancer cells generally over-express these factors, and reducing the production of these factors is an important step in forcing the cancer cell to die.
The final point to make concerns the type of death receptors. This diagram shows the TRAIL death receptor, but the other important one is Fas. Both of them initiate essentially the same pro-apoptotic cascades, but different death receptors tend to be expressed on different types of cancer cells. But there must be sufficient difference in their function since phenoxodiol appears to be far more effective against cancer cells expressing the Fas death receptors than the TRAIL death receptors.
The following diagram summarises the effect of phenoxodiol on the apoptotic cascades.
Rising CoQ10H2 levels in the plasma membrane result in an increase in ceramide levels and an almost complete abrogation of sphingosine-1-phosphate levels. The effect of rising ceramide levels is to promote the intrinsic apoptotic pathway. The effect of falling Sph-1-P levels is to remove the stimulus via the Akt signaling pathway for the production of anti-apoptotic factors.
Primary molecular target
Tumor associated NADH oxidase (tNOX)
Primary biochemical consequences
1. Inhibition of proton pump.
2. Elevated levels of coenzyme Q10H2 in plasma membrane and NADH in cytoplasm.
3. Down-regulation of sphingosine kinase activity leading to a fall in levels of sphingosine-1-phosphate and an increase in levels of ceramide.
4. Increased p21 activity
(4a) Mitotic arrest CYTOSTASIS
(4b) Mitochondrial damage INTRINSIC APOPTOSIS
5. Down-regulation of Akt signalling pathway
(5a) Failure to phosphorylate anti-apoptotic proteins EXTRINSIC APOPTOSIS
An evolutionary perspective
So now to the key question of why on earth would an isoflavonoid drug target an enzyme involved in the proton pump mechanism? A clue to the answer lies in the only isoflavonoid that has been commercialized to date. This is a naturally-occurring isoflavone known commercially as rotenone. Rotenone is found naturally in a number of tropical legumes, the most common being Derris. Gardeners might recognise the name Rotenone or Derris Dust, a common garden pesticide.
Rotenone hasn’t been constructed to be a pesticide…that is its natural function in the plant. Like a number of plant isoflavones, rotenone provides a primitive immune function in defending the plant against attack by predators. Rotenone is able to kill insects by binding to ubiquinone, thereby shutting down the proton pump mechanism within the insect’s cells.
What rotenone does is provide a rational explanation for why phenoxodiol does what it does. Somewhere in the isoflavone heritage of phenoxodiol is an ability of isoflavonoids to target the proton pump mechanism.
The first observed effect of phenoxodiol on cancer cells (within 20 minutes) is the failure of newly dividing cancer cells to enlarge.
The second observed effect (within 12 hours) is a cytostatic effect, with cancer cells failing to divide. phenoxodiol blocks cancer cells in the G1 phase of mitosis (as a result of up-regulation of p21 activity, leading to loss of cdk2 activity).
The third observed effect (within 24-48 hours) is apoptosis. This effect is limited to cancer cells, with non-cancer cells such as cultured human breast cells on the left showing no adverse effects, while the breast cancer cells on the right are rounded-up, detached from their plastic base, and beginning to disintegrate as they undergo apoptosis.
Phenoxodiol has been tested against a wide range of cancer cell types and been shown to be cytotoxic within a fairly narrow band of dosage levels. Cell types known to be sensitive to phenoxodiol include prostate (both androgen-dependent and -independent), breast cancer (both ER+ and ER-), mesothelioma, glioma, rhabdomyosarcoma, lung (both large cell and non-small cell), melanoma and leukaemia (erythroid, myeloid, acute lymphoblastic and multiple myeloma).
Phenoxodiol also is effective against cancer cells that are highly insensitive to standard anti-cancer drugs. The following figure shows the ability of phenoxodiol to kill human ovarian cancer cells that are resistant to high doses of carboplatin and taxol.
Chemo-sensitisation and reversal of chemo-resistance
Chemo-sensitisation refers to the process of enhancing the effect of another anti-cancer drug. Reversal of chemo-resistance refers to the process of restoring sensitivity of a cancer cell to a particular drug after becoming resistant to that drug.
Phenoxodiol does both of these things. It is not clear whether the same mechanism of action underlies both effects, but it seems likely that it is.
These abilities are worth considering here in some detail, because it is far more likely that phenoxodiol will be used for this ability to overcome drug resistance in its early life, than it will as a monotherapy in its own right. With the possible exception of being used as a monotherapy in prostate cancer (as per the current Phase 2 study), the way that it is being tested in the OVATURE study seems to be the way that it will be introduced to the market and go on to establish a main role for itself in that capacity in the short-term.
Irrespective of whether or not there is a common underlying mechanism of action, we still need to consider each effect separately because they represent two quite distinct clinical opportunities.
With chemo-sensitisation, the ability of phenoxodiol to enhance the killing effect of a standard anti-cancer drug could be used with two objectives in mind: (a) to enable a lower dose of the standard drug to be used to achieve the current clinical response but with the result of delivering reduced adverse side-effects, or (b) to accept the current level of adverse side-effects but achieve a much greater clinical effect than is the case currently.
It needs to be emphasised that this effect of phenoxodiol has not been tested to date in any clinical studies, and the Company is unlikely to do so in the foreseeable future. However, I have little doubt that this will become the major way the drug ultimately is used when it reaches the market on any approved basis.
Later we look at another intriguing effect of phenoxodiol which is its ability to protect against neurotoxic complications of chemotherapy. When you combine that protective effect, along with its ability to enhance the anti-cancer effects of standard drugs, plus consider that phenoxodiol has no safety issues in its own right, then that becomes a powerful argument to support its general use in combination with standard anti-cancer drugs as a primary therapy.
The chemo-sensitising effect of phenoxodiol is a synergistic effect rather than a simple additive effect as the presence of phenoxodiol achieves at least a 1000-fold decrease in the dose of the second drug without diminishing its anti-cancer effect as the following two log-scale graphs show.
In each case, phenoxodiol is delivering a 10-5 reduction in the dose of cisplatin and gemcitabine without compromising the ability of either drug to kill 50% of the cancer cells. This same effect applies to paclitaxel and topotecan.
All 4 drugs are damaging cancer cells via different mechanisms, suggesting that phenoxodiol is providing synergy via a common, fundamental mechanism. And it is hard to go past the general and widespread disruption to cell biochemistry following shut-down of the cell’s proton pump as being that fundamental mechanism.
This chemo-sensitising effect, however, is not universal. For instance, it does not occur with the drug, doxorubicin (trade name Adriamycin), and in fact, phenoxodiol appears to antagonise the effect of doxorubicin. I am not aware that the Company has exhaustively looked at the potential for synergy or antagonism across the broad range of anti-cancer drugs (although this is something that certainly will need to be looked at before the drug is marketed), but my guess is that this is a specific effect with doxorubicin (or any member of the anthracycline family of anticancer drugs).
Jim Morre’s team at Purdue University not so long ago showed that doxorubicin inhibits tNOX in much the same way that phenoxodiol does. But the fact that doxorubicin kills cancer cells predominantly via interference with DNA function suggests that tNOX is an incidental effect and not the primary molecular target for this drug as it is for phenoxodiol. Nevertheless the efficiency of the proton pump in cancer cells is affected by doxorubicin, suggesting that the most likely explanation for the antagonism between phenoxodiol and doxorubicin lies in their competition for the tNOX target and highlights that this particular drug combination should never be used.
2. Reversal of chemo-resistance
Reversal of chemo-resistance or restoration of chemo-sensitivity…take your pick of terms. Either way, it is what phenoxodiol does very well.
Just to continue with getting terms right, cancer cells are referred to as either being insensitive to drugs or having acquired resistance. Insensitivity refers to an innate resistance of cancer cells to chemotherapy from the start, while resistance refers to the loss of sensitivity through repeated exposure to a particular drug. For example, taxane drugs such as paclitaxel or docetaxel are the standard form of chemotherapy for ovarian cancer. About 85% of these cancers respond to the initial course of chemotherapy as a result of them being chemo-sensitive; the remainder show no meaningful response and are referred to as being chemo-insensitive. Almost all of the 85% of cases that show an initial response will eventually experience a return of the cancer and will receive a follow-up course of taxane therapy. The response to this second course of therapy normally is weaker than that to the initial therapy, and so it goes with further courses of therapy, with the cancer showing an increasing development of chemo-resistance, until it eventually stops responding to chemotherapy, at which point it is referred to as being refractory to further chemotherapy.
Once a cancer becomes resistant to a particular drug, it generally is resistant to all other drugs. This is known as multi-drug resistance (MDR). MDR is not a particularly well-understood phenomenon. A range of different cellular mechanisms have been described, mostly having to do with the cancer cell developing the means to block the drug from entering the cell, or causing it to be ejected from the cell before it can work, or blocking its passage to its ultimate target within the cell, or learning how to compensate for the anti-cancer effect of the drug. For each drug, any or all of these mechanisms are likely to be behind the development of resistance.
With the exception of doxorubicin (for the reasons cited above), phenoxodiol effectively restores sensitivity in cancer cells that have become resistant to all commonly-used anti-cancer drugs.
The ability of phenoxodiol to overcome carboplatin resistance is a good example of this phenomenon, and it something that we have a reasonable handle on. To understand what is happening here, we need to return to the earlier diagram on the sphingomyelin cycle. To recap, the main structural component of the plasma membrane, sphingomyelin, is in a constant state of dynamic equilibrium with the smaller sphingolipids, ceramide and sphingosine. This produces an ongoing, continuous state of assembly-disassembly of the sphingomyelin. Given that ceramide is a pro-death messenger, and sphingosine (following its phosphorylation to Sph-1-P) is a pro-life messenger, it obviously is in the cell’s best interests to ensure that there is a predominance of Sph-1-P compared to ceramide, but not so much that it would make it impossible for the cell to self-destruct if forced to. The levels of Sph-1-P in the cell, therefore, are tightly regulated. Two enzymes play a key role in this regulation. The first is the enzyme, sphingosine-1-phosphate phosphatase, that cleaves off the phosphate group, returning Sph-1-P to sphingosine. The second enzyme is sphingosine-1-phosphate lyase, which degrades the Sph-1-P to a biologically inactive aldehyde. This latter step is a one-way, irreversible conversion that effectively prevents a build-up of Sph-1-P in the cell.
Resistance in cancer cells to carboplatin is associated with decreased activity of sphingosine-1-phosphate lyase. Without the degradation action of this enzyme, levels of Sph-1-P build up within the cell to the point where there is over-expression of anti-apoptotic proteins, profoundly raising the apoptotic threshold of the cell. Despite the fact that carboplatin is still able to damage the DNA of the cell, the raising of the apoptotic threshhold makes it impossible for that damage to trigger apoptosis. The following diagram shows how phenoxodiol specifically overcomes this effect.
By switching off the proton pump, the resulting increase in CoQ10H2 (quinol) in the plasma membrane and NADH levels in the cytoplasm inhibit the activity of sphingosine kinase, preventing the production of Sph-1-P in the first place, and thereby effectively overriding the carboplatin-resistance effect.
3. Some clinical considerations
One of the big unresolved questions surrounding the whole issue of using phenoxodiol to restore sensitivity to other commonly used anti-cancer drugs, is that of how best to apply the combination. This relates to the so-called temporal use of the two drugs. That is, when should you administer phenoxodiol in order to restore sensitivity to a second drug? …at the same time, or before, or following the second drug?
In the case of the platinum drugs such as cisplatin and carboplatin, it seems that phenoxodiol needs to be given either just before or at the same time as the platinum drug. At least that is what the test-tube and animal studies show. When athymic mice were injected with platinum-resistant human ovarian cancer cells and treated daily with cisplatin, there was no effect on tumor growth. When cisplatin was given in combination with a sub-therapeutic dose of phenoxodiol, tumour growth was reduced by 80%.
And given what we just saw about how phenoxodiol accomplishes this reversal of platinum-resistance, it makes perfect sense to use the two drugs simultaneously. That is, that you would need phenoxodiol to be present in the cell, switching off Sph-1-P production, in order for the DNA-damaging effect of carboplatin to trigger apoptosis.
That simultaneous use of phenoxodiol and carboplatin is the basis for the design of the OVATURE study.
But it may not be a case of one size fitting all. Simultaneous use might not be the optimal approach with other drugs….taxanes being one such case in point. This likelihood arose as a result of clinical observations following a Phase 2 study conducted at the Yale-New Haven Hospital a few years ago. These observations have been reported in the scientific literature. In that study, phenoxodiol monotherapy (that is, phenoxodiol alone) was being evaluated as salvage therapy in patients with taxane-refractory ovarian cancer. That is, the patients came into the study having reached a point where their cancers had failed to respond to taxane (paclitaxel or docetaxel) therapy. Patients remained on phenoxodiol therapy for variable lengths of time depending on how well their cancer responded. But eventually all patients showed cancer progression, leading to them coming off study. At that point, a number of patients were re-treated with docetaxel on a last-chance, salvage basis. Despite all patients being refractory to docetaxel prior to commencing the Phase 2 study, a significant proportion of these patients showed a substantial and entirely unexpected response to the follow-up docetaxel therapy. Making this response even more remarkable was the fact that if phenoxodiol was responsible for this reversal of chemo-resistance, then it had done so despite a gap of up to 3 weeks between the last dose of phenoxodiol and the commencement of docetaxel therapy.
Jim Morre’s group at Purdue subsequently investigated this phenomenon from the perspective of the effect of phenoxodiol on its tNOX target. They conducted in vitro experiments using human cancer cells resistant to docetaxel and subsequently published their findings. What they found was that phenoxodiol not only was most effective in reversing taxane-resistance when there was an interval of at least 24 hours between exposure to phenoxodiol and exposure to a taxane drug, but that the phenoxodiol needed to be absent from the cell’s environment at the time the taxane was introduced. In other words, phenoxodiol appeared to be inducing some change in the cancer cell, presumably within the proton pump mechanism, that left the cell sensitive to the effects of taxanes, but which only operated in the absence of phenoxodiol. Even more curiously, the phenoxodiol-induced change, whatever it is, was transferable between cells, including to cells not previously exposed to phenoxodiol. If that sounds a bit too bizarre, it is worth remembering that this so-called ‘bystander effect with cancer cells has been reported in certain other circumstances, where a ‘learnt’ biological response (such as the development of resistance to radiation) by some cancer cells is transferable to other cancer cells.
The answer to this biological curiosity may well lie in a recent discovery about the cytotoxic mechanism of taxanes. It has long been assumed that the anti-cancer activity of this family of drugs lay solely with their ability to disrupt the structure of the micro-tubules within a cancer cell. However it has now been shown that taxanes can also induce a bystander toxic effect on neighbouring cells through the generation of reactive oxygen species which they accomplish by upregulating the activity of NADH oxidase (NOX) in the plasma membrane [Cancer Res 2007;67(8):3512–7]. A lot more work would need to be done to sort out what is happening here in the case of phenoxodiol and taxane-resistance, but the very fact that both drugs appear to be affecting a common pathway in the proton pump mechanism, with phenoxodiol inhibiting activity of the pump and taxanes enhancing pump activity, suggests that this answer lies somewhere in some fundamental way that a cancer cell is handling its hydrogen and oxygen transfer.
The relevance of this speculation lies in an appreciation that the target of phenoxodiol … the proton pump….is probably more involved in how a cancer cell responds to many chemotherapies and how it develops resistance to those chemotherapies than we hitherto have appreciated. The good news is that that puts phenoxodiol in the box seat when it comes to the opportunity of making the current range of cytotoxic anti-cancer drugs work better. The proton pump is of little consequence with designer drugs such Herceptin and Erbitux, but for the remaining armoury of cytotoxic drugs that are the mainstay of modern chemotherapy, it may well turn out to be a highly significant target. The bad news is that no single rule is likely to apply between phenoxodiol and those other drugs, just as the antagonism between phenoxodiol and doxorubicin shows, and the different temporal relationship between phenoxodiol-carboplatin and phenoxodiol-docetaxel treatment show. Each combination will need to be investigated in its own right.
Protection from neurotoxicity
Damage to peripheral nerves is a relatively common side-effect of chemotherapy, particularly with the taxanes and the platinums. Patients can experience motor dysfunction such as loss of reflexes or weakness, and sensory dysfunctions such as numbness, tingling, pain or burning sensations. Symptoms generally resolve over time once chemotherapy ceases, but for many patients, the symptoms of neurotoxicity can be restrictive and debilitating.
The impetus to investigate whether phenoxodiol might provide any protective effect from adverse side-effects of chemotherapy came from observations in the laboratory that mice treated with combinations of phenoxodiol-carboplatin and phenoxodiol-docetaxel showed less evidence of toxicity that when carboplatin and docetaxel were used alone.
A study was conducted at the University of Melbourne’s Centre for Neuroscience, looking at the ability of phenoxodiol to protect neuronal cells from the toxic effects of cisplatin. Neural cells grown in cell culture extend their axons (or neurites) as shown below.
Cisplatin inhibits this outgrowth of neurites. phenoxodiol blocks this inhibitory effect of cisplatin.
The development of phenoxodiol has faced a number of significant hurdles:
- the uniqueness of its mode of action (making its acceptance by clinicians and investors and regulators challenging);
- early uncertainty over its bio-availability (leading to spending 3 years on the development of an intravenous dosage form, since abandoned);
- the almost universality of its anti-cancer effects (making the selection of an appropriate clinical target difficult);
- the variety of its anti-cancer benefits including use as a monotherapy or as a chemo-sensitiser or as a restorer of chemo-sensitivity (adding to the challenge of selecting an appropriate clinical target).
To a large extent, phenoxodiol has been a victim of its own success, an almost ‘too good to be true’ situation. The first and only drug to kill all forms of cancer, to spare non-cancer cells and to be without side-effects, to synergise the action of most anti-cancer drugs, to reverse chemo-resistance to most anti-cancer drugs, and to protect from the toxic side-effects of commonly used chemotherapies. All of which only starts to make sense when you understand how the drug is working and how that mechanism of action interacts with those of other drugs.
The primary target, viz. a protein associated with regulation of the proton pump of a cancer cell, appears to have an evolutionary basis in a plant’s primitive immune system. Switching off that pump triggers a cascade of biochemical consequences that eventually inhibit the cell’s pro-survival mechanisms, allowing phenoxodiol to kill the cell in its own right or to lower the apoptotic threshold sufficiently to facilitate cell death by other chemotoxic drugs.
As a monotherapy, as the sort of drug that an oncologist might reach for at the first diagnosis of cancer, phenoxodiol is almost certain to enjoy restricted use. All available pre-clinical and clinical data point to prostate cancer being one such use. For patients who fail localised treatment for this cancer, the lack of any suitable alternative chemotherapy at that stage, plus its high safety margin, suggest a likely home for phenoxodiol in that setting with its enormous unmet clinical need.
The only other potential monotherapy application on the horizon is squamous cell carcinoma (SCC). This opportunity is suggested by some dramatic responses seen in patients with multiple cutaneous SCC, where highly aggressive cancers resolved within a matter of days of commencing phenoxodiol monotherapy. A Phase 2 study looking at this application foundered because of the difficulty in recruiting sufficient suitable candidates in a timely manner, and was replaced ultimately with a study of patients with cervical and vaginal cancers.
SCC is a type of cancer that arises in the superficial layers of the skin, the lips and the lining of the mouth, oesophagus, bladder, lung, vagina and cervix. SCC of any of these sites is very poorly responsive to chemotherapy, so as with prostate cancer that has failed localised treatment, SCC in all locations represents a significant unmet clinical need.
But by far the most likely home of phenoxodiol will be in the area of combination therapy. Employing phenoxodiol simply to augment the effectiveness of many standard anti-cancer drugs in early-stage cancers, or using it to reverse chemo-resistance in late-stage cancers, are obvious applications of the drug. The situation with ovarian cancer is a case in point. With approximately 15% of patients showing innate insensitivity to first-line chemotherapy (taxanes) and with no way of identifying such patients, the use of phenoxodiol in combination therapy as a first-line approach in all cases of ovarian cancer would seem an obvious opportunity. Then, with the subsequent development of refractory cancers, the further opportunity is there to use phenoxodiol as a form of salvage therapy in combination with drugs such as carboplatin (as per OVATURE).