Download NOX66 - GENERAL SCIENTIFIC OVERVIEW Oct 2016

NOX66 - GENERAL SCIENTIFIC OVERVIEW
Oct 2016
The steps in this overview are:
1. How a cancer cell differs from a normal cell
2. How frontline cancer drugs work and the mechanisms that cancer cells employ to
become resistant to those drugs
3. How idronoxil works
4. Why NOX66 works.
1.
Cancer vs Normal Cells
A first step in this story is the need to understand the normal. That is the stepping stone to
understanding how idronoxil works on the abnormal.
Cells are herd animals. They can’t function in isolation. They respond to millions of signals
hitting the cell constantly. These signals are coming from neighbouring cells, from distant
cells, and even from themselves (they leave and then come back). The signals are received
by receptors on the surface of the cell as the following diagram of the cell membrane
shows.
The receptors (the coloured ones protruding out of the membrane) have the task of
receiving the different messages, interpreting them, sharing information with other
receptors with related functions, and then sending a single coordinated message to the
cell’s genes. The relevant genes are activated and the cell then responds accordingly.
Between the cell surface receptors and the genes is a complex signaling system that acts as
an interpreter of the thousands of signals heading towards the nucleus. These thousands of
signals are fed into a few central points that act like a telephone switchboard. One of these
key switchboards is known as Akt (shown below).
Akt is known as a pro-survival checkpoint. Its job is to keep the cell alive. It’s sending
messages to the cell’s genes to keep metabolizing, growing, dividing, moving. If a cell needs
to die, it first needs to shut down the Akt switchboard.
In cancer cells, the Akt switchboard is hyper-active. It has been up-regulated enormously,
sending far more than normal rates of signals to the genes to stay alive. As a result, the
cancer cell grows and divides faster than normal and migrates and moves more than
normal. Instead of behaving like a herd animal, dividing when told and only moving when
told, the cancer cell now is a rogue animal, dividing and spreading at will.
Among the myriad of pro-survival mechanisms that Akt is responsible for is DNA repair. The
DNA in our genes is subject to a barrage of damaging agents on a daily basis – carcinogenic
chemicals, environmental carcinogens, carcinogenic viruses, irradiation etc. The cell has a
very competent repair mechanism that quickly comes in and repairs the damage. One of the
many functions that Akt controls is this DNA repair mechanism. In cancer cells, the hyperactivity of Akt means that the cancer cell’s DNA repair mechanism are working overtime.
2. Akt, cancer and drug-resistance
The relevance of a hyper-active Akt switchboard to our story is that frontline cancer
therapies (chemotherapy drugs and radiotherapy) work by damaging DNA. The rationale is
to inflict more damage on the cancer cell’s DNA than the DNA repair mechanisms are able
to repair, in which case the cell at worst will stop dividing, and at best, die.
The problem these frontline therapies face is that their DNA-damaging action is not
discriminatory…. it is just as capable of damaging DNA in a healthy cell as in a cancer cell.
What discrimination there is, is based on the rate of cell division. DNA that is rapidly dividing
is more susceptible to damage than DNA that is not actively dividing. In a cancer patient,
cancer cells are the most actively dividing, and so are proportionately more with lining of
the gut, bone marrow and hair follicles the next most active.
That means that the maximum amount of chemotherapy or radiotherapy that can be
applied is determined by the damage done to healthy tissues such as the gut and the bone
marrow. That is referred to as the Maximum Tolerated Dose (MTD).
So we have a situation with chemotherapy and radiotherapy where we are trying to inflict
as much damage as possible on the DNA of a cell that is fighting back by having ramped up
its DNA repair mechanisms to extraordinarily high levels. All the while, also damaging the
DNA in healthy cells where the rates of DNA repair are much lower.
This means that frontline therapies can never be used at dosages likely to kill all cancer cells.
The proportion of cancer cells that can be killed varies between different cancer types and is
a function of just how much resistance (DNA repair) there is to start with. Cancers of the
pancreas, stomach, brain, gallbladder and liver, for example, have high levels of resistance
from the start, due to very active Akt function. At the other end of the spectrum are certain
leukaemias and cancers of the breast, ovary and large bowel, where initial response rates
can be fairly high.
But irrespective of how a tumour responds initially, in virtually every case of aggressive
cancer, some cells escape being killed because they have higher levels of resistance, and
they return eventually to produce secondary cancers that are even more resistant than the
primary cancers.
The same principles apply in the case of radiation as they do for chemotherapy.
Tackling resistance mechanisms
An obvious target in treating cancer is to knock out the resistance mechanisms. If a cancer
cell was unable to repair its DNA damage, then current dosages of chemotherapy and
radiotherapy should be infinitely more effective, perhaps even curative, by wiping out even
the most highly resistant cancer cells that currently are escaping. That means that the
current MTD dosages might now become highly effective, with the added prospect of being
able to lower chemotherapy and radiotherapy dosages even lower to dosages that would
have little or no effect on healthy cells.
And that means knocking out Akt.
Many have tried, but no-one has yet been successful because Akt in a cancer cell is exactly
the same as Akt in a healthy cell. Up till now there has been no way to discriminate between
the two cells. And Akt is so essential to life, that a non-selective Akt knock-out drug is just
too toxic.
3. Idronoxil and Akt
Idronoxil is the first drug that selectively knocks out Akt in cancer cells. It does not target Akt
directly…its target is a protein that is upstream of Akt and on which Akt is dependent. And it
is a target that is only found in cancer cells and where knocking out that target effectively
inhibits Akt function in cancer cells.
Probably the best way to understand this story of how idronoxil works is to work
progressively back upstream from Akt.
This is like a ladder with 5 rungs. Akt is the bottom (5th) rung.
The next (4th) rung up is sphingosine-1-phosphate (S1P). S1P regulates Akt function. As the
following figure shows, S1P is very much involved in the cancer process, largely (although
not exclusively) through Akt.
S1P levels are excessively high in most forms of cancer, which in turn is directly responsible
for Akt levels also being high in most forms of cancer.
S1P is manufactured within the outer membrane of the cell, and that process is the next
(3rd) rung up in the ladder.
The cell membrane is a fatty structure made up of different types of lipids (fats). The
compound sphingomyelin comprises about 20% of these lipids. It is part of the physical
structure of the membrane, playing a critical role like all the other fatty components in the
membrane in regulating what comes in and leaves the cell. But unlike the other fatty
components, sphingomyelin undergoes an important chain of events that is essentially the
death – survival master switch of the cell.
Sphingomyelin is converted by a series of enzymes to S1P. This is a reversible process that is
in a constant state of flux going either way….from sphingosine to S1P and then back again.
The process involves the production of two intermediate compounds – ceramide and
sphingosine – as the following figure shows.
Ceramide is known as a pro-death factor (activating cell death, or apoptosis) and S1P as a
pro-survival factor. This master switch then effectively controls the fate of the cell, and
survival means maintaining a seesaw tipped towards the production of S1P (in turn
activating Akt). But if the seesaw tips in favour of ceramide, then the double-whammy
happens of pro-survival activity being withdrawn (no S1P = no Akt) at the same time as
increased ceramide levels actively cause the death of the cell.
Within this 3rd rung of the ladder, the activity levels of the enzyme, sphingosine kinase, is
vital in ensuring that enough S1P is being produced to keep the cell alive.
The 2nd rung of the ladder is another activity occurring within this cell membrane known as
the proton pump. Protons are the positive sub-atomic particles in hydrogen atoms, and
these are responsible for powering many of the cell’s biochemical activities, much like a
nuclear reactor. Cells that are active make excess protons, and they need to be eliminated
from the cell or else they would be toxic. This elimination takes place via the proton pump,
which takes protons (shown as H+) and shuffles them from the inside of the cell to the
outside.
It turns out that the function of the enzyme, sphingosine kinase, is dependent on a certain
low level of protons within the cell membrane. If that level rises, then sphingosine kinase
activity shuts down.
Which brings us to the top (1st) rung on the ladder. There is a protein that sits on the outer
edge of the proton pump and which controls the function of the pump. It is known as
External Membrane NADH Oxidase, or ENOX.
This is the target of idronoxil. By binding to ENOX, idronoxil stops it working, which means
that the proton pump shuts down, which means that protons build up inside the
membrane, which means that sphingosine kinase stops working, which means that S1P
stops being made, which means that Akt function is blocked, which means that all prosurvival activities shut down, including the ability of the cell to repair damaged DNA, which
means that the cell is now sensitized to the poisoning effects of chemotherapy and
radiotherapy.
So why does idronoxil work when no other Akt-inhibitor has been able to be used. Why does
idronoxil not affect healthy cells?
The answer is that the ENOX protein on cancer cells is different to the ENOX protein that
regulates the proton pump in healthy cells, and idronoxil only binds to cancer cell ENOX.
Humans have the means to make two main forms of ENOX known as ENOX1 and ENOX2.
They perform the same task (driving the proton pump), but differ in a very tiny way –
ENOX2 drives the proton pump slightly faster than does ENOX1. Think of a 6 HP pump
versus a 5.5 HP pump, because that is about the degree of difference. But that small
difference is important when it comes to how many protons a cell is generating ….. the
more being generated, the more active the proton pump needs to be to expel the protons.
Protons are acidic, and an abnormal build up of protons within the cell would be lethal.
In normal, healthy cells, the standard proton pump is perfectly adequate to handle the
normal rate of proton production, which means that proton pumps in healthy cells are
driven by ENOX1.
But when a cell becomes cancerous, its whole rate of metabolism goes up enormously to
meet the demands of a high rate of growth, and ENOX1 is unable to meet this need to expel
this higher rate of proton production. Under this stress, it appears that cells switch from
ENOX1 to ENOX2 to meet this increased demand. Cancer cells therefore switch from
producing ENOX1 to producing ENOX2. In all forms of cancer tested to date (and this is
across the entire cancer spectrum), ENOX1 has been replaced by ENOX2.
Idronoxil inhibits ENOX2, which is why its anti-cancer effect is across all forms of cancer. And
idronoxil only inhibits ENOX2, which is why it has not effect on healthy cells.
Idronoxil is not unique in binding to and inhibiting ENOX2. A bio-active component of green
tea, epigallocatachin gallate (EGCG), along with capsaicin (from peppers,) also inhibit
ENOX2, although at considerably weaker levels. Idronoxil inhibits ENOX2 some 100 times
greater than any other compound yet identified.
Effect of idronoxil on cancer cells
Working down the ladder:
ENOX2 inhibited
proton pump shuts down
build up of protons in the plasma membrane
sphingosine kinase inhibited
sphingosine-1-phosphate levels fall
Akt function blocked
DNA repair mechanisms blocked
Chemotherapy and radiotherapy DNA lesions not able to be repaired
Cell dies
The result of this sequence of events is that idronoxil exquisitely sensitizes cancer cells to all
common frontline chemotherapies.
The drugs shown to be sensitized by idronoxil are:
 Cisplatin
 Carboplatin
 Paclitaxel
 Doxorubicin
 Gemcitabine.
The cancer cells shown to be sensitized to the above drugs cover all major cancer types.
The level of sensitization varies between 1,000 – 100,000. That is, the amount of drug such
as carboplatin required to kill cancer cells, can be reduced by up to as much as 100,000x in
the presence of idronoxil.
4.
NOX66
NOX66 was developed to protect idronoxil from being inactivated by the body. The
rationale is that if idronoxil could be protected from that inactivation and allowed to reach
the cancer cells intact, then there is no reason why we wouldn’t see in the clinic the sort of
exquisite level of sensitization to chemotherapy that is seen in the test-tube.
The inactivation process is due to a protective detoxification process in the body known as
Phase 2 metabolism. This is not peculiar to idronoxil…it happens to any foreign chemical
entering the body that is not soluble in water. Water-insolubility presents a problem for the
body because it means that it cannot eliminate the foreign chemical quickly in the urine,
something that it wants to do in case the foreign chemical is unsafe. But to get into urine,
compounds need to be water-soluble.
About 10% of all drugs that humans take are not soluble in water. This includes common
drugs such as aspirin, paracetamol and codeine. Idronoxil joins this category.
Phase 2 metabolism aims to make these drugs water-soluble, which it does by attaching
them to other compounds that are themselves soluble in water, the result being that the
combination now is water-soluble. One of the main compounds attached in this way is the
sugar, glucuronic acid. This attachment is conducted by enzymes known as transferases.
This attachment takes place in 2 main locations in the body. The first location is the lining of
the gut, which from an evolutionary point of view is how foreign chemicals traditionally
have entered the body. Transferase enzymes are present all the way from the stomach
down to the large bowel, ensuring that any water-insoluble compound that is absorbed
from the gut is converted into a water-soluble form before it reaches the bloodstream. The
second location is the liver, which will filter out of the bloodstream any water-insoluble
compound that managed to escape the frontline defense in the lining of the gut.
Drugs that are modified in this way by Phase 2 metabolism are inactive. The attached sugar
makes them too big to be able to attach to their target.
Activation of the drug requires the attached sugar to be removed, and just as an enzyme
was required to attach the sugar in the first place, another enzyme is required to remove it.
Most cells in the body contain the necessary uncoupling enzymes. This means that the drug
with its attached sugar needs to enter its target cell (eg a brain cell in the case of
paracetamol), have its attached sugar removed, find its target inside the cell and complete
its function. When it is finished doing its job, the drug leaves the cell, is picked up the liver
and re-attached to glucuronic acid and then passed out in the urine.
In other words, Phase 2 metabolism doesn’t block the ability of drugs like aspirin and
paracetamol to work; it simply ensures that they can be eliminated from the body over a
matter of several hours rather than staying and continuing to work for days because they
can’t be eliminated in the urine.
Idronoxil faces two hurdles that drugs like aspirin and paracetamol don’t. The first hurdle is
that idronoxil’s target is on the outside of the cell, so that it needs to enter the cell, have its
sugar removed, and then exit the cell and locate its target. That is a convolution that
virtually all other drugs subjected to Phase 2 metabolism don’t have to deal it.
But it is the second hurdle that is the clincher, and this is that cancer cells contain very little
enzyme activity for detaching the sugar. This means that once the idronoxil-sugar complex
gets inside the cancer cell, very little of it is released, and even when it is released, it needs
to complete its journey of exiting the cell before it can work. Most of the drug that entered
the cancer cell, however, remains stuck to its sugar and unable to work.
NOX66 has been designed to stop idronoxil being subject to Phase 2
metabolism. The drug is formulated in a fatty mixture designed to prevent
the transferase enzymes in the lining of the gut and in the liver from
attaching a sugar. The result being that the idronoxil theoretically reaches
the cancer cell in a form that is highly active, with its attached fatty construct
meant to actually assist the drug in accessing the cancer cell.