Arsenic and secret antidotes

We’ve just had a new paper published – I got an email today telling me the online version has just gone live.  It’s always nice to get some of our work through the hurdles of academic ‘peer review‘! The hardcopy version won’t be printed in the  Journal of Pharmacy and Pharmacology for a few days, but you can get a look at the abstract here.

So is this piece a brag-blog? Hopefully not. Academic papers don’t really allow us to tell the full story behind a piece of work, the bits that make science fun (as opposed to just important!) So hopefully this post can do a bit of that…

Arsenic as a drug

The drug we were working on, GSAO, is based upon arsenic….yes, the toxic metal of ‘Old Lace’ movies and (maybe) ‘bumping off Napoleon’ fame!  Arsenic has a long history as a medicine: as a traditional Chinese remedy it’s been used for over 2000 years, and in 1906 the famous German scientist Paul Ehrlich, discovered arsphenamine, for the treatment of syphillis. Sold as Salvarsan, arsphenamine became the world’s first ‘blockbuster’ drug. By the mid 1940’s far more ‘selective’ antibiotics like penicillin were available: they killed bacteria better and at the same time had less side-effects. So as the 20th century progressed, arsenic moved from drug to toxin and stayed there for 50 years until arsenic trioxide  (under the tradename Trisenox) became a treatment for leukemia, spearheading a resurgence for arsenic as a cancer therapy.

In 2000, before Trisenox was available, Cancer Research UK (or Cancer Research Campaign as it was at that time) started working up GSAO for Phase I clinical trials. GSAO is the ‘shortname’ for 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid, and was first made by a team of Australian scientists headed by Phil Hogg (who is also one of our paper’s co-authors).  At the Formulation Unit we had the job of discovering a way of producing a stable form of the drug that could be safely given to patients in clinical trials. After this we had to test the drug over several years to prove that it remained chemically intact and still safe for patients to take. This work was published by my colleague, Dr Moira Elliott, last year.

Chemical structure of GSAO
Chemical structure of GSAO

What’s so special about GSAO

GSAO works by throwing a drug spanner in the biochemical works of a cell’s power generator. GSAO is a pro-drug: that is an inactive chemical that is converted, by the body, to an active drug at the ‘site of action’. GSAO enters a cell and is converted to two different chemicals in sequence, eventually becoming CAO (4-(N-(S-cysteinylacetyl)amino) phenylarsonous acid). CAO interferes with the cells powerhouses’, the mitochondria, by binding and crosslinking two cyteines on a particular protein called adenine nucleotide translocase (ANT). This is like putting the plug in the bath while the taps are on full: bottling up ANT clogs the mitochondria, leading to superoxide ‘flooding the kitchen’ and the whole cell just freezes while it tries to save it’s CD collection and call a plumber!

ANT is only active in growing (or proliferating) cells, but it was found is that GSAO worked better in stopping endothelial cells  growing (the cells that make up blood vessels) rather than tumor cells. One of the key features of cancer is a network of irregular and leaky new blood vessels that sprout to feed a growing tumor: this is called ‘angiogenesis’. By stopping angiogenesis it was hoped that GSAO would treat tumors.

Sterility testing

So we needed to formulate and make hundreds of vials of GSAO so that the drug could be injected into patients at the right dose levels. Injectable drugs need to be tested both chemically and biologically, and one key experiment is the ‘sterility test’: are the vials contaminated with micro-organisms that will make the patient sick when they are injected into the blood stream?

The sterility test involves dissolving the drug and letting it sit in biological growth media for 2 weeks: if bugs don’t grow, it’s a pass, but if they do grow, it’s a fail. One part of the test is to make sure that micro-organisms will grow during the test: a vial may be contaminated, but if the bugs that are in it can’t grow (for whatever reason) then the contaminated drug will look like a ‘pass’!  And that’s where our problems started, because – as with other arsenical drugs – GSAO kills bacteria.

How scary it was 60 years ago!

In 1952 at team working at Boots in Nottingham published a paper showing the problems with sterility testing two other arsenical drugs (neoarsephenamine and sulpharsephenamine). Prior to this work it had been believed that arsenicals were ‘self sterilising’ and so were not sterility tested at all after manufacture. However, the Boots microbiologists clearly showed that Clostridia bacteria could survive for at least a year in the presence of neoarsephenamine! (Clostridia is the ‘C’ in C. difficile, but also include the bugs responsible for tetanus and botulism.)

Despite this paper being the only reference anywhere on the sterility testing of arsenical drugs, in the end it wasn’t much use to us – beyond suggesting that we use a method involving digested heart muscle to reduce (not eliminate) GSAO’s anti-bacterial activity.

The smelly bit

We’d tried lots of sulphur containing chemicals to deactivate GSAO without success, and it was during this time that we came across a paper using a chemical called DMP (dimercatopropanol) to measure GSAO levels. This team had allowed DMP bind to the GSAO and then, by measuring how much how much DMP was left, they could calculate how much GSAO has been present originally (the technical term is back-titration). When we tested DMP it deactivated GSAO nicely, but it had two other important properties: DMP didn’t stop bacteria growing (otherwise we’d just be swapping one antibacterial for another!) and we could filter-sterilise it (so it wouldn’t actually introduce extra bacteria into the test). The only minus was that it stinks, really stinks – even a small amount would make the lab unpleasant to work in. (DMP is so smelly, I was planning to use micrograms of it for the WSWEK performance).

Structural formula of dimercaprol, 2,3-dimerca...
Structural formula of dimercaprol, 2,3-dimercaptopropanol. (Photo credit: Wikipedia)

DMP also has a secret past! It was discovered at Oxford in 1940 as an antidote to the chemical weapon Lewisite, which was an arsenic-containing toxic gas. Smelling of geraniums, Lewisite is now obsolete even as a weapon, but residual WWII stocks left over by the Japanese in China still occasionally cause fatalities. DMP is also used a treatment for posioning by other metals and chemicals, leaving the possibility that the process we published in our paper could be useful for the sterility test of lots other ‘inorganic’ drugs.

Our research also confirmed that arsenicals seem to have greater activity against a bacterial species called Staphylococcus aureus, while not ground breaking it’s interesting because these bugs are the ‘SA’ in MRSA.

And GSAO in patients…?

Finally, how did GSAO do in clinic? Well the full trial paper hasn’t been published yet, but Cancer Research UK have put interim results on their website here. GSAO gave stable disease in 8 out of 19 patients and, based on GSAO, the team in Australia has already started clinical trials on a new drug PENAO.

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