Dr Roz Anderson, Professor Paul Groundwater and Mr Pratap Suryadevara, Sunderland Pharmacy School, University of Sunderland
Although the vast range of research on gene therapy is encouraging, it is still a long way from offering a possible treatment, or even cure, for cystinosis. In the meantime, we are working on a strategy to improve the current treatment, cysteamine, using the prodrug approach. Our aims for cysteamine are to make it:
By achieving these qualities, the unpleasant side effects will be decreased, while the bioavailability (the amount of drug actually available after ingestion, metabolism, excretion, and distribution around the body) will be increased. Usually, increased bioavailability results in the advantage of a decreased dose being required, which would further reduce the incidence of side effects and may allow fewer doses per day.
A prodrug is an inactive form of a known drug; it has no medicinal effect itself, but is activated in the body by a defined mechanism and breaks down to produce the active drug. By careful prodrug design, the active drug can be released in a particular location of the body or even over a desired timescale. There are a number of prodrugs already in clinical use; they overcome a range of problems associated with the therapeutic drug, such as poor absorption from the gastrointestinal tract (Pivampicillin is a prodrug of ampicillin, a poorly absorbed penicillin antibiotic) and rapid metabolism and excretion (Bambuterol is a prodrug of terbutaline, an anti-asthmatic drug that suffers from rapid metabolism and excretion when taken orally, which drastically reduces the available dose after ingestion), although some prodrugs are used to target the therapeutic drug to a particular location in the body (Levodopa is a prodrug of L-DOPA, a neurotransmitter that can be deficient in Parkinson’s Disease; this prodrug releases L-DOPA in the brain where it is required).
Usually administered as its bitartrate salt, Cystagon®, cysteamine is pungent, tastes unpleasant and causes undesirable side effects after ingestion, such as nausea, vomiting, halitosis, body odour and stomach ulcers. The problems are exacerbated by the high and frequent dose required for effective treatment, which are also the most probable cause of other side effects noted recently. A considerable amount of the cysteamine dose is lost after absorption from the gastrointestinal tract through liver metabolism and excretion in the urine, such that the amount actually available after an oral dose is significantly lower than that ingested. Serum cysteamine continues to be metabolised after absorption, with a steady decline in cysteamine concentration to lower than the effective therapeutic concentration in about 6 – 8 hours.
Cysteamine is unique in its ability to treat cystinosis effectively, most likely because it can counteract the clinical problems of cystinosis on several levels. First, there is the way in which cysteamine depletes the accumulated cystine from the lysosomes of cells. Cysteamine can enter cells and the lysosome relatively easily; once in the lysosome, it reacts with accumulated cystine to form a mixed disulfide. The disulfide is sufficiently similar in size (Figure 1A), shape and distribution of charge (Figure 1B) to the natural amino acid, lysine, that it can hijack the lysine transporter system, by which it leaves the lysosome.
Figure 1A: Overlay of L-lysine with L-cysteine-cysteamine dimer showing the similarity in the shape, size and relative position of functional groups.
Figure 1B: Comparison of surface charge distribution of L-lysine (left) with the disulfide, L-cysteine-cysteamine dimer (right). [Negatively charged regions shown in red; positively charged regions shown in blue; white represents neutral.]
Although there are a number of molecules that can enter the cell and lysosome and even react with cystine to form the mixed disulfide, the other candidates would not be able to hijack the lysine transporter, so would not have a defined route of excretion and may accumulate in the lysosome, eventually causing another clinical problem.
Secondly, cysteamine is a well-studied antioxidant, reacting with free radicals to inhibit their reaction with cell structures and minimising radical damage, such as that caused by irradiation. There is growing evidence that apoptosis (programmed cell death) is altered in cystinosis; this process is strongly linked to oxidative damage in cells and high concentrations of cellular oxidants can induce apoptosis through several pathways, including alteration of gene expression through modification of protein / DNA binding. There is a possibility that the antioxidant action of cysteamine contributes to its success in treating cystinosis.
With our current knowledge, we cannot improve on cysteamine; we can only improve its ability to reach and act upon the cells that most require its action.
At Sunderland, Pratap Suryadevara’s PhD work is aimed at the design, synthesis and evaluation of a number of prodrugs of cysteamine. We designed the prodrugs to have little or no taste and smell, to cause minimum disruption to the membranes of the gastrointestinal tract, to be rapidly and effectively absorbed, to resist liver metabolism and the accompanying excretion, and to resist breakdown in the serum, but to be absorbed effectively into those cells with an active glutathione cycle, which includes kidney, liver, pancreas, and skin cells, amongst others. We believe that the glutathione cycle is intricately linked to the symptoms and clinical manifestation of cystinosis, if not to the progression of the disease itself, and believe that it is a valid ‘handle’ to target cysteamine to the affected cells.
We have synthesized 8 – 10 prodrug candidates and have developed a reliable and sensitive analytical technique to identify and quantify cysteamine in solution. We are currently evaluating the prodrugs for their ability to enter kidney cells and to release cysteamine intracellularly. The initial results are encouraging: the first prodrug candidate was effectively absorbed by the kidney cells and released cysteamine from the prodrug after internalisation.
In collaboration with other researchers, we will quantify the reduction in taste and smell of the prodrugs in comparison to cysteamine using an electronic nose and electronic tongue.
The next stage of the work, in collaboration with Dr William Van’t Hoff, Great Ormond Street Hospital, will evaluate all of the prodrug candidates in normal human kidney and skin cells for their ability to be absorbed and release cysteamine intracellularly. They will then be tested for the same abilities against cystinotic kidney and skin cells; these experiments are important to ensure that the glutathione cycle remains active in cystinotic cells and that the absorption of prodrug and release of cysteamine are effective in these cells.
After these experiments, we will analyse and rationalise the data to choose the best prodrug candidates to go forward for further testing. Finally, the cystine depleting ability of the best candidates will be evaluated in collaboration with Dr Neil Dalton and his team at Guys Hospital, London.
We are in discussion with Orphan Europe on the development of the best prodrug candidates through pharmacokinetic and toxicological studies towards future clinical trials.