Folate pro-drug of cystamine as an enhanced treatment for nephropathic cystinosis

Article abstract of DOI:10.1016/j.bmcl.2011.02.048

Nephropathic cystinosis is a rare autosomal recessive disease characterised by raised intracellular levels of the amino acid, cystine. If untreated, the disease, progressively deteriorates towards end stage renal disease (ESRD) at the end of the first dec

Full text of DOI:10.1016/j.bmcl.2011.02.048

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2502–2504
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Folate pro-drug of cystamine as an enhanced treatment
for nephropathic cystinosis
Ziad Omran a,b, , Graeme Kay a, Emma E. Hector a, Rachel M. Knott a, Donald Cairns a
⇑
a School of Pharmacy and Life Sciences, Robert Gordon University, Aberdeen AB10 1FR, UK
b School of Pharmacy, The University of Nottingham, University Park, Nottingham, NG7 2RD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Nephropathic cystinosis is a rare autosomal recessive disease characterised by raised intracellular levels
of the amino acid, cystine. If untreated, the disease, progressively deteriorates towards end stage renal
disease (ESRD) at the end of the first decade. The disease is caused by a defect in the lysosomal transport
mechanism for cystine. The treatment of choice is the aminothiol cysteamine which acts as a lysine
mimic. However, cysteamine possesses an offensive taste and smell and irritates the gastrointestinal tract
leading to nausea and vomiting following administration. Furthermore, the rapid metabolism of cyste-
amine requires oral administration every 6 h for life, in consequence, the patient compliance is poor.
As part of our continuing work to obtain new pro-drugs for the treatment of this genetic disease, we have
synthesised a folate derivative of cystamine, the disulfide derivative of cysteamine. This new pro-drug
was non cytotoxic, showed greater ability to deplete intralysosomal cystine than the current treatment,
and, in fact has been the most effective reducer of intralysosomal cystine discovered in our laboratories to
date.
Received 10 February 2011
Accepted 12 February 2011
Available online 23 February 2011
Keywords:
Cystinosis
Cysteamine
Cystamine
Pro-drugs
Folic acid
Ó 2011 Elsevier Ltd. All rights reserved.
Nephropathic cystinosis is a rare, autosomal, recessive disease
with an estimated incidence of 1 case per 100,000 to 200,000 live
births. It is characterised by raised intracellular levels of cystine
(the dimer of the essential amino acid, cysteine). The disease
symptoms are renal tubular Fanconi syndrome, poor growth, hyp-
ophosphatemic rickets, impaired glomerular function, and accu-
mulation of cystine crystals in almost all cells, leading to tissue
destruction. The typical untreated child has short stature, rickets,
and photophobia.1 The responsible gene, CTNS, encodes cystinosin,
a 367 amino acid integral membrane protein that transports cys-
tine out of the lysosome.2
Cysteamine has an offensive taste and smell and irritates the
gastrointestinal (GI) tract leading to nausea and vomiting following
administration. In addition, cysteamine and its metabolites are ex-
creted in breath and sweat, which leads to halitosis and body
odour. Furthermore, some patients exhibit more serious side-ef-
fects, such as neutropenia. As a result of these problems, patient
compliance can be poor. Finally, because of a combination of high
first-pass metabolism and a short half-life, cysteamine only re-
moves cystine crystals for a period of 6 h after the medication
has been taken. This means that the drug must be given every
6 h, every day for life.4
Treatment of cystinosis involves symptomatic and specific
therapy. The symptomatic treatment consists in the administration
of fluids and electrolytes to reverse the effects of Fanconi
In an attempt to overcome these unpleasant side-effects, recent
work in our laboratories has concentrated on the development of
novel pro-drugs of cysteamine and cystamine (the disulphide
counterpart of cysteamine) that have a similar, or enhanced
efficacy than cysteamine, in lowering the intracellular levels of
cystine, while displaying fewer side effects and a longer half-life.5,6
As part of this project, we synthesised a folate derivative of
cystamine 1, whereby two molecules of cystamine were covalently
attached to the carboxylate termini of the folic acid by formation of
amide bonds. Folic acid pro-drugs are reported to increase the
renal uptake of the parent drugs.7 It was envisaged that this would
be advantageous for cystinotic patients as the kidneys are one of
the first organs affected in nephropathic cystinosis.
syndrome, phosphate and vitamin D to treat rickets. L-Carnitine
replacement therapy may also be needed as carnitine concentra-
tions in plasma and muscles are quite depleted in cystinotic
children. The specific therapy for cystinosis is cysteamine
(H2NCH2CH2SH), which acts to lower intracellular levels of cystine
by forming a cysteamine–cysteine mixed disulphide which can
egress the lysosome using the lysine transporter excretion
pathway which remains intact in cystinosis.3 Treatment with
cysteamine is, however, associated with major problems.
The pro-drug 1 was obtained by peptidic coupling8 of folic acid
2 with two molecules of mono-Boc-cystamine.9 The deprotection
⇑
Corresponding author. Tel.: +44 115 9515025; fax: +44 115 9515102.
E-mail address: ziad.omran@nottingham.ac.uk (Z. Omran).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.048

Z. Omran et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2502–2504
2503
Scheme 1. Chemical synthesis of prod-drug 1. Reagents and conditions: (i) tert-butyl 2-(2-(2-aminoethyl)disulfanyl)ethylcarbamate (2.5 equiv), DEC (4 equiv), 12 h, rt; (ii)
4 N HCl in dioxane, 30 min, rt.
of the product so obtained, under acidic conditions5,10 yielded the
attempted water-soluble pro-drug 1 with an overall yield of 92%,
Scheme 1.
The cytotoxicity of pro-drug 1 was determined to confirm that
any change in cystine burden observed on treatment was not a
consequence of cell death or an increase in cell proliferation. The
test was carried out on human cystinotic fibroblasts, the standard
cell line for determination of cystine levels, using the Alamar blue
cell proliferation assay.11 The cystinotic fibroblasts were subjected
to 50 lM of the current treatment, cysteamine, and the compound
1, and cell growth measured over a 72 h period. The results, dis-
played in Figure 1, show that there is no significant difference in
cell growth of the cystinotic fibroblasts through 72 h which con-
firms that cysteamine and our pro-drug 1 have negligible toxicity
at the concentrations and time scales utilised in this Letter.
Intralysosomal cystine was measured using the commercially
available Thiol and Sulfide Quantification KitÒ (Molecular Probes).
Cystinotic fibroblasts were treated for 24 h with 50 lM of cyste-
amine or compound 1. Intralysosomal cystine was isolated from
the lysates, converted to cysteine and the concentration was then
measured on a multiwell plate reader at 410 nm by comparison
to known standards of cysteine.
The results obtained for compound 1 when compared with the
control and cysteamine are displayed in Figure 2. The data are pre-
sented as micromolar cysteine per mg of protein relative to control
as determined by the Bradford method.12 It can be concluded that
Figure 2. Relative cystine depletion in cystinotic fibroblasts measured after 24 h of
incubation with cysteamine and pro-drug 1. The data shown are the mean of five
independent experiments S.E., each measurement was carried out in triplicate. The
level of significance was determined using a modified one tailed Students t-test,
where t = (mean À 1)/SEM at n À 1 d.f., where n = 5 (⁄p <0.05, ⁄⁄p <0.005, #p <0.05).
50
fibroblasts relative to the control and that this depletion is 3–4-
fold greater than that exerted by 50 M of the current treatment,
cysteamine, after 24 h incubation: a result which means this pro-
drug is the most potent compound reported from our laboratories
to date.
In conclusion, a new folate pro-drug of cystamine has been
developed. This new pro-drug is non-cytotoxic up to 72 h of
incubation and is 3–4 times more active in depletion of intralysos-
omal cystine than the current treatment when evaluated in cul-
lM 1 significantly depletes the levels of cystine in cystinotic
tured cystinotic fibroblasts: a result which means 1 is the most
potent prodrug to emerge from our prodrug programme.
Further work is currently underway in our laboratories to ad-
dress the tissue specific nature of 1 and to investigate in vivo phar-
macokinetic parameters using a murine model of cystinosis. If
these studies are successful, we aim to produce an orally active
treatment for cystinosis with a less frequent administration regi-
men. This in turn may lead to improved compliance and a signifi-
cant improvement in the quality of life of patients with
nephropathic cystinosis.
l
Acknowledgements
The authors acknowledge financial support from Cystinosis
Foundation, Ireland. The use of EPSRC funded mass spectrometry
facilities at Swansea, UK is also acknowledged.
Supplementary data
Supplementary data (synthetic procedure for compound 1, Ala-
mar blue and thiol assays protocols) associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2011.02.048.
References and notes
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Figure 1. Alamar blue absorbance at 6, 24, 48 and 72 h intervals for cysteamine and
compounds 1. The data shown are a mean of eight independent experiments S.E,
each measurement was carried out in triplicate.

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