Design of Novel Pro-drugs for the
Treatment of Cystinosis
Dr D. Cairns
Institute of Pharmacy and Chemistry
University of Sunderland,
Sunderland, UK
Nephropathic cystinosis
Rare autosomal recessive metabolic disease
 Characterized by elevated levels of
intracellular cystine
 Caused by defect in lysosomal transporter
mechanism for cystine
 CTNS gene described

Cystinosis - symptoms
Renal Fanconi syndrome (impairment in
proximal tubular function)
 Polyuria (excessive urination)
 Polydipsia (excessive thirst)
 Hypokalaemia (low levels K+)
 Hypophosphataemia (low levels PO4-)

Cystinosis - symptoms
Crystals of cystine present in lysosomes,
bone marrow aspirates, leukocytes, cornea
and conjunctiva
 Photophobia, headaches, burning/itching of
eyes
 Growth retardation, rickets, muscle myopathy
 CNS involvement, hypothyroidism
 Hepatic and GI complications

Cystinosis - Treatment
Administration of electrolytes, glucose etc. to
address imbalance
 Renal dialysis, transplant
 Eye drops, corneal transplant

Drug Treatment
Cysteamine (mercaptamine)
 Cystagon (mercaptamine bitartrate)

NH2
HS
2
COO
H
H
H3N
SH
COO
H3N
COO
NH3
S
Cysteine
S
H
Cystine
NH3
HS
H
COO
NH3
H
H3N
H3N
Lysine
COO
NH3
S
S
Cysteine-cysteamine
mixed disulphide
Cysteamine - problems
Poor patient compliance due to offensive
taste and smell
 Excretion in breath and sweat leads to
halitosis and body odour
 Release in GI tract can cause nausea,
vomiting, irritation etc.
 Reformulate - sustained release, coated
pellets

Pro-drugs of Cysteamine
Pharmacologically inactive molecule
metabolically activated in vivo to yield active
compound.
 Release of cysteamine is intra-cellular
 Avoids GI related side-effects
 Minimises odour problems
 Increased lipophilicity improves uptake into
cells, better PK and PD profiles

O
O
NH
S
O
O
O
O
O
H
N
N
H
S
O
H
N
N
H
S
O
O
2
2
O
O
O
O
O
H
O
NH
S
N
O
S
O
HN
SH
O
HN
HN
S
.HCL
HN
S
S
O
N
S
O
O
O
O
N
S
N
H
N
S
N
H
N
S
O
O
O
O
N
H
H
N
S
O
O
2
N
H
H
N
O
S
O
O
2
N
H
H
N
S
O
2
g Glutamyl Transpeptidase
Provides mechanism for energy-driven
transport of amino acids (AA) into cells
 g GT situated on external cell membrane.
High levels present in kidney cells
 Accepts amino acids (Cys and Met)
 Transfers g Glu from GSH to external amino
acid
 g Glu-AA complex transported into cell

O
O
O
H
N
O
O
HN
O
O
O
S
O
O
HN
SH
O
H
N
O
O
N
2
H
N
O
O
S
O
O
H
N
O
O
HN
H
N
O
SH
O
O
O
O
O
O
H
N
O
O
H
N
HN
O
S
O
O
H
N
O
HN
O
SH
O
O
H
N
O
O
S
O
H
N
O
N
O
O
HN
2
O
O
O
O
O
O
O
S
2
H
N
O
O
N
S
O
O
HN
O
SH
O
N
O
S
Cell culture
Pro-drugs added to CHO Cell lines
genetically modified to express g GT.
 Cytotoxicity and LD50 determined (SRB)
 Cells lysed with Triton X 100 and breakdown
of compound followed by hplc-m/s
 Histochemical detection of g GT using L-gglutamyl-p-nitroanilide and measurement of
absorbance at 530 nm

289 Chlr-CHO
3.0
3.0
2.5
2.5
OD 530nm
OD 530nm
289 K1-CHO
2.0
1.5
1.0
2.0
1.5
1.0
0.5
0.5
0.0
0.0
1
10
100
1000
1
10000
10
Drug Conc M
1000
10000
Drug Conc M
289 5' g GT
289 3' g GT
3.0
3.0
2.5
2.5
OD 530 nm
OD 530 nm
100
2.0
1.5
1.0
2.0
1.5
1.0
0.5
0.5
0.0
0.0
1
10
100
Drug Conc M
1000
10000
1
10
100
Drug Conc M
1000
10000
Results
Pro-drugs synthesized in good yield and fully
characterised spectroscopically
 Pro-drugs taken up into CHO cells
expressing g-GT and detected by hplc
 Levels of pro-drug fall wrt time but…..
 Unable to detect release of cysteamine using
hplc-m/s

Conclusions
g-glutamylcysteamine prodrugs can be readily
synthesized in significant quantities
 These prodrugs are almost entirely non-toxic
to cells and are taken up successfully into
CHO cells in vitro
 The receptor enzyme g-glutamyl
transpeptidase is a valid vehicle for targetting
cysteamine to the cells that most require it.

Acknowledgements
Wendy Cardwell and Dr Roz Anderson,
University of Sunderland (synthesis)
 Prof. Geoff Rowley and Dr Berwyn Owen,
University of Sunderland (formulation)
 Dr Malcolm Coulthard, RVI, Newcastle
 Dr Andy Hall, University of Newcastle (cell
culture)
 Northern Region NHS for funding support
