Design of Novel Pro-drugs for the Treatment of Cystinosis

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Design of Novel Pro-drugs for the Treatment of
Cystinosis

• Dr D. Cairns
• Institute of Pharmacy and Chemistry
• University of Sunderland,
• Sunderland, UK

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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

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Cystinosis - symptoms

• Renal Fanconi syndrome (impairment in proximal
  tubular function)
• Polyuria (excessive urination)
• Polydipsia (excessive thirst)
• Hypokalaemia (low levels K)
• Hypophosphataemia (low levels PO4-)

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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

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Cystinosis - Treatment

• Administration of electrolytes, glucose etc. to
  address imbalance
• Renal dialysis, transplant
• Eye drops, corneal transplant

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Drug Treatment

• Cysteamine (mercaptamine)
• Cystagon (mercaptamine bitartrate)

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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

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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

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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

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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-?-glutamyl-p-nitroanilide and measurement of
  absorbance at 530 nm

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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

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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.

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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

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