Post on 27-May-2015
description
Proline Ester Prodrug of Ara-C
Master’s Thesis Defense: Advisor: Nilesh Mudda Dr. James Wu
Overview
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Introduction
Objective
Methods
Results
Discussions
Conclusions
1.Introduction
Ara-C
Ara-C, a nucleoside analogue, is one of the most effective chemotherapeutic agents used for the treatment of acute myelogenous leukemia and various other hematological malignancies
Why Ara-C cannot be given orally?
Extensive first pass metabolism
Hydrophilic in nature
Low oral bioavailability (20%)
80% of the Ara-U can be recovered in the urine within 24 hours
1.Introduction
Ester Prodrugs
The use of ester prodrugs have reasonably been successful in optimizing the delivery characteristics of drugs
Advantages:
Prevention from metabolic degradation by enzymes such as thymidine phosphorylase and cytidine deaminase
Increase in bioavailability Examples: Valacyclovir Valganciclovir
Amino acid ester prodrugs have shown to be a good substrate for PEPT1 and PEPT2 membrane transporters
1.Introduction
hPEPT1
hPepT1, a human intestinal oligopeptide transporters, is expressed in the apical plasma membrane of the intestinal epithelia
It is normally restricted to small intestine
It has a very broad substrate specificity
PepT1 can also transport a large number of hydrophilic drugs. Examples of PepT1 substrates include the following:
Penicillin and cephalosporin antibiotics ACE inhibitors Anticancer drugs Pro-drugs of levodopa
Objective2.
The objective of this study To synthesize 5’-proline amino acid ester of Ara-C To evaluate in vitro capability of the prodrug to permeate Caco-2 cells To assess enzymatic activation in Caco-2 cell homogenate To check chemical stability at various physiological pH (7.4, 5 and 2) To evaluate the anti-proliferative action of 5’-proline amino acid
prodrug using cancer cells such as HeLa cells and Caco2 cells
Synthesis of Prodrug3.Method
5’ monoester prodrug
3’ monoester prodrug
3’ ,5’ diester prodrug
BOC-PRO-OH
Ara-C
Synthesis of Prodrug3.Method
5’ monoester prodrug
BOC-PRO-OH
Ara-C
: (1:1)
5’-Pro-Ara-C
Hydrolysis Assay Principle3.Method
5’-Pro-Ara-C Ara-C
Prodrug stability at various physiological pH (7.4, 5 and 2) Conversion to the parent drug in the presence of enzymes
Hydrolysis of proline ester of drug to Ara-C
Hydrolysis Assay Procedure3.Method
1% Triton in PBS Maintained at 37OC
Samples were analyzed
200µl of prodrug was added in to the cell homogenate
200µl of the solution was removed and diluted in 800µl of 10% TFA in water
XTT Assay Principle3.Method
XTT assay is based on the ability of metabolic active cells to reduce the tetrazolium salt XTT to orange colored compounds of formazan
XTT Assay Procedure3.Method
Concentrations of Prodrug - 0.1-10µM Concentrations the Drug - 10-500µM
50µl of the XTT solution was added
Measured at 490nm
Suspended cells
96-well plate
Cell Permeability Assay3.Method
> 500 ohm cm2
Apical layer
Basolateral layer Inserts
1.5 ml of the test solution in the apical compartment
200µl of the solution was withdrawn from basolateral compartment and diluted in 0.05N HCl
Samples were analyzed
Hydrolysis study results4.Results
The apparent first-order degradation rate constants of various prodrugs and their parent drugs at 37°C were determined by plotting the logarithm of concentration of prodrug remaining as a function of time.
k = 2.303 × slope (log C vs. time)
The degradation half-lives were then estimated by the equation,
t1/2 = 0.693/k
Hydrolysis study results4.Results
HYDROLYSIS OF PRODRUG IN CACO-2 CELL HOMOGENATE:
Half-life of Prodrug in Caco-2 homogenate :15.35 min
Hydrolysis study results4.Results
HYDROLYSIS OF PRODRUG IN PH 7.4:
Half-life of Prodrug in pH 7.4: 5015.19 min
Hydrolysis study results4.Results
HYDROLYSIS OF PRODRUG IN PH 5:
Half-life of Prodrug in pH 5.0: 1504.55 min
Hydrolysis study results4.Results
HYDROLYSIS OF PRODRUG IN PH 2:
Half-life of Prodrug in pH 2.0: 1003.03 min
Hydrolysis study results4.Results
Estimated Half-life (t1/2)(min)
Prodrug Caco-2 cell pH 7.4 pH 5 pH 2
Pro-Ara C 15.35 5015.19 1504.55 1003.03
XTT study results4.Results
CYTOTOXIC STUDY OF ARA-C IN HELA CELLS
IC50 of Ara-C in HeLa cell: 392.3 µM
XTT study results4.Results
CYTOTOXIC STUDY OF PRO-ARA-C IN HELA CELLS
IC50 of Prodrug in HeLa cell: 2.8506 µM
XTT study results4.Results
XTT study results4.Results
CYTOTOXIC STUDY OF ARA-C IN CACO-2 CELLS
IC50 of Ara-C in Caco-2 cell: 216.761 µM
XTT study results4.Results
CYTOTOXIC STUDY OF PRO-ARA-C IN CACO-2 CELLS
IC50 of Prodrug in Caco-2 cell: 0.27 µM
XTT study results4.Results
Compound IC50 (µM)
HeLa Cell Caco-2 Cell
Ara-C 392.3 216
Proline-Ara-C 2.85 0.27
Where, dQ/dt : rate of appearance of drug on the basolateral side (μM /s) C0 : initial drug concentration on the apical side (μM/cm3) A : surface area of the monolayer(cm²)
In this study, A = 7.536 cm2
Permeability study4.Results
The apical to basolateral permeability of each drug, Ara-C ester prodrug and Ara-C, was calculated using the following equation:
Permeability study4.Results
CACO-2 CELL PERMEABILITY STUDY OF ARA-C:
Papp of Ara-C: 1.34 × 10-6 cm/sec
Permeability study4.Results
CACO-2 CELL PERMEABILITY STUDY OF Proline-ARA-C:
Papp of Proline-Ara-C: 3.98 × 10-6 cm/sec
Permeability study4.Results
Ara-C Proline Ara-C
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Enhanced ratio of Permeability
Discussion5.
In this project, 5’-Pro-Ara-C was synthesized and preliminarily evaluated for their in vitro capability to permeate Caco-2 cells, a surrogate for intestinal transport
Moreover, its chemical stability at various physiological pH (7.4, 5 and 2) and its enzymatic activation in Caco-2 cell homogenate were also assessed
Finally, the possibility of anti-proliferative action of 5’-proline amino acid prodrug was also tested using cancer cells such as HeLa cells and Caco2 cells
Discussion5.
The rate of hydrolysis in buffer solution was stable and was in the order of 7.4 < 5 < 2, which was consistent with previous findings that Proline ester posses high chemical stability at physiological pH
The chemical stability in pH 7.4 and enzymatic activation in Caco-2 cell homogenate was found to be comparable to Valacyclovir and Valganciclovir
Discussion5.
Rate of permeation of prodrug was found to be 3 fold greater than to the parent drug
Furthermore, prodrugs were rapidly hydrolyzed to the parent drugs by the intracellular hydrolysis, following the apical membrane transport
Amino acid ester prodrugs significantly improved the cellular uptake of the parent drugs via peptide transport, PEPT1 mechanism which can be seen by the results of cytotoxicity results
Conclusion6.
5’ proline ester of Ara-c exhibited useful characteristics such as good solution stability and relatively fast enzymatic conversion rates
The anti-proliferative activity of Pro-Ara-C in both the cell lines was found to be significantly higher than the parent drug
These results suggest that Pro-ara-C could effectively improve the oral absorption of Ara-C via the carrier-mediated transport pathways