Proline ester prodrug of cytarabine

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’ ,5’ diester prodrug

BOC-PRO-OH

Ara-C

Synthesis of Prodrug3.Method


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 OF PRODRUG IN CACO-2 CELL HOMOGENATE:

Half-life of Prodrug in Caco-2 homogenate :15.35 min


HYDROLYSIS OF PRODRUG IN PH 7.4:

Half-life of Prodrug in pH 7.4: 5015.19 min


HYDROLYSIS OF PRODRUG IN PH 5:



HYDROLYSIS OF PRODRUG IN PH 2:



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


CYTOTOXIC STUDY OF PRO-ARA-C IN HELA CELLS

IC50 of Prodrug in HeLa cell: 2.8506 µM



CYTOTOXIC STUDY OF ARA-C IN CACO-2 CELLS

IC50 of Ara-C in Caco-2 cell: 216.761 µM


CYTOTOXIC STUDY OF PRO-ARA-C IN CACO-2 CELLS

IC50 of Prodrug in Caco-2 cell: 0.27 µM


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:


CACO-2 CELL PERMEABILITY STUDY OF ARA-C:

Papp of Ara-C: 1.34 × 10-6 cm/sec


CACO-2 CELL PERMEABILITY STUDY OF Proline-ARA-C:

Papp of Proline-Ara-C: 3.98 × 10-6 cm/sec


Ara-C Proline Ara-C

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3.01

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

Education

Proline ester prodrug of cytarabine