Lipophilic Prodrugs of SN38: Synthesis and in Vitro Characterization

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Lipophilic prodrugs of SN38: synthesis and invitro characterisation towards oral chemotherapy Vaskor Bala, Shasha Rao, Peng Li, Shudong Wang, and Clive A. Prestidge Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00785 • Publication Date (Web): 01 Dec 2015 Downloaded from http://pubs.acs.org on December 7, 2015

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Molecular Pharmaceutics

Lipophilic prodrugs of SN38: synthesis and in-vitro characterisation towards oral chemotherapy Vaskor Bala, Shasha Rao, Peng Li, Shudong Wang and Clive A. Prestidge* School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia. *corresponding author; E-mail: [email protected]; Tel: +61 8 8302 3569, Fax: +61 8 8302 3683

Abstract: SN38 (7-ethyl-10-hydroxy camptothecin) is a potent anticancer agent belonging to the camptothecin family; however, its oral delivery is extensively restricted by poor solubility in pharmaceutically acceptable excipients and low trans-mucosal permeability. Lipid-based carriers are well-known for their ability to improve oral absorption and bioavailability of lipid soluble and highly permeable compounds. Thus, this study has focused on improving solubility in lipid excipients, controlling stability and enhancing trans-mucosal permeability of SN38 by specific chemical modification. To achieve these aims, a series of lipophilic prodrugs were designed and synthesised by esterification at the C10 and/or C20 positon(s) of SN38 with dietary fatty acids of diverse hydrocarbon chain lengths. The solubility of these novel prodrugs in long chain triglycerides was increased up to 444-fold, and cytotoxicity was significantly reduced in comparison to SN38. The prodrugs were stable in simulated gastric fluids, but exhibited different rates of hydrolysis (t1/2 < 5 min to t1/2 > 2 hrs) in simulated intestinal fluids (in the presence of enzymes) depending on the alkyl chain length and the position modified. A predictable reconversion of prodrugs to SN38 in plasma was also confirmed. Based on these studies, SN38-undecanoate (C20) was identified as the optimal prodrug. Finally, in vitro permeability and uptake studies in rat intestinal mucosal membrane using an Ussing chamber showed significant improvement in trans-epithelial drug transport and cellular uptake. Together, these results indicate that well designed lipophilic prodrugs have potential for the efficacious and safe oral delivery of SN38. Keywords: Drug delivery; SN38 (7-ethyl-10-hydorxy camptothecin); lipophilic prodrug; simulated gastric/intestinal conditions; cytotoxicity; permeability.

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1

Introduction

Camptothecin (CPT, Figure 1(a)) and its semi-synthetic analogues are a widely studied class of anticancer agents that act by inhibiting DNA topoisomerase 1 (topo-1), the enzyme responsible for adjusting and controlling the topology of DNA.1 CPT binds to topo-1 and DNA, forms a ternary complex in the presence of tyrosine, which prevents DNA religation and replication, and thus causes apoptosis.2-4 Interestingly, CPT cytotoxicity depends on the integrity of the lactone ring, which undergoes pH dependent reversible hydrolysis. At low pH (< 4.5) the lactone is stable and pharmacologically active, whereas at high pH (> 9), it converts into the therapeutically inactive carboxylate form.5 SN38 is one of the most potent CPT derivatives, but it is a challenging class-IV (low solubility and low permeability) compound according to the Biopharmaceutics Classification System (BCS) and direct formulation and delivery is restricted.6 A water soluble prodrug of SN38, irinotecan (CPT-11, Figure 1 (b)), is clinically used as a low pH (~3) intravenous infusion for the treatment of colorectal cancer.7 CPT-11 is metabolized primarily in the liver by carboxylesterases to generate the 100 to 1000 times more potent SN38.8 During metabolic transformation, the majority of CPT-11 and its metabolites are excreted via either biliary or renal elimination.9, 10 In fact, only 2 to 8% (depending on the genetic nature of the patient) of dosed CPT-11 is converted into SN38 and performs its therapeutic action.11 Along with this highly variable inter-patient reconversion, the short plasma half-life, and severe dose limiting diarrhoea and neutropenia are major challenges for a wider therapeutic use.12

Figure 1. Structures of CPT (a), CPT-11 (b), SN38 in the lactone (c) and the carboxylate form (d). Carboxylesterase mediate ester hydrolysis (b to c) of CPT-11 and pH-dependent reversible lactone hydrolysis (c to d) of SN38.

Oral chemotherapy is preferred by both patients and physicians as it significantly improves patients’ quality of life as well as reduces treatment costs.13 Several studies have focused on enabling oral SN38 delivery by synthesising prodrugs and/or nanomedicines; these have shown different degrees of success.12 For instance, a hydrophilic prodrug was synthesized by conjugating SN38 to a macromolecular polyamidoamine (PAMAM) dendrimer; this was Page |2


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stable in simulated gastrointestinal fluids, showed improved aqueous solubility and enhanced in vitro Caco-2 permeability compared to SN38.14, 15 Roger et al.16 encapsulated SN38 into lipid nanoparticles with > 90% encapsulation efficiency; these were stable in simulated gastrointestinal fluids and showed higher permeation across Caco-2 cell lines, however the drug loading was too low (0.04%) for practical use. Recently, pH sensitive polymeric micelles (DDS-06E) of SN38 were developed with drug loading up to 80% and these improved Caco-2 permeability.17, 18 Oral delivery of DDS-06E to an animal model resulted in similar tumour growth regression compared to intravenous CPT-11 at an identical dose. In another study, SN38 was encapsulated into chitosan modified poly lactic-co-glycolic acid nanoparticles (PLGA-SN38) with drug loading of 6.8%.6 An enhanced permeability in Caco2 cell lines and in vivo single pass intestinal perfusion was observed for PLGA-SN38 when compared to SN38. However, no indication was given for the fate of the free drug released in the GIT from those two formulations.

Figure 2. Schematic representation of the fate of SN38 and lipophilic prodrugs upon oral delivery.

Increasing the lipophilicity of BCS class-IV compounds thorough prodrug synthesis can also improve permeability through the intestinal mucosal membrane.19-21 In addition, the increased solubility in lipid based excipients improves encapsulation in lipid based drug carrier systems (e.g. liposomes, nano-emulsions or lipid nanoparticles) and may further improve oral absorption by solubilisation of the lipophilic drugs in the intestinal environments along with lipid digestion.21-23 Importantly, lipidic prodrugs with high logP (> 5), high lipid solubility (> 50 mg/g) and structural similarity to natural lipids are reported to induce lymphatic transport and thus avoid hepatic first pass metabolism.24-26 Furthermore, use of long-chain triglyceride (LCT) based carriers further enhances lymphatic absorption and thus increases systemic exposure of lipophilic drugs.26 A recent study showed an alkyl (C18) ester prodrug of mycophenolic acid in a lipid based formulation enhanced lymphatic drug transport 13 fold Page |3



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compared to the parent drug.27 Therefore, we hypothesise that lipophilic prodrugs of SN38 may improve permeation across the mucosal membrane and thereby improve oral absorption. Prodrug delivery via lipid-based formulations may further improve solubilisation of the lipophilic SN38 in the intestinal environments and increase oral bioavailability by inducing lymphatic transport.21, 22 In the current study, we aim to synthesise lipophilic prodrugs of SN38 and then use a range of physicochemical and in vitro analyses to identify the optimal chemical modification (position and alkyl chain length (i.e. short, medium and long)) to establish the best/lead prodrug with potential for improving oral absorption, increasing lymphatic transport and reducing toxicity (Figure 2). More specifically, the solubility in LCT, log P, and in vitro cytotoxicity in a colorectal carcinoma cell line (HCT-116) were evaluated. Furthermore, prodrug stability in simulated gastrointestinal conditions (in presence of enzymes) and in rat plasma were also determined to identify the ideal prodrug structure. Finally transmucosal permeability and uptake in excised rat gut tissues were determined using an Ussing chamber to confirm the lead prodrug’s potential for oral delivery.

2

Materials and methods

2.1 Materials SN38 (purity 99%) was purchased from Flourish Pharma Ltd (Chai Wan, Hong Kong). N-(3dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), 4-dimethylaminopyridine (DAMP), undecanoic acid, palmitic acid, camptothecin, caffeine, atenolol, pepsin from porcine gastric mucosa, porcine pancreatin extract (activity equivalent to 8×USP specification), monobasic potassium phosphate, sodium chloride and sodium hydroxide pellets were obtained from Sigma-Aldrich (Australia). Propionic acid and dichloromethane (DCM) were purchased from Merck (Australia). Soybean oil was supplied by Southern Cross Science Pty (Australia). All other chemicals were analytical grade or higher quality and used as received. High purity (Milli-Q) water was used throughout the study.

2.2 Chemical synthesis Two approaches were taken to modify SN38 in positions C10, C20 and both C10 & C20 (Figure 3). The synthesis of SN38 ester prodrugs at C10 position was undertaken in a single step reaction (Steglich Esterification).28 Fatty acids react preferentially with C10 hydroxyl group rather than C20 hydroxyl group due to its higher reactivity and less steric hindrance (Figure 3. A). Addition of excess fatty acids subsequently reacted at the C20 position. Dichloromethane (DCM) was used as solvent and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) were added as catalysts for the reaction. SN38 was modified at only position C20 by first protecting the C10 position with di-tert-butyl dicarbonate (BoC) in dichloromethane and pyridine (Figure 3. B).29 The esterification at C20

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was then undertaken as described above. Finally the BoC protection group was removed with 10% trifluoroacetic acid in DCM. Full details are described in supplementary information.

Figure 3. Synthesis of SN38 prodrugs, (A) modification of C10 and both C10 and C20; (B) modification of C20 position.

2.3 Prodrugs solubility in long chain triglyceride (LCT) An excess amount of prodrug was added to 1 mL of LCT (soybean oil) in a glass vial (in duplicate). The suspensions were vortex mixed for 30 sec and continuously tumbled on a rotary mixture (30 rpm, Rotator SB3, Stuart) at room temperature. All samples were inspected every day for drug particles and additional drug added in their absence. After 3 days (72 h) samples were transferred into microtubes and centrifuged at 13,300 rpm for 15 min. The supernatants were diluted accordingly in DMSO and analysed by HPLC as described in section 2.9. The sampling and analysis procedure were repeated daily until less than 2% variation in solubility value was found between subsequent days and that was considered as equilibrium solubility.

2.4 Calculation of logP The prodrugs were designed to have high logP (> 5) values. The conventional octanol/water partition coefficient measurement could not be employed as negligible amounts of compound partitioned into the aqueous phase and hence leading to unreliable measurements.30 Instead, logP values were estimated by using ACD ChemSketch (freeware, Ver. 14.02; Advanced Chemistry Development, Inc. http://www.acdlabs.com) and reported as clogP.

2.5 Cytotoxicity assay Standard 72 hrs-MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma) assays were performed to determine the cell viability of HCT-116 cells, a colon adenocarcinoma cell-line, as previously reported.31 Briefly, cells were seeded at 3×103 cells/well into 96 well plates and incubated overnight at 37˚C, 5% CO2. Tested compounds were diluted from a 10 mM stock solution (in DMSO) with cell culture medium to prepare a series of concentrations (0.1 nM to 10 µM) (final DMSO concentration < 0.01%), added to Page |5



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cells (in triplicates), and incubated at the corresponding time-point at 37°C, 5% CO2. MTT was made up as a 2 mg/mL stock in PBS and added at 20 µL/well and incubated in the dark at 37˚C, 5% CO2 for 3 h. The plate absorbance was read at 560 nm using an EnVision multilabel plate reader (PerkinElmer). Compound concentrations required to inhibit 50% of HCT116 cells (IC50) were calculated using non-linear regression analysis.

2.6 Prodrugs stability in simulated gastrointestinal fluids Simulated gastrointestinal fluids were prepared according to the USP methods. Briefly, gastric fluid, simulated, TS: 20 mg of NaCl, 32 mg of purified pepsin, 70 µL of HCl (32%) and sufficient water to make 10 mL. Intestinal fluid simulated, TS: 136 mg of monobasic potassium phosphate (KH2PO4) in 2.5 mL water, plus 772 µL of 0.2 N NaOH and 5 mL of water. Pancreatin (100 mg) was added and mixed for 15 min and the volume was adjusted to 10 mL. Finally, the pH was adjusted to 6.8 ± 0.1 (with 0.2 N NaOH). Prodrugs and SN38 were dissolved in DMSO and diluted accordingly in PBS to 10 µM solution (the final DMSO concentration was < 1% v/v). These solutions were spiked into freshly prepared (pre warmed to 37°C) simulated gastric/intestinal fluids in microtubes and incubated at 37°C in an orbital shaker (75 rpm). In both conditions, samples were collected at 15, 30, 60 and 120 min and an additional sample at 5 min was taken for simulated intestinal condition. All compounds were tested in triplicate. At each time point 100 µL of sample was withdrawn and immediately mixed with 400 µL of ice cooled acetonitrile which contain 20 ng/mL of CPT as an internal standard (IS) and 0.1% formic acid; vortexed for 10 sec and centrifuged for 10 min at 13,300 rpm. The clear supernatant was analysed by LC-MS/MS (as described in section 2.9).

2.7 Stability in plasma Pooled rat (Dark Agouti) plasma was collected and stored at -80°C until use. The plasma was thawed at 4°C and pre-warmed to 37°C just before the study. The prodrugs were incubated at 37°C at a concentration of 100 nM for 2 hours in triplicate. Plasma samples (50 µl) were collected at 0, 5, 15, 30, 60 and 120 min and mixed immediately with 200 µl of ice cooled acetonitrile which contained 20 ng/mL CPT (as internal standard) and 0.1% formic acid. The samples were then vortex mixed for 10 sec and centrifuged at 13,000 rpm for 10 min. The supernatant was analysed using a LC-MS/MS.

2.8 Permeability and uptake studies (Ussing chamber) A modular Ussing chamber system (EM-CSYS-8, Physiologic Instruments, San Diego, CA) was used for gut transmucosal permeability and uptake studies. Standard Krebs-bicarbonate buffer (KBR) with 10 mM glucose was prepared according to a reported method.32 Scavenged tissue from female Sprague-Dawley rats (300 to 360 g) were used with prior approval from the Animal Ethics Committee, University of South Australia, Australia. The intestine was removed immediately after euthanasia of rats with CO2 and flashed with cold KBR solution. The proximal colon was collected and put into cold KBR (on ice) with Page |6


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continuous carbogen (95% O2 and 5% CO2) flow. Approximately two centimetres of colon were opened along with mesenteric border and both the serosa and muscularis externa were carefully removed under a microscope with continuous carbogen gas flow. Payer’s patches were carefully avoided for the permeability study. Each compartment of the Ussing chamber was filled with 5 mL KBR solution and equilibrated for 30 min at 37°C. Mucosal membranes were mounted in the Ussing chamber with 0.5 cm2 exposure and further equilibrated for 30 min and with continuous carbogen flow. Test compounds were dissolved in DMSO and diluted accordingly to a concentration 10 µM in KBR (DMSO conc. ~1.0%) during the assay. The chambers were connected with 2 pairs of electrode systems (Ag/AgCl electrode filled with 3.5% Agar in 3M KCl) for monitoring the electrical parameters. The transepithelial conductance (Gt), short-circuit current (Isc) and transepithelial voltage potential (Vt) was recorded every 20 seconds over a 2 hour period with a voltage/current clamp (VCC MC8, Physiologic Instruments, San Diego, CA) and handled with the built-for-purpose comprehensive data acquisition and analysis package (Aquire and Analyze, Physiologic Instruments). The transepithelial potential difference (PD) and transepithelial resistance (R) values were calculated by using Ohms law and were given in millivolt (mV) and Ω.cm2 respectively. Acceptance criteria for tissues were set as PD > 6 mV, R > 100 Ω.cm2 and tissues out of this range were omitted from the study.33 Caffeine and atenolol were used as marker compounds to determine the passive paracellular and transcellular permeability respectively. The samples (100 µl of aliquots) were withdrawn from both sides at time 0, 30, 60 and 120 min. At the end of the experiments the mucosal membrane was removed from the chamber and washed three times with fresh KBR buffer and collected for uptake measurement. All samples were flash frozen on dry ice immediately after collection and stored at -80°C until analysis. These samples were thawed at 4°C temperature before drug content analysis. For the permeability study, samples were diluted three times with mobile phase and vortex mixed for 10 sec and centrifuged at 13,000 rpm for 10 min. For the uptake study, the tissue samples were homogenized in KBR. The homogenates (100 µl) were diluted with ice cooled acetonitrile (0.1% formic acid, 300 µl). The mixture was vortex mixed for 10 sec and centrifuged at 13,000 rpm for 10 min. The amount of prodrug remaining and SN38 generated were simultaneously quantified in the supernatant with LC-MS/MS. The apparent permeability was calculated using the following equation.

= × × ……………….. (1)

where, dQ/dt is the rate of drug transport from apical to basolateral, A is the exposed tissue area and Ci is the initial drug concentration at apical side.

2.9 Drug analysis LC-MS/MS: An ABSCIEX TripleTOF 5600 LC-MS system coupled with Simadzu Nexera (Shimadzu Corporation, Japan) were used. Chromatographic separation was obtained by a

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Phenomenex Kinetex C18 column (50 × 3.0 mm; 2.6 µ, 100 Å) with mobile phase A (MP-A) 5:95 acetonitrile : water containing 0.1% formic acid and mobile phase B (MP-B) 95:5 acetonitrile : water containing 0.1 % formic acid. The pH of the MP was ~3 to facilitate lactone ring stabilisation and allow chromatographic separation. A gradient elution was undertaken at a flow rate of 0.4 mL/min, started with 90% MP-A and 10% MP-B for 0.5 min then MP-A was gradually decreased to 0% and MP-B was increased to 100% at 2.2 min and remained for 4 min, then went back to initial conditions. The total run time was 6.5 min. For atenolol, the same gradient was used with a shorter run time (3.2 min). For caffeine an isocratic method was used with 15% MP-B for 3 min. The autosampler temperature was set to 4°C during the analysis. The eluent from the column was directed to the ABSCIEX TripleTOF™ 5600 in positive ionisation mode. The instrumental parameters, limit of detection and limit of quantification are given in the supplementary information. The analytical method was checked and confirmed (according FDA guidelines34) for selectivity, accuracy, precision and recovery. The calibration curve was obtained (correlation R2 > 0.99, 0.5 nM to 250 nM) and used for content analysis in test samples. HPLC-PDA: Compound purity and solubility analysis were carried out using a Shimadzu Prominence ultrafast liquid chromatography (UFLC) system with a CBM-20A communications bus module, a DGU-20A 5R degassing unit, an LC-20AD liquid chromatograph pump, an SIL-20A HT autosampler, an SPD-M20A photodiode array detector, a CTO-20A column oven, and a Phenomenex Kinetex C18 column (250 mm × 4.60 mm, 5µ, 100 Å) using method A (gradient 5 − 95% MeOH containing 0.1% FA over 5 min, followed by 95% MeOH containing 0.1% FA at a flow rate of 1 mL/min) or method B (gradient 5 − 95% MeCN containing 0.1% FA over 5 min followed by 95% MeCN containing 0.1% FA at a flow rate of 1 mL/min). The run time was adjusted from 12 to 30 minutes depending on elution time. Calibration curve was obtained for each compound (R2 > 0.99) and used for solubility determination.

3

Results and discussion

3.1 Synthesis of alkyl ester prodrugs of SN38 A series of lipophilic prodrugs were synthesised by attaching fatty acids to the R1 and/or R2 position of SN38 with high purity (> 95%, Table 1). Three different fatty acids of diverse chain length were employed, i.e. short (propionic acid), medium (undecanoic acid) and long (palmitic acid), and the structure activity relationship was explored as discussed in the sections below. The compound structures were characterised by 1H and 13C NMR spectra and high resolution mass spectra (supporting information).

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Table 1. Summary of structure and biological activity (IC50 on HCT-116)

SN38 SN38-propionate C10 (SN38-pro10) SN38-propionate C20 (SN38-pro20) SN38-dipropionate (SN38-dipro) SN38-undecanoate C10 (SN38-unde10) SN38-undecanoate C20 (SN38-unde20) SN38-diundecanoate (SN38-diunde) SN38-palmitate C10 (SN38-pal10) SN38-palmitate C20 (SN38-pal20)

Structure R1 R2 H H CH3CH2CO CH3CH2CO CH3CH2CO CH3CH2CO CH3(CH2)9CO CH3(CH2)9CO CH3(CH2)9CO CH3(CH2)9CO CH3(CH2)14CO CH3(CH2)14CO

Cytotoxicity (IC50, µM) 0.0053 ± 0.00 0.0059 ± 0.00 0.051 ± 0.00 0.059 ± 0.00 0.0062 ± 0.00 0.632 ± 0.11 7.20 ± 1.20 0.060 ± 0.01 4.82 ± 0.23

SN38-dipalmitate (SN38-dipal)

CH3(CH2)14CO

3.016 ± 1.91

Compound name

CH3(CH2)14CO

Yield (%) -

59.9 38.5 22.9 57.7 97.9 35.2 14.8 47.7 23.5

3.2 Triglyceride solubility and clogP The solubility of SN38 in long chain triglyceride (LCT) was low (0.01 mM) and significantly increased (by 11 to 444 folds) upon modification with fatty acids (Figure 4). Compounds equipped with short chain fatty acids (e.g. SN38-pro10, SN38-pro20 and SN38-dipro) exhibited the least improvement in LCT solubility in comparison to the prodrugs of medium (i.e. SN38-unde10, SN38-unde20 and SN38-diunde) and long chain fatty acids (i.e. SN38pal10, SN38-pal20 and SN38-dipal). Interestingly, SN38 modified with medium chain fatty acids showed higher lipid solubility compared to long chain fatty acids modified prodrugs. Modification at both C10 and C20 positions (bi-substituted) generally resulted in higher solubility in LCT than the mono-substituted prodrugs (except for long chain modified prodrug). The calculated logP or clogP value was dependent on the hydrocarbon chain length and increased as the hydrocarbon chain length increased (Figure 4). The C20 mono-substituted prodrugs have higher clogP values than the respective C10 mono-substituted prodrugs. These findings are consistent with the published literature which demonstrated modification of compounds with fatty acids or triglycerides and significantly increased the clogP value.27, 35 Prodrugs modified at both C10 and C20 positions (bis-substituted) showed the highest clogP. Depending on the solubility and clogP (7.7) data it could be anticipated that SN38 prodrugs of medium chain fatty acids are most suitable for lipid based oral delivery and subsequent lymphatic absorption.

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Figure 4. Prodrug solubility in soybean oil (mean and SD is reported) and cLogP (calculated with ACD ChemSketch)

3.3 Cytotoxicity IC50 values determined by the MTT assay are given in Table 1. The C10 modified (monosubstituted) prodrugs retained comparable IC50 to SN38 (0.0059-0.060 µM vs 0.0053 µM), whilst C20 modified (mono-substituted) prodrugs and both C10 & C20 modified (bissubstituted) prodrugs showed significantly reduced cytotoxicity on HCT-116 cells. The impact of the modification at different positions on the cytotoxicity is consistent with the fact that SN38 exhibits its topoisomerase inhibition activity by forming a ternary complex with DNA at the 1N, 20 hydroxyl, and 17 ketone positions (Figure 1. c).2-4 Minimum impact on the cytotoxicity was observed when the active binding site was not altered (i.e. C10 positon mono-substituted). On the other hand, as the C20 hydroxyl group was esterified, hydrolysis was a prerequisite for the prodrugs to exhibit a strong cytotoxic effect, and this is in agreement with a previous study that showed a C20 modified camptothecin ester showed negligible topoisomerase activity until it reconverted into the parent form.36, 37 Furthermore, for C20 modified prodrugs, a direct correlation was observed between cytotoxicity and hydrocarbon chain length. Increasing the hydrocarbon chain length from 3 to 16 reduced the IC50 from 0.051 µM to 4.82 µM (by ~10 to 1000 fold compared to SN38). It is likely that increasing the fatty acid chain length led to increased steric hindrance thus reduced cytotoxicity.

3.4 Stability in simulated gastrointestinal fluids Stability of the prodrugs in simulated gastrointestinal fluids was determined to predict their fate after oral administration (see Figures 5 and 6). All prodrugs were relatively stable in simulated gastric fluids (SGF), i.e. after 2 hrs of incubation at pH 1.2 in the presence of pepsin enzyme, the reconversion/release of SN38 was < 1.0%, except SN38-pro10, which showed 6.7% reconversion. SN38 reconversion from C10 modified prodrugs was faster than the C20 modified prodrugs and no reconversion/release was observed for the bi-modified prodrugs. Generally the longer chain length prodrugs showed increased ester bond stability.

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Figure 5. SN38-prodrugs stability in simulated gastric fluids (each data point represents mean of three independent replicates ± SD). Only one prodrug, SN38-pro10, showed a reconversion of 6.7% and the rest of the prodrugs showed < 1% reconversion to SN38 after 2 hrs.

The ester prodrugs of SN38 are less stable in simulated intestinal fluids (SIF) in comparison with SGF. Immediately after incubation at pH 6.8 and in the presence of pancreatin enzyme, the reconversion to SN38 was rapid (half-life was less than 5 min) and almost complete for all C10 modified prodrugs (Figure 6). The reconverted SN38 was further metabolised/degraded and ~ 70% remained as SN38 after 2 hrs (this metabolism was also observed for pure SN38 incubated in SIF, data not shown). These findings indicate C10 modified prodrugs are pancreatic enzyme substrates and minimal steric hindrance to the ester bond exposes them to rapid cleavage. In contrast, SN38 modified with short chain fatty acids at C20 and bi-modified at C10 and C20 positions (e.g. SN38-pro20 and SN38-dipro) showed significantly reduced reconversion (3.9% and 6.2% respectively after 2 hrs). Interestingly, medium and long chain hydrocarbon modified prodrugs at C20 and both C10 and C20 positions showed negligible reconversion to SN38 over 2 hrs. This further supports the fact that steric hindrance is the primary factor for the stability of alkyl ester bonds in enzymatic milieu and short chain hydrocarbon modification was not as effective as medium and long chain hydrocarbon.37, 38 The lactone form of SN38 is stabilized by acylation at the C20 hydroxyl position and preserves the therapeutic action until it is cleave off.15, 29, 39, 40 Modification of SN38 at C20 position with medium and long chain fatty acids facilitated stability in both SGF and SIF, hence they are more suitable for oral delivery than C10 modified prodrugs.

Figure 6. SN38-prodrugs stability in simulated intestinal fluids (pH 6.4 and pancreatic enzymes, mean of three independent replicates ± SD).

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3.5 Prodrugs stability in plasma SN38-unde10 and SN38-unde20 were selected for plasma stability studies based on their favourable clogP (> 5) and high lipid solubility comparison with the parent drug, SN38. The reconversion of SN38 from these prodrugs in plasma was similar to that in simulated intestinal conditions. Alkyl esters are prone to hydrolysis at physiological pH and in presence of enzymes. The SN38-unde10 hydrolysed rapidly and completely into SN38 within 15 minutes and then further degraded in a pattern similar to SN38 (Figure 7). This is in agreement with the fact that free SN38 is not stable at higher pH (> 4.5) and/or enzymatic conditions, and transforms into the inactive carboxylate form. On the other hand, SN38unde20 showed sustained reconversion of SN38 over 2 hrs (12%). Plasma esterase enzymes are considered responsible for the release of SN38 from SN38-unde20 and steric hindrance slows down the process. This is contrary to the conversion of SN38 from CPT-11 which requires carboxylesterase enzyme and primarily occurs in the liver.9

% SN38 reconversion from prodrugs

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Figure 7. SN38, SN38-unde10 and SN38-unde20 stability in rat plasma (each data point represents mean of three independent replicates ± SD).

Importantly, CPT’s (e.g. SN38) demonstrate a pH dependent reversible transformation between the active lactone and inactive carboxylate forms. At physiological pH ~75% of CPT-11 remains in the carboxylate form and therefore a significantly higher dose is required for therapeutic effect; this is associated with dose limiting toxicities.29 In contrast, lipophilic SN38 prodrugs (C20 modified) are stable in plasma and slowly release SN38 in the lactone form (the analytical method was optimized to detect lactone only), hence will maximize therapeutic outcomes over an extended period of time.41, 42 Recent clinical studies have shown that an SN38 exposure of as low as 1.2 to 5.9 ng/mL (equivalent of 3 to 15 nM) significantly reduces tumour growth without causing dose related toxicity and other adverse effects.43-45

3.6 Gut tissue permeability and uptake studies SN38-unde20 was selected for gut tissue uptake and permeability studies due to its favourable clogP (> 5), high lipid solubility and stability in simulated gastrointestinal conditions. Excised rat mucosal membranes (from colon) that meet the acceptance quality criteria (PD > 6 mV and R > 100 Ω.cm2 during the study period of 2 h) were used. Caffeine P a g e | 12


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(high permeability) and atenolol (low permeability) were tested to measure the passive paracellular and transcellular permeability of the mucosal membrane, respectively. Permeability values of 19.6 ± 0.12×10-6 cm/sec for caffeine and 1.36 ± 0.19×10-6 cm/sec for atenolol were consistent with literature values thus confirmed the validity of the method.46, 47 Transepithelial uptake and transport of SN38 and SN38-unde20 across rat intestine are reported in Figure 8. SN38-unde20 showed more than 2-fold improvement in permeability compare to SN38 (7.5 × 10-6 cm/sec versus 3.6 × 10-6 cm/sec). In addition, the gut tissue uptake for the SN38-unde20 increased by more than 2.5 fold. Significantly, no measureable reconversion of the SN38-unde20 to SN38 was observed during gut uptake and transport, which further supports the prodrug stability in biologically relevant media.

Figure 8. In-vitro permeability and uptake of SN38, SN38-unde20 through rat intestinal mucosal membrane using Ussing chamber (mean of two replicates ±SD)

The optimal lipophilic prodrug SN38-unde20 facilitated uptake by enterocytes and higher mucosal membrane transport. Based on recent studies which demonstrated that in vivo anticancer efficacy of SN38 prodrugs correlated with higher in vitro permeability and enhanced uptake,48, 49 the SN38-unde20 prodrug is also likely to perform well in vivo, i.e. enhanced lymphatic transport and high uptake in cancer cells/tissue. The lymphatic system is a major route for metastatic spread and a direct delivery of such an anticancer drug could enhance the uptake in metastatic cancer cells and secondary cancer sites.50 In addition, higher enzymatic activity in the tumour microenvironment would further enhance localized reconversion of SN38 from the prodrug51 and potentially lead to targeted uptake. Finally, a combination of fast- and slow-releasing prodrugs (i.e. SN38-unde10 and SN38-unde20) in an optimal lipid-based carrier could provide an initial fast release followed by a sustain release of SN38; which would provide an improved therapeutic outcome.

4

Conclusion

Alkyl ester prodrugs of SN38 at position(s) C10 and/or C20 have been synthesised and demonstrate increased lipid solubility, and controllable cytotoxicity profiles and reconversion rates. SN38 modified at C10 retained higher cytotoxicity and showed rapid reconversion in

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biorelevant fluids. SN38 modified at C20 or both C10 and C20 have low cytotoxicity and slower reconversion in biorelevant media. SN38 modified with medium chain fatty acid indicated highest lipid solubility and a suitable clogP profile for lipid-based oral delivery and subsequent lymphatic absorption. SN38 modified at C20 position with undecanoic acid (SN38-unde20) has increased transmucosal permeability and sustain reconversion to SN38 in plasma and hence is the prodrug with greatest potential for oral delivery.

Acknowledgements: Financial support of the Australian National Health and Medical Research Council project grant scheme (APP1026382) is gratefully acknowledged. We would like to acknowledge Sarah Al Haj Diab, Ben Noll, Dr Mingfeng Yu, Imogen White, Dr Joanne Bowen and Prof Ben Boyd for technical assistance and useful discussion.

Supporting Information: Detailed description of chemical synthesis, isolation and identification are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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5

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Page 19 of 19 Molecular Pharmaceutics BCS-IV ACS ParagonBCS-II Plus(low Environment 1 solubility, low (low permeability) solubility, high permeability)

2 3 Poor oral absorption

Biological barrier

Improved oral absorption

Lipophilic Prodrugs of SN38: Synthesis and in Vitro Characterization

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