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Enabling Oral SN38 based Chemotherapy with a Combined Lipophilic Prodrug and Self-Microemulsifying Drug Delivery System Vaskor Bala, Shasha Rao, Emma Bateman, Dorothy Keefe, Shudong Wang, and Clive A. Prestidge Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00591 • Publication Date (Web): 01 Sep 2016 Downloaded from http://pubs.acs.org on September 5, 2016

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Molecular Pharmaceutics

Enabling Oral SN38 based Chemotherapy with a Combined Lipophilic Prodrug and Self-Microemulsifying Drug Delivery System Vaskor Bala1, Shasha Rao1, Emma Bateman2, Dorothy Keefe2, Shudong Wang1 and Clive A. Prestidge1 1

School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5001, Australia. 2Mucositis Research Group, School of Medicine, University of Adelaide, Adelaide, SA 5001, Australia

Abstract Oral chemotherapy with SN38 is restricted by its poor solubility in gastrointestinal (GI) fluids and low permeability. Here we report the oral delivery of SN38 by a combined lipophilic prodrug and lipid-based formulation strategy. A lead lipophilic prodrug of SN38, SN38undecanoate (SN38-unde20), was incorporated into a self-microemulsifying drug delivery system (SMEDDS) for improved in vitro and in vivo performance. The formulation was purposefully designed and optimised with long chain lipids and lipid-based non-ionic surfactants to maximise drug solubilisation in GI conditions, facilitate trans-membrane permeation, and hence improved oral absorption. SN38-unde20-SMEDDS significantly increased (> 7 fold) drug solubilisation in the aqueous phase compared to unformulated drug during in vitro lipolysis and drug solubilisation studies. In an orally dosed in vivo pharmacokinetics study in a Dark Agouti rat model, the SN38-unde20-SMEDDS formulation confirmed oral absorption of SN38-unde20 and subsequent reconversion to SN38. Importantly, the overall plasma exposure of SN38 (AUC0→∞) was equivalent for orally dosed SN38-unde20-SMEDDS in comparison with a parenteral dose of SN38-unde20-SMEDDS and SN38 at an identical dose (10 mg/kg). The combination of lipophilic prodrug along with an optimal delivery carrier is demonstrated to enable effective oral delivery of challenging chemotherapeutic compounds that are conventionally dosed by injection. Keywords: 7-ethyl-10-hydroxycamptothecin (SN38); SMEDDS; lipophilic prodrugs; lipidbased formulations; oral delivery; chemotherapy; pharmacokinetics (PK).

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1

Introduction

The oral route of drug administration is preferred by both patients and physicians due to the ease of administration (e.g. self-medication), flexibility in dosing schedule, reduced need for hospitalisation, lower cost of treatment and significantly improved quality of life for patients 1

. However, the majority (~90%) of chemotherapeutic drugs are administered through the

parenteral route as either bolus injection or intravenous (i.v.) infusion 2. Over recent years, oral delivery of chemotherapeutics has received increased research interest and as a result, more than 20 oral chemotherapeutic drugs are now available in the clinic 3, 4. Currently, ~25% of all novel anticancer drug formulations are designed for oral delivery 5. SN38 (7-ethyl-10-hydroxycamptothecin) (Figure 1) is one of the most potent anticancer agents of the camptothecin family. A water soluble prodrug of SN38, irinotecan (CPT-11), is currently used clinically as an i.v. infusion for metastatic colorectal cancer 6. However, only 2-8% of infused irinotecan transforms into the active metabolite, SN38, depending on the genetic nature of the patients 7, 8 and thus unpredictable therapeutic responses and severe dose related toxicities are common for CPT-11 therapy 9. SN38 is primarily metabolized in the liver, where uridine 5'-diphospho-glucuronosyltransferase-glucuronosyltransferase 1A1 (UGT1A1) enzyme metabolises SN38 to its water soluble derivative SN38-glucuronide (SN38-G) and elimination is primarily through biliary excretion 10-12. SN38-G is then further metabolised by the gut microbiome and regenerates SN38 which causes late onset of local toxicity, i.e. severe intestinal damage and diarrhoea 13, 14. The major delivery challenges of SN38 include: (i) poor solubility (< 0.5%) in water and pharmaceutically approved solvents 15, (ii) pH dependent hydrolysis of lactone to carboxylate form (Fig 1) thus inactivation (iii) as a P-glycoprotein (P-gp) substrate, it has low absorption across the gastrointestinal tract (GIT) 16, 17, (iv) rapid first-pass metabolism in the liver and (v) exposure of SN38 in the intestine causes severe dose limiting diarrhoea and local toxicity 18

. A variety of approaches ranging from specifically designed prodrugs to state of the art

nanomedicine formulations have been explored to overcome these delivery challenges 19, 20. However, effective oral delivery of SN38 with preclinical/clinical pharmacokinetics evidence is rare. Recent studies have shown that modification at position C20 stabilises the lactone ring (preserving cytotoxic efficacy) and enables a prolonged therapeutic response 21, 22.


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Highly lipophilic drugs are transported through the lymphatic system following oral absorption thus avoid hepatic metabolism 23. In addition, lymphatic absorption has further implication for passive targeting of metastatic cancer cells and to potentially enhance efficacy and reduce toxicity 24-26. Our hypothesis is that an optimally engineered lipophilic prodrug of SN38 can be orally delivered using a specifically designed colloidal lipid-based carrier system to facilitate increased oral absorption and lymphatic drug transport with associated improvements in the efficacy and reduction in the toxicity profile.

Figure 1: SN38 and its analogues. The lactone rings of CPT-11 and SN38 show pH dependent hydrolysis (a→b, c→d) and converts into carboxylate form at high pH (> 6.5), which is pharmacologically inactive. Modification at C20 position (e) stabilises the lactone form, prevents pH dependent hydrolysis and preserves its potency.

We have recently reported on the synthesis of a series of novel lipophilic prodrugs for SN38 and their in vitro characterisation 27. The lead prodrug, SN38-undecanoate (SN38-unde20), was prepared by modifying SN38 at position C20 with the medium-chain fatty acid, undecanoic acid. SN38-unde20 showed significantly improved solubility (up to 444-fold) in lipid excipients and enhanced permeability across the intestinal mucosal membrane compared with the parent drug 27. Furthermore, SN38-unde20 exhibited improved stability (t1/2 > 2 h) in simulated gastrointestinal (GI) fluids, thus prevented the undesirable SN38 reactivation and exposure in the gut prior to drug absorption. In the current study, we developed an optimised Page 3 ACS Paragon Plus Environment


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self-microemulsifying drug delivery system (SMEDDS) for SN38-unde20 based on dispersibility, droplet size and drug loading behaviour, and determined the in vitro and in vivo oral delivery performance. Specifically, in vitro lipid digestion and drug solubilisation was investigated in bio-relevant intestinal conditions, and orally dosed pharmacokinetics of SN38-unde20-SMEDDS and SN38 were determined in healthy rats and compared with equivalent intraperitoneal doses of SN38-unde20 and SN38.

2

Materials and methods

2.1 Materials High purity SN38-undecanoate (SN38-unde20) was synthesised and purified in house according to our previously reported method 27. Briefly, the C10 hydroxyl group of SN38 was protected with di-tert-butyl dicarbonate (as it is more reactive than the C20) and the C20 position was esterified with undecanoic acid. The protecting group was removed to obtain the final compound. Labrafil (Oleoyl polyoxyl-6 glycerides), Labrasol (Caprylocaproyl polyoxyl-8 glycerides), Peceol (glyceryl monooleate), Maisine 35-1 (LC mono-, di-, and triglycerides, LC mixed glycerides) and Transcutol HP (diethylene glycol monoethyl ether) were generous gifts from Gattefosse (France). Capmul MCM (medium chain mono-, di-, triglycerides and mixed glycerides) and Captex 300 (medium chain triglycerides) were generous gifts from Abitech Corporation (USA). Soybean oil (long chain triglycerides), olive oil (long chain triglycerides) and Miglyol 812 (Caprylic/capric triglycerides) were obtained from Hamilton Laboratories (Adelaide, Australia). Camptothecin (CPT), Tween 80, Cremophor RH (PEG 40 castor oil), d-α tocopherol polyethylene glycol 1000 succinate (TPGS), sodium taurodeoxycholate (NaTDC), trizma maleate, type X-E L-α-lecithin (approximately 60% pure phosphatidylcholine, from dried egg yolk), porcine pancreatin extract (tributyrin units (TBU) activity of 8X USP specification), 4-bromophenylboronic acid (4-BPB), calcium chloride dihydrate, PEG400 and sodium hydroxide pellets were purchased from Sigma-Aldrich (Australia). Ethanol, HPLC/LC-MS grade acetonitrile and formic acid were obtained from Merck (Australia). All other chemicals were analytical grade or higher and used as received. High purity (Milli-Q) water was used throughout the study.


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2.2 Determination of prodrug solubility in pharmaceutical excipients The SN38-unde20 prodrug solubility was measured in a number of generally recognised as safe (GRAS) excipients according to a method described previously with minor modifications 27. Briefly, an excess amount of SN38-unde20 was added to 1 ml of each excipient in a glass vial (in triplicate) and closed with Teflon-sealed screw cap. The mixtures were vortexed for 30 sec (except for TPGS, which was liquefied at 40°C before mixing) and left with continuous mixing at 75 rpm in a thermostated chamber at 37°C (KS 4000i control, IKA) for 5 days to ensure equilibrium solubility. After the experimental period (120 h) samples were transferred into Eppendorf microtubes and centrifuged at 13,300 rpm (17,000 × g) for 30 min. The supernatants were analysed using HPLC (according to the method described below) after appropriate dilution in DMSO and mobile phase. This method was also employed to measure solubility of SN38-unde20 in the SMEDDS pre-concentrate.

2.3 SMEDDS formulation development Preparation of SMEDDS pre-concentrates: All excipients (e.g. lipid, surfactant, cosurfactant and solvent) were accurately weighed into glass vials, closed with Teflon-sealed screw caps and thoroughly mixed. The mixtures were left overnight in a thermostated chamber (KS 4000i control, IKA) at 37°C for equilibrium and these are considered as the SMEDDS pre-concentrates. For drug loading, an appropriate amount of drug (below the equilibrium solubility) was added to SMEDDS mixture before placing in the thermostated chamber. A systematic approach was followed for identification of a specific self-emulsifying region within a ternary phase diagram (without drug). In a typical ternary diagram the apexes represent 100% of lipids (natural or modified triglycerides and mixed glycerides/fatty acids), surfactant and cosolvent respectively. Each combination of lipid (5-40%), surfactant (PEGylated lipids, 40-90%) and cosolvent (5-20%) was investigated by keeping the ratio of two components fixed and then the third was altered. In the final SMEDDS pre-concentrate the cosolvent was restricted to 10% because of the potential incompatibility with gelatine capsules (not used in this study). The selection criteria for SMEDDS pre-concentrate were (i) isotropic mixture at room temperature, (ii) disperses in aqueous media within 2 min and (iii) particle size and polydispersity index (PDI) less than 200 nm and 0.4 respectively. After



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SMEDDS optimisation, Vitamin E TPGS (1-10%) was incorporated in the optimised preconcentrate for further optimisation and reanalysed against selection criteria. The self-emulsification capacity was investigated by dispersing an approximate amount (~1 g) of SMEDDS pre-concentrate in 250 ml of milli-Q water by gentle hand shaking and visual inspection. A clear to bluish/cloudy solution was considered as acceptable and the particle size distribution and polydispersity index (PDI) of emulsion droplets in aqueous medium were determined by dynamic light scattering (Malvern Nano-ZS Zetasizer, UK) without any further sample treatment. The particle size and distribution of SMEDDS pre-concentrates were also measured at pH~1.2 (0.1N HCl) to simulate gastric conditions.

2.4 In vitro lipolysis and drug solubilisation Preparation of lipid digestion medium: Simulated human intestinal conditions were prepared to predict lipid digestion in SMEDDS formulation(s) and the subsequent impact on drug solubilisation, based on an established procedure with minor modifications 28. Briefly, a mixed micellar solution was prepared by dissolving phospholipid (egg lecithin, 1.25 mM) in chloroform in a round bottom flask and a thin film was formed by evaporating the organic solvents under reduced pressure (BÜCHI Rotavapor RE, Switzerland). The film was hydrated with NaTDC (5 mM) and digestion buffer (50 mM Trizma maleate (pH 7.5), 150 mM NaCl, and 5 mM CaCl2.2H2O) and stirred overnight (~12 hrs) to form a clear micellar solution. This solution was stored bellow 8°C and used within two weeks. Pancreatin extracts (pancreatic lipase, co-lipase and non-specific lipolytic enzymes e.g. phospholipase A2) were freshly prepared in digestion buffer (200 mg/ml) by magnetic stirring for 15 min followed by centrifugation (at 4,500 rpm and 4°C for 15 min). The supernatant was kept on ice until use. Digestion experimental procedure: Lipolysis of ~600 mg of formulation was carried out for 60 min using a TitraLab® 854 pH-stat titration apparatus (Radiometer Analytical, Copenhagen, Denmark). The formulation was dispersed in digestion medium (18 ml) in a thermostated glass vessel at 37°C. The pH of the mixture was adjusted to 7.45 ± 0.01 with 0.1 M HCl or NaOH. The lipid digestion was started by adding pancreatin extracts (2 ml, 2000 TBU lipase activity). A constant pH (7.45) of the medium was maintained by auto-titration of free fatty acids (FFA) produce from the lipid digestion with 0.2 M NaOH. The phase partitioning of the drug was measured by collecting 1 ml of sample at different time points (e.g. 1, 5, 15, 30 and 60 min). The lipid digestion was terminated by 10 µl of 4-BPB (0.5 M Page 6 ACS Paragon Plus Environment

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in methanol) which was prefilled in the sample collection tubes. The phases (e.g. oil, aqueous and pellet) were separated by centrifugation at 37°C and 43,300g for 60 min. The aqueous phase was collected and diluted with DMSO for drug content analysis using HPLC. The pellet phase was extracted with Folch solution (2:1 v/v chloroform: methanol), acidified (0.1 M HCl) and then diluted with DMSO for analysis. Collection and analysis of the oil phase was not feasible due to its low volume.

2.5 In vivo pharmacokinetic studies All animal experiments were approved by the University of Adelaide Animal Ethics Committee (No. 15355). The rats had free access to a standard rat diet and tap water at all times during the studies. Each group of four Dark Agouti (female) rats (weighing ~200g) were dosed either by the intraperitoneal (IP) or oral (PO) route at a dose of 10 mg/kg. For IP dosing, drug was solubilised in DMSO: PEG-400 (1:4) and diluted in isotonic saline before dosing. Blood samples were collected from the tail vain before dosing and 0.25, 1, 2, 3, 6, 24 and 48 h post dosing. The samples were centrifuged at 10,000 rpm for 5 min and plasma supernatants harvested and placed in dry ice immediately after collection for flash freezing. The frozen plasma samples were stored at -80°C until the drug content analysis.

2.6 Drug analysis A Shimadzu LC-MS/MS system (LC-8050) coupled with Nexera X2 (Shimadzu Corporation, Japan) were used for simultaneous determination of SN38-unde20 and SN38 in blood plasma samples. After thawing the plasma samples on ice, one volume of plasma was treated with three volume of ice cooled acetonitrile which contained 20 ng/ml CPT (as an internal standard, IS) and 0.1% formic acid. Thereafter, the mixture was vortex mixed for 10 sec, centrifuged for 10 min at 13,300 rpm and the clear supernatant of each sample was analysed as follows. Chromatographic separation was obtained by a Phenomenex Kinetex C18 column (50 × 3.0 mm; 2.6 µm, 100 Å) with mobile phase (MP-A) LC-MS grade acetonitrile (0.1 % formic acid) and mobile phase (MP-B) Milli-Q water (0.1 % formic acid). Gradient elution was started at a flow rate of 0.4 ml/min with 90% MP-A and 10% MP-B for 0.5 min then MP-A was gradually decreased to 5 % and MP-B was increased to 95 % at 2.2 min and maintained for 1.2 min, then reverting to the initial condition. The total run time was 4.0 min. The auto-



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sampler temperature was set to 4°C during the analysis. Multiple reaction monitoring (MRM) was used in positive ionisation mode with the parameters as listed in the Table 1. Blank plasma (from untreated animal) was spiked with known amounts of drug and treated similarly to the unknown samples to generate a calibration curve. The analytical method was checked and confirmed for selectivity, accuracy, precision and recovery according to FDA guidelines 29

. The lower limits of detection and quantification were also determined according to FDA

guidelines 29 for SN38-unde20 and SN38, and were found as 0.1 ng/ml, 0.25 ng/ml and 0.1 ng/ml, 0.5 ng/ml, respectively. The peak area ratio of SN38-unde20 or SN38 to those of CPT were used to generate a linear calibration curve (R2 > 0.99) with weight regression (1/x2) analysis in the range of 0.25 to 100 ng/ml for SN38 and 0.5 to 250 ng/ml for SN38-unde20. Table 1: LC-MS/MS instrument parameters and transitions for SN38-unde20, SN-38 and CPT (IS). Compound SN38-unde20 SN38 CPT

Retention time (min) 3.33 2.32 2.38

MRM (m/z) 561.1 > 375.1 392.9 > 349.2 349.1 > 305.2

Dwell time (msec) 100 100 100

Q1 pre bias (V)

Collision energy (V)

Q3 prebias (V)

-20.0 -20.0 -18.0

-32.0 -26.0 -25.0

-28.0 -26.0 -23.0

Abbreviations: MRM, multiple reaction monitoring; V, volt; m/z, mass to charge ratio.

SN38-unde20 solubility analyses in different excipients, SMEDDS pre-concentrate and during lipid digestion were measured by using a Prominence HPLC system (Shimadzu Corporation, Japan). The instrumentation consisted of a series of LC-20ADXR pumps, SIL20ACXR auto sampler, CTO-20AC column oven, and SPD20A PDA detector (set at 380 nm). A reverse phase C18 column (Alltima®, 250 × 4.6 mm, 5 µm, 100 Å) was employed for chromatographic separation with mobile phase consisting of a mixture of 80:20 v/v acetonitrile and 10 mM ortho-phosphoric acid (pH 3.0) and the compound eluted at a flow rate of 1.5 ml/min (injection volume: 20 µl, run time: 8 min). The lower limit of detection and quantification for SN38-unde20 were 5 ng/ml and 10 ng/ml respectively. A linear calibration curve was established for concentration range of 10 to 1000 ng/ml (R2 > 0.99).


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3

Results and discussion

3.1 Preparation and physicochemical properties of SMEDDS for SN38unde20 SN38-unde20 (clogP 7.7) is a highly lipophilic compound and practically insoluble in water 27

. For successful formulation development and optimisation, knowledge of a novel drug’s

solubility in relevant pharmaceutical excipients is essential. SN38-unde20 solubility in a range of pharmaceutically approved lipid-based excipients was measured and is presented in Figure 2. The natural long chain triglycerides (LCT), i.e. olive oil and soybean oil, showed the lowest solubilisation for SN38-unde20 (< 0.2 mg/ml). PEG-functionalised LCT and mixed long chain glycerides (LCG), e.g. Labrafil and Maisine 35-1, provided increased solubilities. Overall, medium chain triglycerides (MCT), i.e. Myglyol 812, Captex 300 and Capmul MCM provided a greater solubilisation capability for SN38-unde20. Similarly, mixed MC glycerides or PEGylated MCT increased solubility by more than 4 fold compared to modified LCT/mixed LC glycerides. SN38-unde20 solubility in non-ionic surfactants (Tween 80 and Vitamin-E TPGS) was comparable with mixed glycerides and was highest in the cosolvents Transcutol HP (13.5 mg/ml) and ethanol (5.6 mg/ml); this is in agreement with previous findings for other drugs 30.

Solubility (mg/ml)

14 13 6.0 5.0 4.0 3.0 1.5 1.0 0.5 0.0 O C So liv re yb e m e o M pho an il ai r oi si e l ne R H La 35 br -1 M P af ig ec il C ly e C ap ol 8 ol ap te 1 m x 2 ul 3 0 La MC 0 Vi b M t-E ra Tr T TPsol an we G sc en S ut 8 o 0 Et l H ha P no l

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Figure 2: SN38-unde20 solubility (mg/ml) in different lipid based formulation excipients (mean ± SD, n = 3).

Based on the high solubility of SN38-unde20 in the mixed MCT Capmul MCM, lipid-based surfactant Labrasol and co-solvent Transcutol; these were predicted to provide maximum drug loading as a SMEDDS pre-concentrate. However, they failed to meet the selection Page 9 ACS Paragon Plus Environment


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criteria (as described in section 2.3) for SMEDDS and similar incompatibility (i.e. lack of self-emulsification) has been reported previously 31. Alternatively, a compatible oil Labrafil was used along with Labrasol and Transcutol to obtain the maximum drug solubilisation/loading in the final SMEDDS. The self-emulsifying region in a ternary phase diagram was established and is presented in Figure 3. Labrafil

Labrasol

Transcutol

Figure 3: Ternary phase diagram showing self-emulsifying region (♦) of SMEDDS based on Labrafil, Labrasol and Transcutol.

A minimum of 40% Labrasol (lipid-based surfactant) was required for self-emulsification, and 60% Labrasol was required for a uniform particle size distribution (PDI < 0.3). As shown in the Figure 3 the self-emulsification of the formulation depends on the Labrasol:Labrafil ratio. A maximum of 20% Labrafil was required to enable self-emulsification with a uniform particle size distribution. Further optimisation suggested that 10% Labrafil can form monodispersed fine droplets (182.2 nm, PDI 0.261). However, subsequent SMEDDS dispersion in 0.1N HCl (gastric pH) revealed that the particle size and PDI dramatically increased (particle size > 10 µm; PDI ~1) and self-emulsification capacity was lost. To mitigate this instability behaviour, vitamin-E TPGS (TPGS) which has high surface activity and is stable at low pH was used 32. While SMEDDS prepared with Labrafil, Labrasol and Transcutol were not stable at a gastric pH of 1.2, addition of as little as 0.25% TPGS stabilized the formulation during dispersion in 0.1N HCl (pH~1.2). As shown in the Table 2, using 2 and 3% of TPGS showed optimal performance in terms of smaller particles and narrow size distribution in both in water (data not shown) and in 0.1N HCl.


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Table 2: The composition of SMEDDS pre-concentrate and its dispersion at different pH. SMEDDS composition (Lf:Ls:Tr:TP) 10:80:10:0 10:80:10:0 10:79:10:1 10:78:10:2 10:77:10:3 10:75:10:5

Dispersant

Droplet size (d, nm)

Poly dispersity index (PDI)

Water (pH 7.0) 0.1N HCl (pH 1.2) 0.1N HCl (pH 1.2) 0.1N HCl (pH 1.2) 0.1N HCl (pH 1.2) 0.1N HCl (pH 1.2)

182.2 ± 10.8 > 10,000 355.4 ± 78.2 168.5 ± 7.6 124.2 ± 5.4 109.8 ± 9.8

0.261 ± 0.014 1.000 0.413 ± 0.051 0.226 ± 0.024 0.264 ± 0.043 0.378 ± 0.066

Equilibrium solubility (mg/g) of SN38-unde20 5.36 ± 0.20 5.36 ± 0.20 5.43 ± 0.04 5.79 ± 0.12 5.69 ± 0.09 5.19 ± 0.15

Abbreviations: Lf, Labrafil; Ls, Labrasol; Tr, Transcutol; TP, vitamin-E TPGS.

The determined equilibrium solubility values of SN38-unde20 in the SMEDDS are also included in Table 2 and show little variation with composition and TPGS inclusion levels. Based on this data, 2-3% TPGS levels and a 5 mg/g or 0.5% (w/w) drug loading (< 90% equilibrium solubility) was selected for subsequent studies. Drug inclusion had no detrimental influence on dispersion (droplet sizes and distribution) of the optimal SMEDDS in gastric conditions, see Table 3. The polyethylene glycol (PEG 1000) chain of TPGS is longer than Labrsol (PEG 8) and therefore considered to be present at the lipid/water interface and stabilises the formulation in acidic pH 33. Table 3: Dispersion of SMEDDS with or without SN38-unde20 (0.5%) in 0.1N HCl. Composition Lf:Ls:Tr:TP = 10:78:10:2 Lf:Ls:Tr:TP = 10:78:10:2 Lf:Ls:Tr:TP = 10:77:10:3 Lf:Ls:Tr:TP = 10:77:10:3

Drug loading Size (nm, Mean ± SD) PDI (Mean ± SD) Blank 168.1 ± 6.7 0.226 ± 0.024 0.5% 147.6 ± 4.9 0.243 ± 0.012 Blank 124.2 ± 5.4 0.264± 0.043 0.5%

115.9 ± 7.2

0.369 ± 0.022

Abbreviations: Lf, Labrafil; Ls, Labrasol; Tr, Transcutol; TP, vitamin-E TPGS.

3.2 In vitro lipid digestion and drug solubilisation For lipid-based delivery systems, lipid digestion plays a critical role in drug solubilisation and precipitation in the GIT and subsequent absorption 34, 35. The phase partitioning of SN38unde20 in simulated GI conditions is shown in Figure 4. The regeneration of SN38 during this study was not monitored, as our previous study has confirmed negligible reconversion of SN38 from SN38-unde20 over 2 hr in similar conditions 27. SN38-unde20 has inherently poor aqueous solubility (clogP 7.7), and only ~1% of drug partitioned into the aqueous phase at 5 min when exposed in simulated intestinal conditions. The aqueous solubility increased Page 11 ACS Paragon Plus Environment


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gradually to 6% at 30 min and plateaued at 60 min (Figure 4, A). Being highly lipophilic the pure pro-drug solubilised slowly over time as the lipid digestion progressed. As expected the amount of drug in pellet phase was significantly higher for pure drug than the SMEDDS formulation (Figure 4, C). Incorporating SN38-unde20 into SMEDDS significantly improved aqueous drug solubilisation in simulated intestinal conditions (> 60% of the dose) (Figure 4, D). The encapsulation of SN38-unde20 in SMEDDS avoided the conventional dissolution step of raw drug. As the digestion of the digestible components (i.e. Labrasol and Labrafil) progressed, the amount of drug in the aqueous phase decreased from 40% at 1 min to 28% at 60 min, indicating the drug is less soluble in the digestion products (Figure 4, B). Importantly, the solubilised drug concentration was more than 7-fold higher than the pure drug for the entire 1h. This result was expected since colloids of digestion medium (e.g. mixed micelles) facilitate the solubilisation of lipophilic drug 30.

Figure 4: SN38-unde20 partitioning in pellet phase (■ brown) and aqueous phase (■ blue) during in vitro lipolysis of (A) Pure drug and (B) SMEDDS. The solubility profile of SN38-unde20 (C) drug in pellet phase and (D) drug in aqueous phase during the digestion of pure drug (square) and SMEDDS (circle) following digestion of 1mg/ml of drug in digestion medium in the both cases (mean ± SD, n = 3).

3.3 In vivo pharmacokinetic studies The oral absorption of SN38-unde20 from the SN38-unde20-SMEDDS and SN38 solution were investigated and compared with an equivalent IP dose. The formulations were well


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tolerated following oral (PO) and IP dosing and no adverse effects were observed. The plasma concentration of SN38-unde20 and SN38 versus time profiles following PO and IP treatment of identical SN38 doses (10 mg/kg) are illustrated in Figure 5 and Figure 6, respectively. The corresponding PK parameters are provided in Table 4 and Table 5, respectively. Following IP administration, the drug dispersed slowly in the peritoneal cavity from the site of administration and acted as a depot thereby required longer time to reach blood circulation (i.e. Tmax: 360 min); this correlates with poor in vitro aqueous solubilisation (Figure 4, D). In contrast, the absorption following the oral administration of SN38-unde20SMEDDS was rapid within the first hour (Tmax: 180 min), and this is in agreement with the rapid drug solubilisation in the in vitro simulated intestinal conditions (see Figure 4, D); the plasma SN38-unde20 concentration increased gradually from 1h to 3h before decreasing slowly to 4 ng/ml at 24h and 2 ng/ml at 48h. Plasma SN38-unde20 conc. (ng/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1000

100

10

1 0

2

4

6

8

24

48

Time (hr)

Figure 5: Plasma exposure of SN38-unde20 following IP dosing of SN38-unde20 (●) and PO dosing of SN38-unde20-SMEDDS (■) (10 mg/kg) (mean ± SD, n = 4). Table 4: Pharmacokinetic parameters of SN38-unde20 following IP dosing of SN38-unde20 (10 mg/kg), and PO dosing of SN38-unde20-SMEDDS (10 mg/kg); (mean ± SD, n = 4). Drug

Formulation/route

SN38-unde20

SMEDDS/PO Solution/IP

Tmax (min)

Cmax (ng/ml)

180.0 ± 0.00 24.54 ± 4.37 360.0 ± 0.00 138.24 ± 34.94

AUC0→∞ (min*µg/ml) 19.83 ± 3.74 129.32 ± 29.24

The long plasma exposure was possibly due to the lymphatic absorption, given the high lipophilicity (clogP 7.7) of the SN38-unde20 34, 36. The area under the curve (AUC) value of the plasma SN38-unde20 concentration versus time profile was significantly higher (more Page 13 ACS Paragon Plus Environment


than 6-fold) when SN38-unde20 was given as an IP dose in comparison with the orally dosed SN38-unde20-SMEDDS. The orally dosed pharmacokinetics of unformulated SN38-unde20 was not investigated. Based on the 7-fold difference observed in the in vitro solubilisation level, it is likely that the absorption will be much lower than that observed for the SMEDDS formulation. Furthermore, vitamin E-TPGS (TPGS), which has been widely used to enhance trans-membrane permeation (as P-gp efflux pump inhibitor) may play a role in enhancing the oral absorption of SN38-unde20 by reducing the P-gp efflux 32. 100

Plasma SN38 conc. (ng/ml)

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SN38 (Unde20 IP)

SN38 (Unde20 PO)

SN38 (IP)

SN38 (PO)

10

1

0.1 0

2

4

6

8

24

48

Time (hr)

Figure 6: Plasma concentration of SN38 following IP dosing of SN38 (□) and SN38-unde20 (▲); PO dosing of SMEDDS-SN38-unde20 (▼) and SN38 (○) at a dose of 10 mg/kg; (mean ± SD, n = 4) Table 5: Pharmacokinetic parameters of SN38 following IP dosing of SN38 and SN38unde20 (10 mg/kg); and PO dosing of SN38-unde20 and SN38 (10 mg/kg); (mean ± SD, n = 4). Drug

Formulation/route

Plasma exposure of SN38

SN38-unde20SMEDDS/PO SN38-unde20 solution/IP SN38 solution/IP SN38 solution/PO

Tmax (min)

Cmax (ng/ml)

AUC0→∞ (min*µg/ml)

195.0 ± 113.6

6.59 ± 1.80

8.18 ± 3.28

180.5 ± 129.6

11.62 ± 3.17

7.96 ± 2.73

37.5 ± 25.9

19.81 ± 11.89

7.50 ± 1.57

120.0 ± 0.0

0.44 ± 0.16

0.07 ± 0.04

Interestingly, in spite of significant differences in the AUC (SN38-unde20) values between SN38-unde20-SMEDDS/PO and SN38-unde20/IP, the AUC of the active metabolite, SN38, following oral or IP delivery of SN38-unde20 formulation were comparable. It is likely that Page 14 ACS Paragon Plus Environment

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the orally absorbed SN38-unde20 more readily converted into SN38 and thus showed a direct correlation to SN38 exposure in plasma. In contrast, the higher plasma exposure of SN38unde20 following IP dose is more likely to be incompletely converted into SN38. Both PO or IP dosed SN38-unde20 show 100-fold higher AUC (SN38) in comparison with that following the oral administration of an unformulated SN38 solution, and are comparable with the IP administered unformulated SN38 solution. A schematic mechanism aimed to explain the different oral absorption pathways of unformulated SN38 and SN38-unde20-SMEDDS is presented in Figure 7. Oral absorption of SN38 is restricted by its poor solubility, low permeability and first-pass metabolism in liver 19. In addition, exposure of SN38 causes local toxicities to the gut 14. The optimal prodrug, SN38-unde20, has higher permeability and low toxicity in comparison with SN38 27. Furthermore, the lipid-based formulation (SMEDDS) along with lipid digestion enhance the solubility in GI conditions and increase permeability across the mucosal membrane thus improved oral bioavailability 37. Recent clinical studies have suggested that the longer exposure of SN38 at a low concentration (1.2 to 5.9 ng/ml) in plasma provided improved therapeutic response without causing dose-limiting toxicity in human subjects 38-40. Therefore, the prolonged SN38 exposure observed following the oral administration of SN38-unde20-SMEDDS proves the concept that the combined lipophilic prodrug and nanomedicine approach offers potential for convenient and safe SN38 based chemotherapy.



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Lipid based (SMEDDS) formulation SN38 Not lipophilic

Lipophilic prodrug GI Digestion (no SN38)

P-gp efflux

In aqueous media

M cell

Mucosal toxicity

Enterocyte

Chylomicron First pass hepatic metabolism Low bioavailability

Lymphatic absorption Improved bioavailability

Systemic circulation and tumours Figure 7: Schematic representation (size not to scale) illustrating formulation and the possible fate of SMEDDS in GIT. Poor solubility and P-gp efflux pump limit intestinal absorption of SN38 and glucuronisation in the liver further reduce its systemic exposure. The lipophilic prodrug in an optimal SMEDDS formulation enhances solubilisation in GIT and resulting colloidal solution which is believed to increase direct passive diffusion. Moreover, TPGS in the formulation inhibits P-gp efflux pump, thus facilitates absorption. Long chain triglycerides in formulation and high clogP (7.7) of the prodrug may induce lymphatic transport, thereby significantly enhance oral bioavailability and longer systemic exposure of SN38.

4

Conclusions

A SMEDDS formulation containing a lipophilic prodrug of SN38, SN38-unde20, for oral delivery has been reported. The ratio of LC lipid, co-surfactant and co-solvent were carefully optimized based on dispersibility, droplet size and maximum drug loading. The resulting SMEDDS formulation demonstrated significantly improved SN38-unde20 solubilisation under simulated intestinal digesting condition. The systemic exposure of SN38 following oral SMEDDS-SN38-unde20 was comparable with an identical IP dose of SN38-unde20SMEDDS, and was higher than an identical IP dose of SN38 solution. The current study


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suggests, a combined lipophilic prodrug and optimal SMEDDS formulation play a synergistic role for enabling oral delivery of challenging chemotherapeutic molecules.

Acknowledgements: Financial support of the Australian National Health and Medical Research Council project grant scheme (APP1026382) is gratefully acknowledged.



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5

References

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SN38 (BCS IV)


Lipid based

Aqueous

formulation

media

Lipophilic Prodrug (BCS II)

Self emulsification

First pass metabolism Low bioavailability

High oral bioavailability ACS Paragon Plus Environment

≥ Increased systemic exposure

Enabling Oral SN38-Based Chemotherapy with a ... - ACS Publications

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