Synthesis of a Gemcitabine Prodrug for Remote Loading into

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Synthesis of a gemcitabine pro-drug for remote loading into liposomes and improved therapeutic effect Jonathan May, Elijus Undzys, Aniruddha Roy, and Shyh-Dar Li Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00619 • Publication Date (Web): 16 Dec 2015 Downloaded from http://pubs.acs.org on December 23, 2015

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Bioconjugate Chemistry

Synthesis of a gemcitabine prodrug for remote loading into liposomes and improved therapeutic effect Jonathan P. May, †,‡, Elijus Undzys‡, Aniruddha Roy †,‡ and Shyh-Dar Li†,‡* † Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC Canada ‡ Drug Discovery Program, Ontario Institute for Cancer Research, Toronto, ON Canada

KEYWORDS: Gemcitabine, prodrug, liposomal drug delivery, remote loading

ABSTRACT: The chemotherapeutic, gemcitabine, was actively and stably loaded into lipid nanoparticles through the formation of a prodrug. Gemcitabine was chemically modified to increase the lipophilicity and introduce a weak base moiety for remote loading. Several derivatives were synthesized and screened for their potential to be good liposomal drug candidates for remote loading by studying their solubility, stability, cytotoxicity and loading efficiency. Two morpholino derivatives of GEM (22 and 23) were chosen as the preferred prodrugs for this purpose as they possessed the best loading efficiencies (100% for drug-to-lipid ratio of 0.36 w/w). This is a considerable improvement over a passive loading strategy where typical loading efficiencies are in the order of ~10-20% for a drug-to-lipid ratio of ~0.01.

ACS Paragon Plus Environment

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Liposomes loaded with these two prodrugs were studied in an s.c. tumour model in vivo and showed improved therapeutic effect over free GEM (~2-fold) and saline control (8- to 10-fold). This work demonstrates how chemical modification of a known hydrophilic drug can lead to improved loading, stability and drug delivery in vivo.

INTRODUCTION Gemcitabine

(GEM),

2’,2’’-difluoro-2’-deoxycytidine,

is

a

well-tolerated,

potent

chemotherapeutic.1 However, part of the reason for its low systemic toxicity is the fact that it is readily metabolised to the less effective difluoro-uridine derivative and undergoes rapid renal elimination.2-3 This has led us and others to investigate whether liposomal delivery of GEM could lead to a formulation which was well-tolerated, but more stable to degradation. Liposomes are nano-sized lipid vesicles consisting of a lipid bilayer surrounding an aqueous core. Their use in drug delivery stems from the improved pharmacokinetics and biodistribution, partly due to the EPR-effect,4 which has been described for tumours, whereby the rapidly growing tumour forms leaky vasculature, allowing increased uptake for nanoparticles in the 50-200 nm range. Liposomal encapsulation of GEM has often been performed via a passive loading strategy,5-6 taking advantage of the drug’s high aqueous solubility (22.3 mg/mL). Passive loading performed by ourselves and others for liposomes of a size suitable for EPR-effect (~100 nm) has delivered encapsulation efficiencies of around 10% for solutions of 10-20 mg GEM/mL, leading to final liposomal GEM concentrations of 1-2 mg/mL and a corresponding drug-to-lipid ratio of 0.010.02. However, loading efficiency is generally much lower for passive loading than for a remote loading strategy, a method which could also increase the stability of the drug in the liposome.7 Remote loading, refers to where the drug is “actively loaded” into the liposome core via a pH or


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ion gradient (e.g. ammonium sulfate) across the liposome membrane, which is best represented in the case of Doxorubicin (DOX) with the DOXIL® liposome formulation (Figure 1).8-9

Figure 1: Schematic for remote loading with a weak base drug (e.g. DOX). Crucially DOX contains a weak base amine which becomes protonated once inside the ammonium sulfate containing liposome core, forming a sparingly soluble DOX sulfate salt that crystallizes out while liberating ammonia gas that can escape through the liposome membrane. GEM however, is lacking some of the key physical properties of DOX that make it suitable for this remote loading strategy. For example, the pKa is higher for DOX (primary amine pKa 8.9), relative to GEM (pyrimidine pKa 3.6). This means that the pH inside the liposomes needs to be strongly acidic (pH 0.05) observed for liposomal formulations of drugs such as DOX. Another technique used to improve the pharmacokinetics of GEM has been the formation of GEM prodrugs, usually with lipophilic chains added to the N4 position, such as stearoyl and squalene, which in some cases are used for insertion into hydrophobic nanoparticles or liposome membranes.14-22 There is also a case where short isoprene polymers have been grown from a GEM anchor.23 This strategy also provides chemical protection of the N4 position of the cytosine base, which is susceptible for the rapid hydrolysis to uracil via cytidine deaminase present in body fluids. Other prodrugs have been investigated including a recent example of a theranostic GEM derivative with a redox sensitive disulfide linker and RGD targeting moiety,24 a PEGylated folate-targeted GEM derivative25 and an antibody conjugated derivative.26 Here we investigated whether prodrugs of hydrophilic drugs, such as GEM, can enable their remote loading with a high efficiency and stable encapsulation. We hypothesized that for a


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hydrophilic drug, modifications may be necessary to temporarily increase the logP to a value more suitable for passage across a lipid membrane (logP >0). To achieve this, we decided to modify the N4 position with a small alkyl group to increase the prodrug lipophilicity. A weak base appendage was then conjugated to a 3’ or 5’ hydroxyl via a cleavable ester linkage to form a difunctionalized GEM derivative with suitable physical properties for remote loading. Modifications in these positions should be labile to enzymes – for the N4 modification a carboxylesterase-2 has been described,27 and for the 5’-ester there are myriad examples of primary ester hydrolysis in many cells including those of tumour tissue.28 We then sought to investigate the solubility, stability, cytotoxicity, liposomal loading, and preliminary efficacy of the liposomal drug to elucidate the validity of this technique for improving the delivery of GEM.


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RESULTS Synthesis of gemcitabine derivatives Six derivatives of GEM were synthesized incorporating both a lipophilic tail attached to the cytosine base to increase lipophilicity and an ionizable weak base appendage attached to the 5’hydroxyl of the furanose ring to enable remote loading into liposomes. These derivatives differed by the identity of the weak base (N-methyl-piperazine, or morpholine) and the length of the carbon chain linker between the 5’-hydroxyl point of conjugation and the weak base moiety.

Scheme 1. Synthetic scheme for the preparation of weak base appendages 1-6. Initially the 6 different weak base appendages were prepared with minor modifications to the preparations described in the literature (Scheme 1).29 This afforded three morpholino and three N-methyl-piperazino derivatives, each with either 2, 4 or 5 carbon chain length linkers. These amino acids were isolated as their HCl salts in the form of white crystalline compounds (overall yields for the two steps ranged from 70-85%).


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Scheme 2. Preparation of 4-N-octanoyl-2’-difluoro-deoxycytidine, 8. Addition of the lipophilic tail to the 4-amino group of the nucleoside base of GEM was achieved using a transient protection of the 3’ and 5’-hydroxyls with trimethyl silane (Scheme 2). The amide was formed through reaction with the acid chloride derivative of octanoic acid. Following removal of the transient protection and a number of aqueous washing steps, including a wash with a dilute solution of phosphoric acid, the derivative was acquired in good yield and relatively high purity even without a purification step (90%). However, purification with a short silica gel column improved the purity and afforded the desired GEM derivative 8 in good yield (80%). The nucleoside base modified derivative 8 was then orthogonally protected (Scheme 3) in the 5’position with TBDMS (97%) and at the 3’-position with THP (73%). This allowed deprotection of the silyl group with fluoride (either as the pyridine or trimethylamine complex) to yield compound 11 (87%).30 The 5’-hydroxyl that was exposed during this last deprotection was then coupled with the six different weak base appendages (1-6), and after isolation of the crude products followed by a series of short deprotections of the acid labile THP groups with TFA, products 18-23 were afforded. All final compounds were purified by automated silica gel chromatography (overall yield for 5 steps 38-50%) and characterized by NMR and LC-MS to confirm the desired identity and purity.


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O

R1

O

NH

N

O

O

ii) Tos-pyr, CH2Cl2

N

Si O

N

HF.NEt3 O

N

HO

O

O

F O

8

F O

F O

O

O

F F

R1 NH

N

i) TBDMS-Cl, pyr

O OH

R1 NH

N HO

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10

F 11

O

N R2

OH.HCl n O

1-6 EDC, NEt3, CH2Cl2

O

O

R1

NH

NH # 18: 19: 20: 21: 22: 23:

R1 C7H15 C7H15 C7H15 C7H15 C7H15 C7H15

R2 NMe O NMe O NMe O

n 3 3 1 1 4 4

R1

N

N N R2

n O

N

O

O

TFA

N

O F OH

CH2Cl2

R2

N

O

12-17

O

O F O

F

18-23

n O

F O

Scheme 3. Synthetic route to the 5’-weak base modified-4-N-octanoyl-2’-difluoro-deoxycytidine derivatives, 18-23. Evaluation of GEM prodrug solubility Once the compounds were in hand, they were tested for aqueous solubility over a range of pH (pH 3, pH 4, pH 5, pH 6, pH 7). Compounds were readily soluble at lower pHs (pH 3-5) at >20 mg/mL (the maximum concentration tested), but a solubility limit of ~10 mg/mL was reached at pH 6-7.


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Testing the stability of gemcitabine derivatives All final compounds were tested for their stability in the neutral and weakly acidic buffers used for drug loading. To test the stability of each of the GEM derivatives (18-23) a study was performed at 37°C with buffers at pH 5 and pH 7; two relevant pHs for liposome loading and stability. Compounds were incubated for up to 24 h in these buffers and samples were checked for the degree of decomposition by LC-MS (Figure S1). All samples studied were significantly more stable at pH 5 than at neutral pH. In fact, most compounds showed excellent stability at pH 5, with the majority still remaining greater than 90% intact by the end of the 24 h study period. The 2 carbon linker with a morpholine base attached (21) appeared to be the most labile at pH 5 (~12 % decomposition), while the 5 carbon linkers in compounds 20 and 23 and the 2 carbon linker with N-methyl-piperazine (18) were essentially stable during the study period (Figure 2). Interestingly, it appears the ester bond at the 5’position of the GEM derivative was particularly labile when the 4 carbon linker was used, no matter which basic moiety was tethered to the other end (19 and 22). The ester cleavage was most accelerated at neutral pH, and following 24 h incubation only 17% or 29% was still intact for compounds 19 and 22, respectively.


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Figure 2. Summary of GEM derivative (18-23) stability based on the percent remaining after incubation at 37°C for 24 h at either (a) pH 5 (NaOAc buffer) or (b) pH 7 (HBS). In vitro cytotoxicity (IC50) study In order to assess the relative cytotoxicity of the GEM derivatives against tumour cells, multiple cell viability studies were performed. Initially, the murine mammary cancer cell line EMT-6 was studied with GEM, the base-modified GEM (8) and the six weak-base GEM derivatives (18-23), with all results being repeated in at least 3 independent experiments (Table 1).

Table 1. Average IC50 values for the GEM derivatives against the EMT-6 murine breast cancer cell line from >3 separate experiments. Mean ± S.D. For the raw data please check the supplementary information Figure S2.


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Sample

Average IC50 (nM)

GEM

30.5 ± 2.7

GEM-oct (8)

43.3 ± 2.7

p2Go (18)

166.6 ± 20.1

p4Go (19)

132.6 ± 22.7

p5Go (20)

201.4 ± 51.8

m2Go (21)

219.9 ± 76.2

m4Go (22)

146.9 ± 27.7

m5Go (23)

152.8 ± 26.0

The IC50 values of the GEM derivatives (18-23) are higher than that found for native GEM (31 nM), correlating to a 4- to 7-fold reduction in potency. However, it should be noted that the potency for the octanoyl derivative 8 (43 nM) is almost equal to that of the native drug. Two of the more potent GEM derivatives (22 and 23) were also studied with the multidrug resistant cell line, EMT6-AR1 (P-glycoprotein overexpression) and similar IC50 values were obtained (Table 2). Table 2. Average IC50 values for the GEM derivatives against the EMT6-AR1 multi-drug resistant murine breast cancer cell line. See supplementary information for curves, Figure S3.

Sample

Average IC50 (nM)

GEM

14.1 ± 0.5

GEM-oct (8)

39.0 ± 5.1

m4Go (22)

150.5 ± 12.7

m5Go (23)

78.5 ± 3.7


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Loading of gemcitabine derivatives into liposomes Preliminary screening of drug loading was performed with a non-PEGylated liposome (DSPC/Chol, 55:45) prepared with an ammonium sulfate gradient and acetate external buffer (pH5) as the derivatives were found to show greater stability and solubility at this pH. Different drug-to-lipid ratios were screened as well as incubation time to assist in optimization of the method. All samples were studied for drug loading using ultrafiltration to separate encapsulated drug from non-encapsulated drug, followed by LC-MS detection (Figure S4). This proved to be a robust method and was able to indicate quantities of drug decomposition products if that occurred at a significant rate during the loading procedure.

Figure 3. Loading of GEM derivatives on a small scale (100 µL) with DSPC/Chol liposomes at different drug-to-lipid ratios (w:w) for a) m4Go (22); and b) m5Go (23). The initial loading data represented in Figure S4 suggested that the morpholine derivatives m4Go (22) and m5Go (23) were loaded rapidly at high drug-to-lipid ratios. To further investigate


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the limitations of loading for these derivatives the drug-to-lipid ratios were increased and experiments were performed multiple times with incubation at 60°C for 60 mins (Figure 3).

Figure 4. Loading of GEM derivatives on a small scale (100 µL) at different drug-to-lipid ratios (w:w) with a PEGylated liposome for a) p4Go (19); b) p5Go (20); c) m4Go (22); and d) m5Go (23). Once optimal conditions were identified, the GEM prodrugs were tested for loading efficiency with a PEGylated liposome (DSPC/Chol/DSPE-PEG, 56:39:5) also prepared with an ammonium sulfate gradient and acetate external buffer (pH 5). This is similar to the liposome used in the DOXIL® formulation and is known to be a long circulating liposome. As before, several drug-tolipid ratios (3:1, 4:1, 5:1; w:w) were studied in order to establish the optimal system (Figure 4). Liposomes were incubated with drug derivative in acetate buffer at pH 5 at 60°C for at least 30 min, and all samples were then processed with ultrafiltration and followed by quantification with LC-MS to calculate encapsulated drug concentrations (Figure 4). These figures clearly show that the loading of the piperazine derivatives (19 and 20) was less effective (~40-60% loading efficiency) than for the morpholine derivatives (22 and 23), even at the lower drug-to-lipid ratios (0.18 and 0.24). The morpholine derivative 22 showed particularly efficient loading (>80%) even


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at a D:L of 0.3. The morpholine derivative 23 also had a good loading efficiency at lower drugto-lipid ratios, but dropped to ~50% at a drug-to-lipid ratio of 0.3. Table 3. Physical parameters of liposomes (L22 and L23), following loading with GEM derivatives 22 and 23, purification by dialysis and sterile filtration for use in vivo.

Sample

Z-ave

Polydispersity

composition

(nm)

index (PDI)

100.1 ± 0.9

0.04 ± 0.00

-

-

98.4 ± 0.3

0.06 ± 0.01

100

0.15

101.2 ± 2.0

0.04 ± 0.01

100

0.15

Liposome

DSPC/Chol/DSPE-PEG

pre-loading

(56:39:5)

L22

L23

Final

Liposome

DSPC/Chol/DSPE-PEG (56:39:5) DSPC/Chol/DSPE-PEG (56:39:5)

% Loading

D:L (w/w)

Once an optimal drug-to-lipid ratio had been found, the drug loading was scaled up (from 100 µL to 1 mL) to generate dose for in vivo studies (Table 3). A drug-to-lipid ratio of 0.16 was selected to achieve >90% drug encapsulation, followed by dialysis (HBS pH 7.4) to remove any unencapsulated drug giving a final D:L of 0.15. Liposomes were routinely prepared with diameters of 95-105 nm, low polydispersity (1500 mm3 tumour volume, or symptoms of significant discomfort (severe piloerection / withdrawn behaviour) were observed.

Cells were grown in DMEM as described above and harvested in FBS free media for inoculation in Balb/c mice. The multidrug resistant cell line, EMT6-AR1 (1 x 105 cells) was inoculated s.c. into the right flank of the 2-3 week old mice (18-20 g). By around 1 week after inoculation the tumours had grown to a palpable size (~10 mm3) and the animals could be injected i.v. with the chosen treatment. Four groups were chosen for comparison: Saline, Gemcitabine (40 mg/kg), Liposomal formulation of GEM derivative 22 (40 mg drug/kg), Liposomal formulation of GEM derivative 23 (40 mg drug/kg). Treatment dosing was given twice weekly (day 0, day 3, day 7 etc.). Tumour size and body weights were measured every 1-2 days.

Statistical analysis All data are expressed as mean ± standard deviation unless noted otherwise. Statistical analysis was conducted with the two-tailed unpaired t-test for two-group comparison or one-way ANOVA, followed by the Tukey multiple comparison test for three or more groups (GraphPad Prism). Values of p < 0.05 were considered to be statistically significant.


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ASSOCIATED CONTENT Supporting Information. GEM derivative stability study; Cytotoxicity plots; Preliminary GEM derivative liposomal loading screen; Liposome stability study; NMR and LC-MS characterisation. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author: *Shyh-Dar Li, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC Canada. [email protected]; tel. 604-827-0675; fax. 604822-3035. ACKNOWLEDGMENT We would like to acknowledge the Canadian Institutes for Health Research (CIHR) for assistance with funding for this project, through a combination of CIHR proof-of-principle (PPP130153) and CIHR operating grants (MOP-119471). SDL also received a CIHR New Investigator Award and a Young Investigator Award from the Prostate Cancer Foundation. The Ontario Institute for Cancer Research (Funded by the Government of Ontario) and the University Health Network, Toronto are also acknowledged for providing the facilities and equipment necessary to conduct this research. ABBREVIATIONS Chol, cholesterol; DOX, doxorubicin; DMEM, dulbecco’s modified eagle’s medium; DMSO, dimethylsulfoxide; DSPC, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine; DSPE-PEG, 1,2distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000]; DTX, docetaxel; EtOAc, ethyl acetate; FBS, fetal bovine serum; GEM, gemcitabine; HBS, hepes buffered

saline;

HPLC,

high

pressure

liquid

chromatography;

LC-MS,

liquid


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chromatography/mass spectrometry; MeCN, acetonitrile; NEt3, trimethylamine; pyr, pyridine; TBDMS, tert-butyldimethylsilyl; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, Thin layer

chromatography;

XTT,

2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-

carboxanilide; REFERENCES 1. Cham, K. K., Baker, J. H., Takhar, K. S., Flexman, J. A., Wong, M. Q., Owen, D. A., Yung, A., Kozlowski, P., Reinsberg, S. A., Chu, E. M., et al. (2010) Metronomic gemcitabine suppresses tumour growth, improves perfusion, and reduces hypoxia in human pancreatic ductal adenocarcinoma. Br J Cancer 103, 52-60. 2. Shipley, L. A., Brown, T. J., Cornpropst, J. D., Hamilton, M., Daniels, W. D. and Culp, H. W. (1992) Metabolism and disposition of gemcitabine, and oncolytic deoxycytidine analog, in mice, rats, and dogs. Drug Metab Dispos 20, 849-855. 3. Veltkamp, S. A., Pluim, D., van Eijndhoven, M. A., Bolijn, M. J., Ong, F. H., Govindarajan, R., Unadkat, J. D., Beijnen, J. H. and Schellens, J. H. (2008) New insights into the pharmacology and cytotoxicity of gemcitabine and 2',2'-difluorodeoxyuridine. Mol Cancer Ther 7, 2415-2425. 4. Maeda, H. (2001) The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul 41, 189-207. 5. Bornmann, C., Graeser, R., Esser, N., Ziroli, V., Jantscheff, P., Keck, T., Unger, C., Hopt, U. T., Adam, U., Schaechtele, C., et al. (2008) A new liposomal formulation of Gemcitabine is active in an orthotopic mouse model of pancreatic cancer accessible to bioluminescence imaging. Cancer Chemother Pharmacol 61, 395-405. 6. Graeser, R., Bornmann, C., Esser, N., Ziroli, V., Jantscheff, P., Unger, C., Hopt, U. T., Schaechtele, C., von Dobschuetz, E. and Massing, U. (2009) Antimetastatic effects of liposomal gemcitabine and empty liposomes in an orthotopic mouse model of pancreatic cancer. Pancreas 38, 330-337. 7. Cern, A., Golbraikh, A., Sedykh, A., Tropsha, A., Barenholz, Y. and Goldblum, A. (2012) Quantitative structure-property relationship modeling of remote liposome loading of drugs. J Control Release 160, 147-157. 8. Haran, G., Cohen, R., Bar, L. K. and Barenholz, Y. (1993) Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta 1151, 201-215. 9. Barenholz, Y. (2012) Doxil(R)--the first FDA-approved nano-drug: lessons learned. J Control Release 160, 117-134. 10. Xu, H., Paxton, J., Lim, J., Li, Y., Zhang, W., Duxfield, L. and Wu, Z. (2014) Development of high-content gemcitabine PEGylated liposomes and their cytotoxicity on drugresistant pancreatic tumour cells. Pharm Res 31, 2583-2592. 11. Gravem, H. Gemcitabine-containing liposomes. University of Tromso, 2006. 12. Mokelby, T. Active loading of gemcitabine into liposomes. University of Tromso, 2009.


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Synthesis of a Gemcitabine Prodrug for Remote Loading into

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