Imaging Agent to Bile Acid

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Tethering of Chemotherapeutic Drug/Imaging Agent to Bile Acid-Phospholipid Increases the Efficacy and Bioavailability with Reduced Hepatotoxicity Vedagopuram Sreekanth, Nihal Medatwal, Sandeep Kumar, Sanjay Pal, Vamshikrishna Malyla, Animesh Kar, Priyanshu Bhargava, Aaliya Naaz, Nitin Kumar, Sagar Sengupta, and Avinash Bajaj Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00564 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017

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Bioconjugate Chemistry

Tethering of Chemotherapeutic Drug/Imaging Agent to Bile Acid-Phospholipid

Increases

the

Efficacy

and

Bioavailability with Reduced Hepatotoxicity

Vedagopuram Sreekanth,†,‡ Nihal Medatwal, Sanjay Pal,

†,¶

†, ‡

Sandeep Kumar,

†,‡

Malyla Vamshikrishna,† Animesh Kar,† Priyanshu

Bhargava,† Aaliya Naaz,† Nitin Kumar,§ Sagar Sengupta,§ and Avinash Bajaj†,❉

†: Laboratory of Nanotechnology and Chemical Biology, Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone Faridabad-Gurgaon Expressway, Faridabad, Haryana, 121001, India. ‡: Manipal University, Manipal, 576104, India. ¶: KIIT University, Bhubaneswar, Odisha, 751024, India. §: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.

❉

Corresponding Author:

Email: [email protected], Ph: +91-124-2848831, Fax: +91-124-2848803

ACS Paragon Plus Environment

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Abstract: Weakly basic drugs display poor solubility and tends to precipitate in stomach’s acidic environment causing reduced oral bioavailability. Tracing of these orally delivered therapeutic agents using molecular probes is challenged due to their poor absorption in the gastrointestinal tract (GIT). Therefore, we designed a gastric pH stable bile acid derived amphiphile where Tamoxifen (as a model anticancer drug) is conjugated to lithocholic acid derived phospholipid (LCA-Tam-PC). In vitro studies suggested the selective nature of LCA-Tam-PC for cancer cells over normal cells as compared to parent drug. Fluorescent labelled version of the conjugate (LCA-TamNBD-PC) displayed an increased intracellular uptake compared to Tamoxifen. We then investigated the antitumor potential, toxicity, and median survival in 4T1 tumor bearing BALB/c mice upon LCA-Tam-PC treatment. Our studies confirmed a significant reduction in the tumor volume, tumor weight, and reduced hepatotoxicity with a significant increase in median survival on LCA-Tam-PC treatment as compared to parent drug. Pharmacokinetic and bio-distribution studies using LCATam-NBD-PC witnessed the enhanced gut absorption, blood circulation, and tumor site accumulation of phospholipid-drug conjugate leading to improved antitumor activity.

Therefore,

our

studies

revealed

that

conjugation

of

chemotherapeutic/imaging agents to bile acid phospholipid can provide a new platform for oral delivery and tracing of chemotherapeutic drugs.


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Introduction Organ toxicity is one of the key parameters that limit the dosage regimens of chemotherapeutic drugs.1 Therefore, development of new chemotherapeutic modifications with reduced toxicity is much needed for effective cancer therapy.1 Few chemotherapeutic drugs like Tamoxifen (Tam), a non-steroidal estrogen receptor modulator, is used orally in pre- and post-surgical management of breast cancer as an adjuvant therapy.2 Tam is an aromatic drug with a N,N′-dimethylaminoethanol side chain; and oral absorption of Tam is hindered by its low dissolution in gastric media. Moreover, poor bioavailability of Tam is exacerbated by its intestinal and hepatic first pass metabolism. Therefore, higher dose administration of Tam is required for effective treatment leading to hepatotoxicity.3 Encapsulation of Tam in emulsions,4 lipids,5 polymeric nanoparticles,6 or in phospholipid-drug complexes7 have been attempted to enhance its oral bioavailability. Tracing of chemotherapeutic drugs can help in scheduling the proper dosage regimens and can provide advantages in reducing the drug-induced toxicity.8 Therefore, imaging agents are being integrated with chemotherapeutic drugs for cancer treatment.8 Fluorescent dyes

provided the benefits for determination of

pharmacokinetics and bio-distribution of Mitomycin C-Phospholipid complexes.9 Luminescent biodegradable polymers with π-conjugated core were attempted for doxorubicin delivery and cellular imaging.10 Oral delivery of the imaging probes to monitor the bioavailability of drugs is challenging due to multiple biological barriers of gastrointestinal tract (GIT) like GIT efflux, acidic pH of stomach, gut microbiota, and presence of proteolytic enzymes that can destabilize the delivery vehicles.11 Therefore, engineering of biomaterials that has the ability to deliver the cytotoxic


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drugs in combination with imaging agents with enhanced oral bioavailability will be highly useful for scheduling the proper chemotherapeutic dosages.12 Lipid-drug conjugates offer advantages of better cellular penetration, controlled drug release, enhanced bioavailability, and improved tumor accumulation leading to desired therapeutic effects with reduced toxicity.13,14 Phospholipids have traditionally been part of the clinically approved drug formulations.15 They can be safely administered by oral, systemic, or other parenteral routes owing to their biocompatible nature; and ability to withstand different stress conditions like pH, efflux, and enzymatic hydrolysis.15-17 Therefore, tethering of cytotoxic drugs to phospholipids can improve the stability and activity of these drugs with reduced toxicity.16,17 Chlorambucil-phospholipid conjugates are reported to be highly effective;18 and palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine-cisplatin conjugate (PGPC-cisPt) exhibited reduced nephrotoxicity.19 Bile acids present interesting scaffolds for engineering of materials for different biomedical applications due to their biocompatible nature.20-22 Presence of free hydroxyl and carboxyl groups provide additional advantages for tethering of drugs and imaging agents.23 In earlier studies, we have synthesized Tam conjugated bile acid amphiphiles with acid and amine head groups.24 We have shown that conjugation of three Tam molecules to cholic acid backbone with amine head group executed strong membrane interactions with high therapeutic efficacy.24 It has also been witnessed that tethering of dimethylaminopyridine head group to lithocholic acid-tamoxifen conjugate enhanced its cytotoxicity against breast cancer cells.25 We have recently reported the engineering of bile acid derived phospholipids,26 their ability to form supramolecular self-assemblies, their interactions with model


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membranes,27 and their use for drug delivery.28 These scaffolds can be used for conjugation of chemotherapeutic drugs and imaging agents. Therefore, in this manuscript, we present the synthesis of a drug-conjugated amphiphile where Tam is tethered to lithocholic acid derived phospholipid (LCA-TamPC). Bile acids are known to form mixed micelles with dietary fat, phospholipids, and hydrophobic endogenous/drug molecules due to their facial amphiphilic character; and help in oral absorption of lipophilic materials.29 Therefore, we hypothesize that amphiphilic nature of LCA-Tam-PC would aid in forming mixed micelles in the GIT; and assist in better absorption of the conjugate over parent drug. We also synthesized a phospholipid derived chimeric amphiphile having an imaging probe and Tam conjugated to bile acid phospholipid that would allow easy tracing of the amphiphile to determine the pharmacokinetics and bio-distribution of drugs.

Results and Discussion Design of amphiphiles: Lithocholic acid is a suitable steroidal moiety for synthesis of bile acid derived phospholipid-drug conjugates as it has the ability to get absorbed in the GIT.23 In addition, presence of a free hydroxyl and a carboxyl group makes it a suitable lipid carrier for tethering of drugs, and head groups.25 Phosphocholine, providing an amphiphilic character to lipids, is the most compatible head group due to its abundance in cell membranes.30 Therefore, we hypothesize that conjugation of Tam to lithocholic acid using amide linkage will provide the gastric pH stability, and tethering of phosphocholine head group will help in its absorption at GIT (Figure 1A). Tam (1, Figure 1B) itself is a prodrug that gets metabolized by phenyl ring hydroxylation using CYP2D6 enzyme; and by N-demethylation using CYP3A4 and CYP3A5 enzymes to endoxifen.31 Therefore, we conjugated lithocholic acid and N-


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desmethyltamoxifen using amide bond followed by introduction of phosphocholine head group at 3’-OH terminal of lithocholic acid (2, LCA-Tam-PC) (Figure 1B). Under neutral conditions, there is a high-energy barrier for deformylation of this amide bond.32 Protonation of amide under acidic conditions lowers the energy barrier for its degradation by Cytochrome P450s that can metabolise N-methyl-amides in acidic conditions.32 Therefore, this amphiphile can have a therapeutic effect as it is or can slowly be degraded by cytochromes to active drug. For delivery of imaging probes in combination with Tam; we developed a chimeric amphiphile (4, LCA-Tam-NBD-PC) by conjugation of nitrobenzoxadiazole (NBD) and Tam to lithocholic acid phospholipid using lysine linker (Figure 1B). As a control, NBD was also conjugated to Tam to make Tam-NBD (3). Synthesis of amphiphiles: For synthesis of LCA-Tam-PC (2), lithocholic acidtamoxifen conjugate (5, LCA-Tam) was synthesized as reported earlier.25 LCA-Tam (5) was then reacted with 2-chloro-1,3,2-dioxaphospholane-2-oxide to produce reactive intermediate (6) along with triethylammonium hydrochloride salt (NEt3.HCl) (Scheme 1). Reaction mixture was then filtered through celite and immediately reacted with trimethylamine gas in a pressure tube. Purification of reaction mixture using reverse phase C18 silica combi flash column chromatography gave LCA-TamPC (2) in 78% yield that was characterized by 1H NMR, 31P NMR, and HRMS.


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Figure 1. Design of amphiphile: A) Schematic presentation of the study showing the design of Lithocholic Acid-Tamoxifen derived Phospholipid (LCA-Tam-PC) for gastrointestinal pH stability and enhanced absorption at GIT; B) Molecular structures of Tamoxifen (1, Tam), LCA-Tam-PC (2); and their fluorescent derivatives Tam-NBD (3) and LCA-Tam-NBD-PC (4) highlighting the chemotherapeutic drug (Tam) and imaging probe (NBD).


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Scheme 1

Reagents, reaction conditions, and yields: (i) 2-chloro-1,3,2-dioxaphospholane-2oxide, triethylamine, 0 oC to RT, 10h; (ii) trimethylamine gas, acetonitrile, reflux, 48h, 78%.

We synthesized fluorophore conjugated Tam (3, Tam-NBD) where desmethylated Tam was directly conjugated to 4-chloro-7-nitrobenzofurazan (NBD-Cl) using earlier reported methods.25 Chimeric fluorophore conjugated amphiphile (4, LCA-Tam-NBDPC) was synthesized using a lysine linker (Scheme 2). First desmethyltamoxifen was conjugated with tandem amine protected lysine (7, Fmoc-Lys(Boc)-OH) to give conjugate (8) that upon treatment with acid released Boc and provided free ω-amino group (9). Free ω-amino group was then reacted with 4-chloro-7-nitrobenzofurazan (NBD-Cl) to give NBD derivative of α-amino protected lysine-tamoxifen conjugate (10). Fmoc was deprotected with piperidine to compound 11 with free α-amino group. Compound 11 was reacted with lithocholic acid to provide Tam and NBD conjugated

lithocholic

acid

(12).

Reaction

of

12

with

2-chloro-1,3,2-

dioxaphospholane-2-oxide followed by trimethylamine gas in a pressure tube provided conjugate 4. Purification of reaction mixture using reverse phase C18 silica flash column chromatography yielded LCA-Tam-NBD-PC (4) that was characterized by 1H NMR, 31P NMR, and HRMS.


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Scheme 2

Reagents, reaction conditions and yields: (i) N-desmethyltamoxifen, DCC, DMAP, DMF, RT, 12h, 89%; (ii) Dioxane.HCl, DCM, 0 oC to RT, 4h, 99%; (iii) NBD-Cl, triethylamine, DCM, RT, 12h, 82%; (iv) Piperidine, DCM, RT, 4h, 85%; (v) Lithocholic acid, EDC, HOBt, DMF, RT, 12h, 79%; (vi) a) 2-chloro-1,3,2-dioxaphospholane-2oxide, triethylamine, 0 oC to RT, 3h; b) trimethylamine gas, acetonitrile, reflux, 24h, 72%.

In vitro activities: Stability of drugs in stomach and intestinal media is critical for absorption of drugs from oral dosage forms. We therefore tested the stability of LCATam-PC in simulated gastric fluid (SGF) and Simulated Intestinal Fluid (SIF).33 SGF is a solution of NaCl and pepsin with the pH of ~1.2; and SIF is a solution of monobasic potassium phosphate and pancreatin with the pH of ~6.8.33 We tested the stability of LCA-Tam-PC in SGF conditions for 2 hours and in SIF conditions for 4 hours simulating the normal gastro-intestinal emptying time.33 HPLC chromatogram


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of SGF and SIF incubated LCA-Tam-PC revealed that LCA-Tam-PC is stable in both stomach and intestinal media conditions (Figure 2A). Tam, due to its lipophilic character, disrupts the biological membranes apart from its interactions with intracellular receptors.34,35 Therefore, it is being used for treatment of early stages estrogen receptor (ER) positive and ER negative breast tumors.36 We tested the anticancer activities of Tam and LCA-Tam-PC against murine (4T1), and human ER positive (MCF-7) and ER negative (MDA-MB-231) breast cancer cell lines using trypan blue cell viability assay (Figure 2B-2E). We observed an increase in IC50 value of LCA-Tam-PC in all the three cell lines over Tam irrespective of their ER status (Figure 2B). The higher IC50 of LCA-Tam-PC as compared to Tam might be either due to lower cytotoxicity of the molecule as it is; or slow release of the active Tam from LCA-Tam-PC conjugate. As expected, there was ~1.5-fold increase in IC50 value of Tam and LCA-Tam-PC for triple-negative MDA-MB-231 cells over ER positive MCF-7 cells due to absence of ER receptors in triple-negative breast cancer cells (Figure 2B, 2D). We also tested the cytotoxicity of Tam and LCA-Tam-PC against normal C2C12 murine myoblast cells. Tam and LCA-Tam-PC are ~2-fold less toxic to normal C2C12 cells than MCF-7 cells. LCA-Tam-PC is ~1.5 fold is less toxic than Tam to C2C12 cells suggesting the selectivity of the complex for cancer cells over normal cells (Figure S1). We then analysed the fate of cells in different phases of cell cycle on treatment with Tam and LCA-Tam-PC in 4T1 cells. Concentration dependent increase in number of cells in sub G0 phase of cell cycle was observed confirming the arrest of cells before entering cell cycle (Figure 2F). Apoptosis assay in 4T1 cells using AnnexinFITC/Propidium Iodide (PI) assay revealed the concentration dependent increase in apoptosis on LCA-Tam-PC treatment as we witnessed ~9-fold increase in number of


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total apoptotic (early and late) 4T1 cells (Figure 2G). Similarly, concentration dependent increase in percentage of ROS generating cells was observed. We found ~10-fold increase in ROS positive cells after 3h of treatment (Figure 2H). This enhanced ROS levels induces disruptions in mitochondrial membranes and cause arrest of cells in sub-G0 phase leading to apoptosis. We then investigated the relative intracellular uptake of Tam and LCA-Tam-PC by MCF-7 cells using their fluorophore derivatives Tam-NBD and LCA-Tam-NBD-PC. Firstly, cytotoxicity of Tam-NBD and LCA-Tam-NBD-PC against MCF-7 cells was investigated. It was observed that Tam-NBD (IC50 = 18.08 ± 1.02 µM) and LCA-TamNBD-PC (IC50 = 24.74 ± 1.03 µM) possessed the similar toxicity profiles as that of Tam (IC50 = 14.99 ± 1.03 µM) and LCA-Tam-PC (IC50 = 25.12 ± 1.04 µM) (Figure S2). Confocal micrographs revealed a uniform and augmented distribution LCA-TamNBD-PC in MCF-7 cells as compared to poor accumulation of Tam-NBD in the cells (Figure 3A). Intracellular mean fluorescence measurements from confocal images confirmed a significant increase in uptake of LCA-Tam-NBD-PC compared to TamNBD (Figure 3B). We also quantified the cellular uptake of Tam-NBD and LCA-TamNBD-PC by fluorescence after treatment of MCF-7 cells with Tam-NBD and LCATam-NBD-PC at different concentrations. A concentration dependent increase in intracellular levels of LCA-Tam-NBD-PC was observed in comparison with Tam-NBD (Figure 3C). Phosphocholine group in LCA-Tam-NBD-PC might help in building its interactions with cell membranes whereas bile acid moiety would assist in easy penetration of the amphiphile using hydrophobic interactions with membrane lipids. Therefore, these results witnessed that conjugation of bile acid phospholipid helps in enhanced cellular uptake of the lipid-drug conjugates.


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Figure 2. In vitro activities: A) HPLC chromatogram showing the simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) stability of LCA-Tam-PC conjugate; B) Table showing comparison of IC50 (µM) values (Mean ± Standard error) of Tam and LCA-Tam-PC in breast cancer cell lines; C-E) Concentration dependent in vitro cytotoxicity of Tam and LCA-Tam-PC in breast cancer cell lines MCF-7 (C), MDAMB-231 (D), and 4T1 (E); F) Percentage of 4T1 cells in different phases of cell cycle on treatment with Tam and LCA-Tam-PC at different concentrations showing sub-G0 arrest, G) Percentage of apoptotic cells using Annexin-FITC-V assay on treatment of 4T1 cells with Tam and LCA-Tam-PC at different concentrations; H) Percentage of ROS positive cells after treatment of 4T1 cells with Tam and LCA-Tam-PC at different concentrations.


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Figure 3. Intracellular uptake studies: A) Confocal micrographs of MCF-7 cells treated with Tam-NBD and LCA-Tam-NBD-PC showing uniform and increased cellular uptake of LCA-Tam-NBD-PC over Tam-NBD; B) Mean fluorescence intensity from confocal micrographs showing enhanced cellular uptake of LCA-Tam-NBD-PC in comparison to Tam-NBD; C) Concentration dependent uptake of Tam-NBD and LCA-Tam-NBD-PC in MCF-7 cells as quantified by fluorescence.


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In vivo anticancer activities: We then evaluated the anticancer activity, toxicity, and survival potency of LCA-Tam-PC suspensions in 4T1 murine breast cancer model.37 Sodium carboxymethyl cellulose (CMC) and polysorbate-80, known as GRAS (Generally recommended as safe) agents approved by FDA, are most commonly used suspending agents. They are widely used in paediatric oral formulations,38 eye lubricants, in food products;39 and to test the efficacy of investigational molecules and drugs by oral route in animal models.40 Therefore, Tam and LCA-Tam-PC (as per 10 mg/Kg equivalent of Tam) were suspended in 0.2 mL (for each mice) of suspending vehicle (0.5% CMC and 0.1% polysorbate-80 in distilled water) for oral delivery. Tumor bearing mice were randomized into four groups of ten animals each. At palpable stage of tumors; mice groups were administered orally with vehicle control (suspending agent without drugs); Tam or LCA-Tam-PC suspension at an equivalent Tam dose of 10 mg/kg for three weeks (5 days/week); and one group of mice was left untreated. Tumor volume and body weight of mice were measured on alternate days. Figure 4A-4C presents the kinetic growth of tumor volume for untreated, Tam, and LCA-Tam-PC treated mice revealing the significant decrease in growth kinetics of tumors on LCA-Tam-PC treatment as compared to Tam treated mice. We observed ~60% reduction in tumor volume upon treatment with LCA-Tam-PC as compared to untreated or vehicle control; and LCA-Tam-PC was ~2-fold more effective in comparison to Tam (Figure 4D, 4E). We witnessed a ~2.5-fold decrease in the tumor weight on LCA-Tam-PC treatment as compared to Tam after 21 days of dosage regimen (Figure 4F). Survival studies revealed a significant improvement in mice survival where LCA-Tam-PC treated mice showed a ~8 day increase in median survival over control groups (Figure 4G) without any significant change in body weight (Figure 4H).


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Figure 4. In vivo anticancer activity: A-C) Kinetics of tumor volume of individual mice in untreated control (A), Tam (B), and LCA-Tam-PC (C) treatment groups; D) Change in the tumor volume (Mean ± S.E.M.) of mice groups after above mentioned treatments, E) Representative images of tumor samples isolated from each group on day 21; F-H) Weight (Mean ± S.D.) of the tumor tissues excised after 21 days (F) Median survival (G), and percentage body weight change (Mean ± S.E.M.) (H) of 4T1 tumor bearing mice after oral administration of Tam and LCA-Tam-PC.


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We then performed Hematoxylin and Eosin staining of the tumor sections after different treatment regimens to see the effect of these treatment on tumor pathology. Histology analysis (H&E) of tumor samples in general showed even Hematoxylin staining of nuclei and eosin staining of cytoplasmic proteins. We observed a significant increase in necrotic regions on LCA-Tam-PC treatment with diminished Hematoxylin stain as compared to untreated, vehicle, and Tam treated tumors suggesting the increased cellular death on LCA-Tam-PC treatment (Figure 5A). Ki67 is an excellent marker for the determination of cellular proliferation in histology samples that is abundantly present in the nuclei of highly proliferating tumor cells.41 LCA-Tam-PC treatment induced substantial decrease in the tumor proliferative activity compared to parent drug as witnessed by reduced number of Ki-67 positive tumor cells (Figure 5B). Chronic oral administration of Tam in breast cancer patients is often linked to hepatotoxicity as evident from increase in hepatic specific biomarkers like alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP).42 We therefore estimated and compared the hepatic ALT, AST, and ALP levels in mice serum after three weeks of treatment with Tam and LCA-Tam-PC in 4T1 tumor bearing mice. There was significant increase in circulatory levels of ALT (Figure 5C) and ALP (Figure 5E) above the normal levels in mice after Tam treatment. In contrast, we observed normal levels of ALT (Figure 5C), AST (Figure 5D) and ALP (Figure 5E) in LCA-Tam-PC treated mice. These results suggested that oral administration of LCA-Tam-PC is safe over Tam in chronic treatment schedules.


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Figure

5.

Histochemistry

and

toxicology

studies:

A-B)

Representative

micrographs of tumor sections after H&E (B) and Ki-67 (C) staining showing significant increase in necrosis and low proliferation on LCA-Tam-PC treatment; C-E) Toxicological parameters showing the levels of ALT (C), AST (D) and ALP (E) after Tam and LCA-Tam-PC treatment (Red dotted lines present range of normal levels).


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Pharmacokinetic and tissue-distribution studies: Fluorescent molecular probes allow easy tracing of delivery vehicles and chemotherapeutic drugs especially in pharmacokinetic and tissue-distribution profiling over cumbersome HPLC and mass spectrometry methods.43 It also helps in scheduling treatment regimens, thereby avoiding unwanted toxicities due to increased dose administration.44 We therefore used NBD fluorescent analogues Tam-NBD and LCA-Tam-NBD-PC to estimate the kinetics of Tam in plasma of 4T1 tumor bearing BALB/c mice. Tumor bearing mice were randomized into three groups: (a) untreated control group (n = 3), (b) Tam-NBD (n = 3 per time point), and (c) LCA-Tam-NBD-PC (n = 3 per time point). Control untreated group was used to subtract any background fluorescence. Mice in groups (b) and (c) were orally treated with 15 mg/kg (Tam equivalent) of Tam-NBD and LCATam-NBD-PC; and pharmacokinetic parameters were estimated by extravascular non-compartment method. LCA-Tam-NBD-PC demonstrated enhanced AUC0-24h (840.1 vs. 615.5 ng/mL*h) over Tam-NBD (Figure 6A, 6B). Relative bioavailability of LCA-Tam-NBD-PC was approximately 136.5% in comparison to Tam-NBD confirming the enhanced oral bioavailability of the phospholipid conjugate (Figure 6B).


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Figure 6. Pharmacokinetic and bio-distribution studies: Comparison of (A) plasma pharmacokinetics, and (B) pharmacokinetic parameters of LCA-Tam-NBDPC, Tam-NBD upon oral administration of single dose (15mg/kg, Tam equivalent) of Tam-NBD and LCA-Tam-NBD-PC. C-E) Comparison distribution of Tam-NBD, and LCA-Tam-NBD-PC in different organs after 0.5, 3 and 24h of oral administration of single dose of Tam-NBD and LCA-Tam-NBD-PC. F) Confocal micrographs showing the absorption of Tam-NBD and LCA-Tam-NBD-PC through stomach, small intestine, colon of mice GIT.


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We then estimated the distribution of Tam-NBD and LCA-Tam-NBD-PC in tumor tissues and other organs after 0.5, 3.0, and 24h of dosing. Tumor bearing mice (n = 3/group) were administrated with 15 mg/kg (Tam equivalent) of Tam-NBD and LCATam-NBD-PC; and mice were sacrificed at different time points to collect the tumor tissues and organs (Figure 6C-6E). Fluorescence based quantification revealed a ~2-fold increase in NBD concentration at the tumor site post 3h of treatment of LCATam-NBD-PC as compared to Tam-NBD (Figure 6D). We also observed a ~2.3- and ~1.2-fold increase in NBD concentration in stomach and small intestine as compared to Tam-NBD (Figure 6D). We then observed the gastrointestinal tissues (stomach, small intestine, colon) for the presence of NBD fluorescence under confocal microscope after co-staining with Hoechst 33258. Confocal micrographs witnessed the enhanced fluorescence in the stomach and small intestine tissue sections of LCA-Tam-NBD-PC treated mice as compared to Tam-NBD whereas colon sections show a very minimal presence of NBD fluorescence (Figure 6F). These results suggested that maximum amount of LCA-Tam-NBD-PC is absorbed from the stomach and small intestine of GIT as confirmed by bio-distribution studies as well. Conclusions: In summary, we engineered a lithocholic acid derived phospholipidTamoxifen amphiphile for oral drug delivery and its fluorescent analogue that allowed the easy tracing of the chemotherapeutic drug. Cytotoxic studies revealed that LCATam-PC is highly selective for cancer cells over normal cells as compared to Tamoxifen.

Phospholipid-drug

conjugate

showed

enhanced

intracellular

accumulation as compared to Tamoxifen. In vivo anticancer activities established the significant reduction in in 4T1 tumor burden in mice on LCA-Tam-PC treatment with reduced hepatotoxicity and increase in median mice survival. Pharmacokinetic and


20

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bio-distribution studies using traceable fluorescent analogues confirmed the increased circulatory and tumor-site drug concentrations as compared to parent drug. Therefore, this study opens the newer insights into design of repertoire of bile acid phospholipid derived drug conjugates for future cancer therapeutics. Experimental Procedures: Materials:

Lithocholic

nitrobenzofurazan),

acid

(LCA),

Tamoxifen,

NBD-Cl

(4-Chloro-7-

2-chloro-1,3,2-dioxaphospholane-2-oxide,

N,N'-

Dicyclohexylcarbodiimide, EDC.HCl, Dioxane-4M HCl, and Fmoc-Lys(Boc)-OH, Cell culture media (RPMI-1640 and DMEM), Annexin-FITC-V, Pepsin, Pancreatin, Dichlorofluorescein Diacetate (DCFDA), 0.4% Trypan blue solution, Hoechst 33258 were purchased from Sigma-Aldrich. Antibiotic solution, and fetal bovine serum (FBS) were purchased from Hyclone. Cryomatrix-OCT solution is purchased from Thermo Fisher Scientific. NMR spectra were recorded on Brucker-Avance-400 MHz FT-NMR spectrometer. Chemical shifts are reported in δ ppm reference to respective deuterated solvent. Mass spectra were recorded with AB SCIEX Triple TOF 5600 system. Flow cytometry was performed on FACS Verse (Becton Dickinson, Mountain View, CA). Methods: Synthesis of amphiphiles: Detailed synthetic protocols of LCA-Tam-PC, Tam-NBD and LCA-Tam-NBD-PC amphiphiles are presented in supporting information. Gastric pH stability and solubility of LCA-Tam-PC:33 LCA-Tam-PC (1 mM in 1 mL PBS) was incubated with 9 mL of simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) in a 25 mL round bottom flask with magnetic stirring bar for 2 h for SGF or 4 h for SIF. The composition of simulated gastric fluid (0.2% w/v NaCl, 0.32% w/v pepsin, 0.7% v/v HCl with pH 1.2), simulated intestinal fluid (0.68% w/v KH2PO4,


21


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Page 22 of 36

1.0% w/v pancreatin, 7.7% v/v 0.2 N NaOH with pH 6.8) and time interval was selected based on the average residence time in stomach. After 2 h, an aliquot (1 mL) from incubation mixture was evaporated to dryness; and was extracted using 200 µL of methanol (for LCA-Tam-PC) and 200 µL of acetonitrile (for Tam) each by vortexing for 5 min. followed by centrifugation at 14,000 rpm for 5 min at 4 ˚C. Supernatant liquid was immediately transferred to fresh tube and the solvent was completely evaporated using speed-vac. Final residue was dissolved in 100 µL of methanol and transferred to HPLC vials. Samples were analysed by Waters HPLC system equipped with Octyl-80 TS C8 column (S0005) 4.6 X 250 mm (5µm) with UV detection parameters for Tam set at 235 nm. A HPLC isocratic 15 minute run program was set using 30:70 mixtures of solvent-A (acetonitrile) and Solvent-B (methanol) at room temperature at a flow rate of 1 mL/min. For solubility studies, 1 mg of Tam and LCA-Tam-PC were used to check the dissolved amount in SGF and SIF by HPLC. Cell culture: Murine (4T1) and human (MCF-7, MDA-MB-231) breast cancer cells were maintained as monolayers for experiments in RPMI-1640 (for 4T1 cells) or in DMEM (for MCF-7, MDA-MB-231, and C2C12 cells) media containing 10% (w/v) FBS, penicillin (100 µg/mL), streptomycin (100 U/mL), gentamicin (45 µg/mL) at 37 ˚C in a humidified atmosphere with 5% CO2. Subcultures were made by trypsinization and reseeded for experiments. Trypan blue cell viability assay: Cytotoxicity of Tam and LCA-Tam-PC in human (MCF-7, MDA-MB-231) and murine (4T1) breast cancer cell lines; and normal murine myoblast cells (C2C12) were studied using trypan blue assay. Typically, cells (0.5 X 105) in 1 mL media per well were seeded in a 12-well plate for 24h. Cells were then treated with different concentrations of Tam and LCA-Tam-PC. After 48 h, media


22

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having dead cells and remaining attached cells were pooled using trypsin-EDTA (100 µL). Cell pellets were collected by centrifuging the media at 5000 rpm; washed twice (2 X 1 mL) with cold DPBS; and finally re-suspended in 0.5 mL DPBS. Cells were then stained with trypan blue (0.4%) for 3 min. at room temperature and counted using haemocytometer. Percentage of live cells was plotted against the respective concentration of either Tam or LCA-Tam-PC. Cell cycle and apoptosis assay: Cells (4T1) were plated at density of 2 x 105 cells per well in a 6 well plates for 24h; and then treated with Tam and LCA-Tam-PC at different concentrations for 24h. Cells were then harvested using trypsin-EDTA, washed twice with ice cold PBS, and fixed in chilled 70% ethanol at 4 oC. Cells were washed two times with PBS to remove supernatant; and then incubated with RNAse (40 µg/mL) in PBS at 37 oC for 1 h. Cells were stained with propidium iodide (10 µg/mL) at room temperature for 30 min at 4 oC and counted on flow cytometry. Percentages of cells in SubG0, G0/G1, S, and G2/M phases of the cell cycle were determined using ModFit LT software (Verity Software House, Topsham, ME). For apoptosis assay, treated harvested cells were re-suspended at a density of 1 x 106 cells/mL in 1X binding buffer (as provided with the kit) and stained simultaneously with FITC labelled Annexin-V and Propidium iodide using Annexin V-FITC (Fluorescein Isothiocyanate) labelled apoptosis detection kit as per manufacturer's protocol (Sigma/Aldrich). Cells were then analysed using a flow cytometer, and data was analysed with Cell Quest software. ROS

generation

assay:

Intracellular

ROS

levels

were

measured

using

Dichlorofluorescein Diacetate (DCFDA, Sigma/Aldrich). Generation of ROS oxidizes DCFDA and covert it to Dichlorofluorescein (DCF) that yields green fluorescence. 4T1 cells (5 X 105 cells/ well) were seeded in 6 well plates for 24 h. Cells were then


23


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Page 24 of 36

washed thrice with DPBS containing 0.2% FBS; and incubated with DCFDA (25 µM) in DPBS containing 0.2% FBS for 30 min at 37 oC. Cells were then treated with Tam (12.5 and 25 µM) and LCA-Tam-PC (37.5 and 75 µM) for 3h. Cells were analysed using a flow cytometer, and data were analysed using cell Quest software. Percentages of ROS generated fluorescent cells were plotted against respective conditions.

In vitro uptake by confocal microscopy and fluorescence spectroscopy: MCF-7 cells were seeded on coverslips in 24-well plates (0.5 X 105 cells/well) and cultured for 24 h to get adhered. Intracellular uptake of Tam-NBD and LCA-Tam-NBD-PC was tested at 10 µM Tamoxifen equivalent concentration. Post 6 h of treatment, media was removed and cells were washed with DPBS. Nuclei were stained with Hoechst 33258 (1: 10,000 concentration of a 5 mg/ml stock solution), washed with DPBS for 1 minute; and fixed with 4% paraformaldehyde. Cover slips were mounted on glass slide with prolong gold™, and dried for overnight before being observed under Leica confocal microscope SP5 using 63X oil immersion objective. All acquisition settings were kept identical for control as well as test samples. Acquired images were processed using Leica offline image analysis software (LAS) and mean fluorescence was plotted as arbitrary units against respective samples. For concentration dependent quantification,45 MCF-7 cells were seeded in 96 well plate at density of 10,000 cells per well and grown. After 24h, cells were washed with DPBS, and fresh media with different concentrations (12.5, 25, 50 µM) of Tam-NBD and LCA-Tam-NBD-PC was supplemented to cells. After 2h, the media was removed and cells were washed twice with DPBS. Cells were then lysed with Ethanol:DMSO (1:1) to solubilize the compounds. Fluorescent intensity in each well was measured


24

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using Spectra max plate reader M5 (Molecular Devices). Final fluorescent intensity was plotted against different concentrations after the blank corrections.

In vivo anticancer and toxicological studies: All animal experiments were done with the approval of Institutional Animal Ethical Committee (IAEC), NII, New Delhi. Murine breast cancer (4T1) cells (1.5 x 106) in 200 µL FBS were injected subcutaneously into right flanks of 5-week-old female BALB/c mice. Mice were randomized into four groups (n = 10 mice each group) after they develop palpable tumors on day 3; and subjected to different treatments a) untreated control group, b) Vehicle treated group, c) Tam suspension (10 mg/Kg), d) LCA-Tam-PC suspension (10 mg/Kg, Tam equivalent). Tam (1 mg/mL) and LCA-Tam-PC (2.35 mg/mL) suspensions. All suspensions were prepared in suspending vehicle (0.5% sodium carboxymethyl cellulose with 0.1% polysorbate-80 in distilled water). Briefly 50 µL ethanolic solution of Tamoxifen (20 mg/mL) or LCA-Tam-PC (47.2 mg/mL) or TamNBD (42 mg/mL) or LCA-Tam-NBD-PC (94.6 mg/mL) were added drop wise in 1 mL of suspension media (sodium CMC + tween-80) on vigorous vortexing. All dosages are administered orally through an oral gavage (0.2 mL per mice) for five days in a week for three weeks. Tumor measurements were made every alternate day using a digital caliper; and tumor volume was calculated using formula L×B2/2 where L and B are length and breadth of the tumor. After completion of the treatment schedule on day 21; three mice from each group were sacrificed. Tumors were collected from these mice, weighed, and processed for histopathological analysis. Blood samples collected from the mice were analysed for important hepatotoxic markers such as alanine

transaminase

(ALT),

aspartate

transaminase

(AST)

and

Alkaline

phosphatase (ALP). All the blood samples were processed and evaluated for the important toxicological parameters at a certified pathological laboratory (Newmax


25


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Page 26 of 36

care pathological laboratory at New Delhi). Survival study was continued with rest of mice till the last mouse was found live in all groups. Pharmacokinetics and bio distribution studies: Tumor (4T1) bearing mice (200 mm3) were randomized into three groups a) control group (n= 3 mice), b) Tam-NBD (n = 3 per time point), c) LCA-Tam-NBD-PC (n = 3 per time point). Mice in group b and c were administered with a single oral dose (equivalent to 15 mg/Kg of Tamoxifen) of Tam-NBD and LCA-Tam-NBD-PC respectively. Animals were anesthetized at respective time point using intraperitoneal injection of freshly mixed ketamine (40 mg/Kg). and xylazine (5 mg/Kg). Serial bleeds were collected in ACDmicro centrifuge tube through orbital sinus under mild ketamine-xylazine anaesthesia at 0.08, 0.25, 0.5, 1, 3, 6, 12 and 24 h after oral dosage. Plasma was separated from collected blood samples by centrifuging them at 13000 rpm at room temperature. Plasma samples were stored at -80 ˚C until analysis. For bio-distribution studies; at time points of 0.5, 3, and 24 h; mice were sacrificed; and tumor, stomach, small intestine, large intestine, liver, spleen, kidneys, lungs, and heart were removed after blood withdrawal. Organs were washed with ice cold PBS, weighed, and stored at 80 ˚C until analysis. Fluorophore content present in 100 µL of plasma or 100 mg of respective tissue samples were extracted using 500 µL extracting solvent (90% isopropanol with 10% DMSO). Tissue samples with extracting solvent were placed in 2 mL homogenization tubes and homogenized with zirconia beads by bead beating at 5000 rpm. Plasma samples were transferred to 2 mL tubes and mixed with 500 µL of extracting solvent. Homogenized tissue samples and plasma samples were stored at 4 ˚C overnight to extract the drug. Samples were then centrifuged at 14,000 rpm for 5 min at 4 ˚C. Supernatant liquid was transferred immediately to fresh tube and solvent was


26

Page 27 of 36

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evaporated completely using speed-vac. Final residue was dissolved in 200 µL extracting solvent and transferred immediately to 96-well plates. Analysis was performed spectrofluorimetrically using Molecular devices M5 instrument with λex at 460 nm; and λem of 535 nm. A calibration curve was prepared for Tam-NBD and LCATam-NBD-PC using concentrations from 5-5000 ng/mL in Isopropanol:DMSO (9:1). Final fluorescence values were obtained by subtracting the background fluorescence from each tissue type by using control untreated group. A graph was plotted against concentration

of

respective

compound

versus

different

time

points

for

pharmacokinetics; and concentration of respective compound versus each tissue type at different time points for bio-distribution studies. Plasma drug concentration data was fitted and pharmacokinetic non-compartment analysis were performed by extravascular non-compartment method using WinNonLin (Version 6.4).46 Relative bioavailability (F) of LCA-Tam-NBD-PC to Tam-NBD was calculated using the following equation: Relative bioavailability (F) = (AUC LCA-Tam-NBD-PC)/ (AUC Tam-NBD) X 100. Histopathological analysis: Tissue samples stored in 10% buffered formalin were embedded in paraffin molds; and ~3-4 µm thin sections were cut using Cryomicrotome. Processing of paraffin embedded samples, sectioning and staining with H&E or proliferation marker Ki-67 were done at a certified histological laboratory (Singh histology processing centre at New Delhi). H&E and Ki-67 stained slides were observed using Nikon fluorescence microscope under color camera mode with 60X oil objective. For imaging of stomach, small intestine and colon; mice were sacrificed at 3 h time point after the treatment of Tam-NBD, LCA-Tam-NBD-PC. Stomach, small intestine and colon were removed and washed thoroughly to remove any food particles from


27


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Page 28 of 36

lumen of the intestine and placed in formalin for overnight. These formalin-preserved samples were embedded in OCT (Cryomatrix) molds and ~3 µm thin sections were made using Cryomicrotome. After fixation on the slides; samples were counterstained with Hoechst 33258 (1: 10,000 concentration of a 5 mg/ml stock solution). Villi, epithelial and inner regions of stomach, small intestine and colon samples were observed under Leica confocal microscope SP5 using 63X oil immersion objective and images were processed using LAS software. Acknowledgements: We thank RCB and NII for intramural funding; and Department of Biotechnology (DBT) for supporting this project. VS, NM, SP, AK, and SK thank RCB, UGC, and CSIR for research fellowship. We thank Dr. Aniruddha Sengupta and Mr. Mallik Samarla for helping us in analysing pharmacokinetic data. We acknowledge Servier Medical Art for cartoon representation of GIT and Villi. We thank Dr. Sam J. Mathew, at Regional Centre for Biotechnology for providing us the C2C12 cells. Author contributions: AB conceived the idea, and supervised the project. VS and AB designed the studies. VS, SK, MV, and PB synthesized the molecules. VS, NM, AK carried out the cell culture studies. VS and SP carried out the in vivo studies. SS guided the in vivo experiments. VS and AB analysed the data and wrote the manuscript. Supporting information: Detailed synthesis protocols of amphiphiles; Figure S1 (Cytotoxicity of Tam and LCA-Tam-PC in C2C12); Figure S2 (Cytotoxicity of TamNBD and LCA-Tam-NBD-PC in MCF-7 cells); and 1H NMR,

31

P NMR, Mass spectra

of LCA-Tam-PC and LCA-Tam-NBD-PC are reported in supporting information. Conflict of Interest Disclosure: The authors declare no competing financial interest. References:


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TOC Figure


36

Imaging Agent to Bile Acid

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