Effect of paclitaxel content in the DHP107 oral formulation on oral bioavailability and antitumor activity

Accepted Manuscript Effect of paclitaxel content in the DHP107 oral formulation on oral bioavailability and antitumor activity Yura Jang, Min Kyung Ko, Ye Eun Park, Jung Wan Hong, In-Hyun Lee, Hye Jin Chung, Hesson Chung PII:

S1773-2247(18)30496-9

DOI:

10.1016/j.jddst.2018.09.014

Reference:

JDDST 775

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 11 May 2018 Revised Date:

23 August 2018

Accepted Date: 14 September 2018

Please cite this article as: Y. Jang, M.K. Ko, Y.E. Park, J.W. Hong, I.-H. Lee, H.J. Chung, H. Chung, Effect of paclitaxel content in the DHP107 oral formulation on oral bioavailability and antitumor activity, Journal of Drug Delivery Science and Technology (2018), doi: https://doi.org/10.1016/ j.jddst.2018.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Research Article

RI PT

Effect of paclitaxel content in the DHP107 oral formulation on oral bioavailability and antitumor activity

SC

Yura Janga,1, Min Kyung Koa, Ye Eun Parka, Jung Wan Honga,b, In-Hyun Leeb, Hye Jin

a

M AN U

Chungc,*, and Hesson Chunga,**

Center for Neuro-Medicine, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil,

b

TE D

Seongbuk-gu, Seoul 02792, Republic of Korea

Department of Product Planning and Development, Daehwa Pharmaceutical Co. Ltd., 2038,

c

EP

Nambusunhwan-ro, Gwanak-gu, Seoul 08805, Republic of Korea College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang

*

AC C

National University, 501, Jinju-daero, Jinju, Gyeongnam 52828, Republic of Korea

Corresponding author: H. J. Chung, College of Pharmacy and Research Institute of

Pharmaceutical Sciences, Gyeongsang National University, 501, Jinju-daero, Jinju, Gyeongnam 52828, Republic of Korea. E-mail address: [email protected] (H. J. Chung) 1

ACCEPTED MANUSCRIPT **

Corresponding author: H. Chung, Center for Neuro-Medicine, Korea Institute of Science

and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea.

1

RI PT

E-mail addresses: [email protected] (H. Chung) Present address: Department of Biochemistry, University of Nebraska–Lincoln, Lincoln, NE

AC C

EP

TE D

M AN U

SC

68588, USA.

2

ACCEPTED MANUSCRIPT

ABSTRACT DHP107 is a novel lipid-based oral paclitaxel formulation for the treatment of various

RI PT

cancers. In the present study, to increase the treatment effect of DHP107, we aimed to improve this formulation. We optimized the paclitaxel content in DHP107 formulation, evaluated its antitumor activity, and compared its activity with that of the intravenous

SC

paclitaxel formulation, Taxol®, in various mouse cancer models. The oral bioavailability of

M AN U

paclitaxel increased dose-dependently up to 50 mg/kg. However, the paclitaxel absorption from the DHP107 formulations containing ≥ 1.5% (w/v) paclitaxel decreased with the increase in DHP107 dose as paclitaxel was crystallized when exposed to water. Therefore, the

TE D

optimized DHP107 formulation containing 1% (w/v) paclitaxel was selected, and it exhibited similar antitumor activity as Taxol®. However, the DHP107 showed a better survival rate

EP

with low toxicity than that of Taxol®. The concentration of paclitaxel in the tumor tissues of mice in the DHP107 was also higher than that in Taxol®. The optimized DHP107 formulation

AC C

might reduce the possible toxic effects of the formulation, with antitumor activities similar to those of intravenous paclitaxel Taxol®, by enhancing the distribution to tumor tissues. Furthermore, it might be beneficial to use DHP107 for patients with hypersensitivity reactions to intravenous Taxol®.

Keywords: Paclitaxel, oral formulation, DHP107, formulation optimization, antitumor 3

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

activity, tumor distribution

4

ACCEPTED MANUSCRIPT

1. Introduction

RI PT

Paclitaxel is one of the most widely used anticancer drugs. It is effective against various types of cancers, including ovarian cancer, breast cancer, head and neck cancer, melanoma, non-small-cell lung carcinoma, and Kaposi’s sarcoma [1-4]. However, paclitaxel is difficult

SC

to administer orally owing to its poor oral bioavailability (BA) [5]. The intravenous (iv)

M AN U

formulation of paclitaxel contains organic solvents as pharmaceutical excipients to overcome the low aqueous solubility of paclitaxel; however, the iv formulation exerts side effects, such as hypersensitivity reactions, caused by the organic solvents [4].

TE D

There have been several attempts to develop oral formulations of paclitaxel to reduce toxicity and to improve its oral BA, given the various advantages of oral administration [1, 3-

EP

7]. A novel oral paclitaxel formulation, DHP107, which is composed of an edible lipid and a US-FDA approved emulsifier, was developed by our research group, and this lipid-based

AC C

formulation has been proven to be safe [8-11]. DHP107 has also exhibited enhanced oral BA and remarkable antitumor activity with low toxicity in animal studies [10, 12] and clinical trials [13-16]. Although DHP107 has already undergone clinical trials in patients, further nonclinical studies are necessary to better understand the properties of the formulation and provide important data for further clinical studies and drug development. We aimed to identify the optimized maximum paclitaxel content in the DHP107 formulation to increase 5

ACCEPTED MANUSCRIPT

paclitaxel absorption and reduce dosing volume. We hypothesized that the DHP107 formulation with increased paclitaxel content can

RI PT

improve patient compatibility as it can reduce dosing volume. For example, if the treatment dose is 200 mg/m2 bid, which is one of the dosing regimens followed in clinical trials [13-16], a 70-kg adult male patient with a body surface area of 1.74 m2 [17] would require 69.6 mL of

SC

this lipid-based formulation per day. It might be difficult for some patients to swallow a large

M AN U

volume of the DHP107 formulation, which is mucoadhesive in the presence of water and can adhere to the oral cavity after administration [10, 11, 18]. Moreover, some patients might also need to take doses higher than 200 mg/m2 bid. Therefore, higher strengths of the DHP107

TE D

formulation have to be developed. If paclitaxel content in the DHP107 formulation is optimized to the maximal loading capacity with the best paclitaxel absorption in vivo, the

formulation.

EP

dosing volume of DHP107 can be reduced without modifying the composition of vehicle

AC C

In this study, we optimized the drug content in the formulation. The maximum loading capacity of paclitaxel in the DHP107 formulation with good stability was determined. We also investigated whether higher paclitaxel contents in the DHP107 formulation affect the oral absorption of paclitaxel in mice. Antitumor activity and drug distribution to tumor tissues were also evaluated using the optimized DHP107 formulation in various murine tumor models. 6

ACCEPTED MANUSCRIPT

2. Materials and methods

RI PT

2.1. Materials

Paclitaxel was obtained from Samyang Genex (Seoul, Korea). Taxol® (6 mg/mL) and Abraxane® (5 mg/mL) were purchased from Boryung Pharmaceutical Co. (Seoul, Korea) and

SC

Celgene Co. (Summit, NJ, USA), respectively. Distilled monoolein (RYLO™ MG 19, > 90%

M AN U

purity) was purchased from Danisco Ingredients (Copenhagen, Denmark). Polysorbate 80, tricaprylin, ammonium formate, carbamazepine, methylene blue, thioflavin T, methyl orange, crystal violet, pepsin, and sodium taurodeoxycholate hydrate were obtained from Sigma-

TE D

Aldrich (St. Louis, MO, USA). Cyclosporin A was purchased from Chong Kun Dang Pharmaceutical Co. (Seoul, Korea). Acetonitrile, methanol, and methyl t-butyl ether (HPLC

AC C

2.2. DHP107

EP

grade) were purchased from JT Baker (Phillipsburg, NJ, USA).

DHP107 is a lipid-based oral solution, which forms a mucoadhesive sponge phase in the presence of water and therefore can adhere to mucosal cells [10, 18]. A paclitaxel-free vehicle formulation (F109) of DHP107 was prepared by mixing monoolein, tricaprylin, and polysorbate 80 in the ratio of 1:0.5:0.3 (v/v/v) at approximately 40 °C. The DHP107 formulation is a semi-solid wax at room temperature and a liquid above 30 °C [18]. The 7

ACCEPTED MANUSCRIPT

DHP107 formulation containing 3% (w/v) paclitaxel was prepared by dissolving paclitaxel in F109 with sonication for 1 min [10-12]. The DHP107 formulations containing 0.5%, 1%,

RI PT

1.5%, 2%, and 2.5% (w/v) paclitaxel were prepared by diluting DHP107 containing 3% (w/v) paclitaxel with F109, and stored at 4 °C. As the formulations were in semisolid state at room temperature, they were melted by warming up to body temperature to form an oily liquid

M AN U

SC

before use [10, 11].

2.3. Cell cultures

TE D

EMT-6 (murine mammary carcinoma cell line) was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Waymouth’s MB 752/1 medium

EP

(Gibco, Grand Island, NY, USA) with 15% fetal bovine serum (FBS; Gibco) and 1% antibiotic-antimycotic solution (ZellShield™; Minerva Biolabs, Berlin, Germany). MDA-

AC C

MB-231 (human breast mammary gland cell line) was purchased from the Korean Cell Line Bank (Seoul National University, Seoul, Korea) and maintained in Roswell Park Memorial Institute 1640 medium (Welgene, Gyeongsan, Korea) supplemented with 10% FBS and 1% ZellShield™. MX-1 (human breast adenocarcinoma cell line) was purchased from the Cell Line Service (Eppelheim, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) and nutrient mixture F-12 Ham medium (Gibco) in the ratio 1:1 with 15% FBS and 8

ACCEPTED MANUSCRIPT

1% ZellShield™. B16F10 (murine melanoma cell line) was purchased from the Korean Cell Line Bank and maintained in DMEM (PAA Laboratories, Pasching, Austria) supplemented

RI PT

with 10% FBS and 1% ZellShield™. All the cell lines were incubated at 37 °C and 95% humidity with 5% CO2.

M AN U

SC

2.4. Animal handling

Seven-weeks-old female BALB/c and BALB/c nude mice (18–21 g) were purchased from Nara Biotech (Seoul, Korea). Seven-weeks-old female C57BL/6 mice (18–21 g) were

TE D

purchased from Orient Co. (Seongnam, Korea). They were acclimated for one week before using them in the experiments. The mice were housed in groups of five mice per cage (260

EP

mm × 200 mm × 130 mm; Jeung Do Bio & Plant Co., Seoul, Korea) or nine to ten mice per cage (420 mm × 260 mm × 180 mm) with autoclaved corncob bedding (Harlan Teklad,

AC C

Madison, WI, USA) under a 12-h light/dark cycle from 08:00 h to 20:00 h. They were fed irradiated pellet food (Harlan Teklad) and sterilized drinking water ad libitum. The mice were euthanized using CO2 after the completion of experiments. All the procedures in the study involving experimental animals were performed in accordance with the ethical standards of the European Community Guidelines and the Animal Care and Use Committee of Korea Institute of Science and Technology (approval number: 2015-002). 9

ACCEPTED MANUSCRIPT

RI PT

2.5. Measurement of droplet-size distribution

The droplet-size distribution of DHP107 in deionized water was measured using a particle size analyzer (Photal Otsuka Electronics, Osaka, Japan) [5, 19]. Three samples,

SC

prepared by shaking, vortexing, and sonicating for 1 min, respectively, were subjected to the

the stability of the formulation.

TE D

2.6. Polarized light microscopy

M AN U

size measurement. The droplet size was measured again 24 h after preparation to determine

EP

The crystallization of paclitaxel in the DHP107 formulations containing 0% (F109), 0.5%, 1%, 1.5%, and 2% (w/v) paclitaxel was observed by polarized light microscopy (Olympus

AC C

BX51 microscope; Olympus Co, Tokyo, Japan) at ×200 magnification after diluting 1.5, 5, and 20 times (v/v) in simulated gastric juice and bile. All the samples were mixed by vortexing for 1 min, and then immediately observed. The simulated gastric juice was prepared by dissolving pepsin at 0.3 mg/mL in saline and the pH was adjusted to 2.0 with HCl. [20]. The simulated bile was prepared by dissolving sodium taurodeoxycholate in water to a concentration of 100 mM. 10

ACCEPTED MANUSCRIPT

2.7. Effect of dose, paclitaxel content, and dosing volume of DHP107 on paclitaxel

RI PT

absorption in mice

Different contents of paclitaxel in the DHP107 formulations ranging from 0.5% to 3%

SC

(w/v) were prepared and tested to optimize paclitaxel content in the formulation for enhanced

M AN U

paclitaxel absorption in mice. To evaluate whether paclitaxel absorption is affected by paclitaxel content in the formulation, 0.5% and 1% (w/v) paclitaxel formulations were administered to mice at various doses and volumes (n = 5 per time point, Table 1). In another

TE D

experiment to compare the effect of various paclitaxel contents in the DHP107 formulation on paclitaxel absorption, DHP107 containing 0.5%–3% (w/v) paclitaxel was administered to

EP

mice at an equal volume of 5 mL/kg (n = 5 per time point). To determine the concentration of paclitaxel in the plasma of mice, 1 mL of blood sample from each mouse was collected at 0.5,

AC C

1, 2, 4, 8, 12, and 24 h after the oral administration of DHP107 by cardiac puncture with the mice under ether anesthesia. The plasma sample was obtained by the centrifugation of blood. These samples were stored at –70 °C until further analysis.

2.8. Tumor growth inhibitory effects of DHP107 in the mice transplanted with various cancer cell lines 11

ACCEPTED MANUSCRIPT

To evaluate the in vivo antitumor activity of DHP107, it was orally administered to

RI PT

various tumor-bearing mice. EMT-6 cells (5 × 104 cells in 0.05 mL of injection volume per mouse) were transplanted subcutaneously on the dorsal side of BALB/c mice on day 0 [10]. MDA-MB-231 and MX-1 cell lines (5 × 106 cells in 0.05 mL of injection volume per mouse)

SC

were transplanted subcutaneously into the dorsal flank of BALB/c nude mice on day 0 [21,

M AN U

22]. The mice were randomly divided into the following six groups: saline iv, F109 peroral (po), Taxol® iv, Abraxane® iv, DHP107-25 mg/kg po, and DHP107-50 mg/kg po (n = 10 per group). Taxol® was diluted six times with saline and infused via the tail vein of mice at a

TE D

dose of 12.5 mg/kg (at a volume of 12.5 mL/kg or 250 µL for mice weighing 20 g) [10]. Abraxane® was infused intravenously into mice at a dose of 12.5 mg/kg (at a volume of 2.5

EP

mL/kg or 50 µL for mice weighing 20 g). DHP107 was orally administered to mice at doses of 25 and 50 mg/kg. Saline was infused intravenously into control mice at a volume of 12.5

AC C

mL/kg, which is equal to the volume for intravenous Taxol® administered at a dose of 12.5 mg/kg. F109 was administered to mice at a volume of 5 mL/kg, which is equal to the volume of oral DHP107 administered at a dose of 50 mg/kg. The drugs were administered to mice once a day for three 5-day cycles separated by 2-day intervals. The tumor size (tumor volume = length × width × height × 0.5236) [23], body weight, body temperature, and survival rate of mice were measured after the inoculation of tumor cells. The body temperature was measured 12

ACCEPTED MANUSCRIPT using an infrared thermometer (Gilwoo Trading Co., Giltron®, Seoul, Korea), which was in contact with the body surface [24].

RI PT

B16F10 cells (8 × 105 cells in 0.05 mL of injection volume per mouse) were injected subcutaneously on the dorsal side of C57BL6 mice on day 0 [4, 25]. The mice were randomly divided into the following three groups: Taxol® iv, F109 po, and DHP107 po (n = 10 per

SC

group). Taxol® was diluted six times with saline and infused intravenously into the mice at a

M AN U

dose of 10 mg/kg. F109 (5 mL/kg) and DHP107 (50 mg/kg) were orally administered to the mice. The treatment commenced on the day following inoculation, and the drugs were administered to mice once every two days for 14 days. Tumor volume was measured and the

TE D

survival rate of mice was monitored. In an additional experiment, B16F10 cells (8 × 104 cells in 0.05 mL of injection volume per mouse for lung tumor weight measurement and 9 × 104

EP

cells in 0.05 mL of injection volume per mouse for survival rate calculation) were injected intravenously into the tail vein of C57BL/6 mice to establish a mouse pulmonary metastatic

AC C

melanoma model (n = 10 per group) [2, 25]. The mice were randomly divided into the following two groups: F109 po and DHP107 po. F109 (5 mL/kg) and DHP107 (50 mg/kg) were orally administered to the mice. The treatment was started on the day following the injection of tumor cells, and the drugs were administered once every two days for 14 days. The mice were sacrificed 30 days after inoculation, and the lungs were isolated. After weighing the lungs, the tumor mass, which appeared as black spots in the lungs, was 13

ACCEPTED MANUSCRIPT

collected and weighed. In a separate experiment, the survival rate was monitored in the F109 and DHP107 groups under the same conditions after the inoculation of melanoma cells. The

RI PT

administration schedule of all antitumor activity studies was chosen based on previous studies [10, 11, 26].

SC

2.9. Paclitaxel distribution to the tumor tissue after the oral administration of DHP107 in

M AN U

melanoma mice

B16F10 cells (6 × 105 cells in 0.02 mL of injection volume per mouse) were implanted

TE D

subcutaneously into the dorsal flank of C57BL6 mice as mentioned in section 2.8. and grown for 7 days. C57BL6 mice with tumor volumes of 200–300 mm3 were selected. The mice were

EP

randomly divided into the following two groups: Taxol® iv and DHP107 po, on day 8 after the inoculation of melanoma cells (n = 5 per group). Taxol® was diluted six times with saline

AC C

and infused intravenously into the mice with melanoma at a dose of 10 mg/kg. DHP107 was orally administered to mice at a dose of 50 mg/kg. To determine paclitaxel concentration in the plasma and tumor tissue after the administration of drugs, blood and tumor tissue were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 12 h after drug administration from the mice in the Taxol® iv group and at 0.5, 1, 2, 4, 8, and 12 h after drug administration from the mice in the DHP107 po group by cardiac 14

ACCEPTED MANUSCRIPT

puncture, with the mice under ether anesthesia. The plasma sample was obtained by the centrifugation of blood. The tumor samples were homogenized in deionized water (20%, w/v)

samples were stored at –70 °C until further analysis.

M AN U

SC

2.10. LC-MS/MS analysis

RI PT

using a homogenizer (Ultra-Turrax T25 basic; IKA Labortechnic, Staufen, Germany). These

The concentration of paclitaxel in the plasma and tumor tissues was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) [10, 12, 27]. Carbamazepine was

TE D

used as an internal standard (0.1 µg/mL in acetonitrile); 50 µL of the internal standard was added to 50 µL of plasma or 100 µL of tumor sample, and vortexed. The samples were then

EP

extracted with 1 mL of methyl t-butyl ether and centrifuged at 10,000 × g for 10 min at 25 °C. The upper organic layer was transferred to a clean tube and evaporated under a stream of

AC C

nitrogen. The residue was reconstituted in the mobile phase. The reconstituted solution was vortexed and centrifuged, and then the supernatant was transferred to a high-performance liquid chromatography (HPLC) vial for analysis. The supernatant (5 µL) was injected into the LC-MS/MS system. The LC-MS/MS system consisted of an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) and an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies) equipped with an electrospray ionization source. The 15

ACCEPTED MANUSCRIPT Waters XTerra® MS C18 (3.5 µm, 2.1 mm × 50 mm) was used as an analytical column. The mobile phase consisted of aqueous 5 mmol/L ammonium formate (pH 6.0) and 90% (v/v)

RI PT

acetonitrile containing 5 mmol/L ammonium formate (pH 6.0). A gradient elution method was used with a total run time of 6 min. The chromatographic retention times were 2.94 and 2.66 min for paclitaxel and the internal standard, respectively. To detect the analytes, the

SC

transition of precursor to product ions was monitored at m/z 853.8→285.9 for paclitaxel and

M AN U

m/z 237.4→194.4 for the internal standard in the multiple reaction monitoring mode. The data were acquired using the Agilent Mass Hunter Workstation software (Agilent Technologies). The pharmacokinetic parameters were determined from the mean

EP

2.11. Statistical analyses

TE D

concentration data using the WinNonlin program (Pharsight Co., Mountain View, CA, USA).

AC C

All data are presented as mean ± S.D. A two-tailed Student’s unpaired t-test was performed using the GraphPad Prism 5.0 software. A P value < 0.05 was considered statistically significant.

3. Results 3.1. Droplet-size distribution 16

ACCEPTED MANUSCRIPT

The droplet-size distribution of DHP107 dispersed in water was measured using a

RI PT

particle size analyzer (Fig. 1). We aimed to mimic the digestive process at various strengths of peristalsis. As we attempted to emulate mixing forces with different degrees of mixing, we compared size distribution after manual shaking, vortexing, and sonicating the samples.

SC

Depending on the stomach conditions, the formulation can undergo mild to severe mixing

M AN U

with the gastric or intestinal juice. We aimed to observe the emulsification process, the possible formation of liquid crystalline structures, and the precipitation of paclitaxel after the mixing process. The size distribution of the formulation after shaking and vortexing showed

TE D

bi- and tri-modal patterns, containing micron-sized particles. After sonication, the size distribution of the droplets exhibited a unimodal pattern, and the mean droplet size was 187

EP

nm, which did not change over a period of 24 h. The droplet size of the manually shaken or

AC C

vortexed samples could not be measured due to phase separation at 24 h.

3.2. Crystallization of paclitaxel from DHP107 in simulated gastric juice and bile

The solubility of paclitaxel in F109 was approximately 3% (w/v) in our previous study [11]. To study the physical stability of the oral paclitaxel formulation depending on paclitaxel content, the formulations containing 0% (F109) to 2% (w/v) of paclitaxel were prepared in 17

ACCEPTED MANUSCRIPT

F109. These DHP107 formulations were dispersed in simulated gastric juice (Fig. 2) and bile (Fig. 3) by diluting 1.5, 5, and 20 times (v/v). Paclitaxel crystals precipitated from DHP107

RI PT

containing 1.5% (w/v) paclitaxel in both simulated gastric juice and bile. With respect to the samples containing DHP107 diluted 20 times (v/v) in simulated bile, the paclitaxel crystals were not precipitated from DHP107 containing 0.5% and 1% (w/v) paclitaxel until 20 h at

SC

room temperature (Fig. S1). Although we could not quantify paclitaxel that precipitated, it

M AN U

was higher in the formulations containing 2.5% and 3% (w/v) paclitaxel than in the formulation with 2% (w/v) paclitaxel; however, they were all microscopically similar.

TE D

3.3. Comparison of paclitaxel concentration in the plasma after the oral administration of

EP

DHP107 containing 0.5% and 1% (w/v) paclitaxel

To evaluate whether paclitaxel absorption is influenced by paclitaxel content in the

AC C

DHP107 formulation and to optimize the drug content in the DHP107 formulation for enhanced paclitaxel absorption, paclitaxel concentration-time profile was obtained after the oral administration of DHP107 containing 0.5% and 1% (w/v) paclitaxel at doses of 25–125 mg/kg for the formulation with 0.5% (w/w) paclitaxel and 25–150 mg/kg for the formulation with 1% (w/v) paclitaxel at different volumes to BALB/c mice (Fig. 4 and Table 1, n = 5 per time point). The administered volumes of 0.5% and 1% (w/v) paclitaxel formulations were 5– 18

ACCEPTED MANUSCRIPT

25 and 2.5–15 mL/kg, respectively. The relative BA was calculated by comparing the dosenormalized area under the plasma concentration-time curve (AUC) of each formulation with

RI PT

the AUC of paclitaxel iv infusion Taxol® at a dose of 10 mg/kg, and the AUC value was 10.4 µg·h/mL.

When DHP107 containing 0.5% (w/v) paclitaxel was administered to mice at various

SC

doses, the BA increased dose-dependently up to 75 mg/kg; however, it decreased from 100

M AN U

mg/kg. The dose-normalized Cmax and AUC, and BA at the dose of 75 mg/kg were the highest compared with those of other doses. The AUC at 100 mg/kg was similar to that at 75 mg/kg; therefore, the BA at 100 mg/kg was lower than that at 75 mg/kg. This might be due to

TE D

saturable absorption. In the case of 1% (w/v) paclitaxel formulation, the Cmax and AUC increased when paclitaxel doses were increased up to the highest dose tested. However, the

EP

AUC did not increase dose-dependently, and the BA increased up to 100 mg/kg. The reason for the increase in AUC not being dose dependent might be due to saturable cytochrome P450

AC C

(CYP) metabolism and/or P-glycoprotein (P-gp) efflux at high doses of paclitaxel [28-30]. However, the AUC increased dose dependently from 75 mg/kg. The BA from 75 to 150 mg/kg was similar (± 2.1%). The absorption of paclitaxel after the oral administration of 1% (w/v) paclitaxel formulation was higher than that after the administration of 0.5% (w/v) paclitaxel formulation at all doses tested (1.2–9.6 times, Table 1). DHP107 containing 1%

19

ACCEPTED MANUSCRIPT

(w/v) paclitaxel was found to be better than that containing 0.5% (w/v) paclitaxel according

RI PT

in this regard.

3.4. Comparison of paclitaxel concentration in the plasma after the oral administration of

SC

DHP107 formulations with different paclitaxel contents at the same volume

M AN U

In the previous experiment (section 3.3.), we found that the formulations with high paclitaxel content enabled better paclitaxel absorption. Therefore, to verify whether paclitaxel absorption can be enhanced further, we prepared formulations with higher paclitaxel contents

TE D

up to 3% (w/v) and administered them at the same volume of 5 mL/kg corresponding to the dose of paclitaxel up to 150 mg/kg to mice. The plasma paclitaxel concentration was

EP

determined after the oral administration of these DHP107 formulations at various doses to BALB/c mice (Fig. 5 and Table 1, n = 5 per time point). The AUC and BA of the 1% (w/v, 50

AC C

mg/kg) paclitaxel formulation significantly increased when compared with those of the 0.5% (w/v, 25 mg/kg) paclitaxel formulation. However, the BA decreased gradually from 75 mg/kg for the 1.5% (w/v) paclitaxel formulation. The time to reach the maximum concentration (Tmax) at 25 and 50 mg/kg was 2 h; however, this changed to 0.5 h at 75 mg/kg. The AUC at 125 and 150 mg/kg significantly decreased compared with that at 100 mg/kg, despite an increase in the dose; accordingly, the BA also decreased. 20

ACCEPTED MANUSCRIPT

3.5. Tumor growth inhibitory effect of oral DHP107 on mice transplanted with breast cancer

RI PT

cell lines

In the previous experiment (section 3.4.), there was a limit up to which paclitaxel content

SC

in the DHP107 formulation could be increased due to its crystallization at 1.5% (w/v).

M AN U

Therefore, the maximum paclitaxel absorption was observed after the administration of 1% (w/v) paclitaxel formulation among the 0.5%–3% (w/v) paclitaxel formulations. Hence, the administration of 1% (w/v) paclitaxel formulation at less than 7.5 mL/kg was considered

TE D

optimal. To evaluate the effect of the optimized DHP107 formulation on the treatment of breast cancer, the antitumor activity and toxicity of oral DHP107 were examined by

EP

measuring tumor volume, body weight and temperature, and survival rate of mice subcutaneously inoculated with EMT-6, MDA-MB-231, and MX-1 cell lines (Fig. 6, n = 10

AC C

per group). The saline iv and F109 po groups were used as negative controls. The Taxol® and Abraxane® iv groups were used as positive controls. The rate of increase in tumor volume in the saline and F109 groups was faster than that in the Taxol®, Abraxane®, and DHP107 groups for all tumor cell lines. The tumor volume rapidly increased from approximately 20 d after inoculation into the mice of the negative control groups. In the case of EMT-6 tumor-bearing mice, the inhibition of tumor growth in 21

ACCEPTED MANUSCRIPT the 25 and 50 mg/kg DHP107 groups was similar to that in the Taxol® and Abraxane® groups, respectively. In the case of MDA-MB-231 tumor-bearing mice, the inhibition of tumor

RI PT

growth in the 50 and 25 mg/kg DHP107 groups was similar to that in the Taxol® and Abraxane® groups, respectively. In the case of MX-1 tumor-bearing mice, the inhibition of tumor growth in the 50 mg/kg DHP107 group was similar to that in the Taxol® and

SC

Abraxane® groups. The DHP107 treatment groups exhibited tumor growth inhibition similar

M AN U

to that of the Taxol® and Abraxane® treated groups in all types of cell lines tested. The body weight of mice in the DHP107 groups was similar to that of the mice in the saline and F109 groups as negative control groups in all types of tumor cell lines tested

TE D

during the experimental period. However, the body weight of almost all mice in the Taxol® group decreased during the treatment period in all types of cell lines, and it took 15‒20 d for

EP

them to recover to their normal body weight, which was similar to the body weight of mice in the negative control groups, after the treatment. The body weight of EMT-6 and MX-1 tumor-

AC C

bearing mice in the Abraxane® group also decreased during the treatment period. The body temperature of all tumor-bearing mice in the DHP107 groups did not decrease, and they were similar to that of the mice in the negative control groups during the experimental period. However, the body temperature of all tumor-bearing mice in the Taxol® group dropped by 3%–10% compared with that of the mice in the saline group, and it recovered to the normal value more slowly than the body weight. 22

ACCEPTED MANUSCRIPT Only 60% of the EMT-6 tumor-bearing mice treated with Taxol® survived during the treatment period; however, more than 90% mice survived in the other groups. The survival

RI PT

rate of mice in all the treatment groups bearing MDA-MB-231 was > 90% during the experiment. The survival rate of mice in all the treatment groups bearing MX-1, except the Taxol® group, was also > 90% during the experiment; however, half of the mice in the Taxol®

M AN U

SC

group died within 26 d after inoculation.

3.6. Tumor growth inhibitory effect of oral DHP107 on mice transplanted with melanoma

TE D

and pulmonary metastatic melanoma cells

The antitumor activity of oral DHP107 containing 1% (w/v) paclitaxel administered at a

EP

dose of 50 mg/kg (5 mL/kg) was also evaluated by measuring the volume of tumor and monitoring the survival of mice bearing B16F10 melanoma cells (Fig. 7A, n = 10 per group).

AC C

The Taxol® and F109 treatments were used as positive and negative controls, respectively. The inhibition rate of tumor growth in the DHP107 group was similar to that in the Taxol® group during the experimental period. However, the survival rate of the DHP107 group was higher than that of both the positive and negative control groups. Four of 10 mice in the DHP107 group survived till day 60; however, all mice in the Taxol® and F109 groups died by

23

ACCEPTED MANUSCRIPT

days 60 and 41, respectively. In addition, the number of survived mice in the DHP107 group was maintained from day 41 until the end of the experiment.

RI PT

The pulmonary metastatic B16F10 melanoma-bearing mouse model was employed to determine the activity of DHP107 against metastatic cancer (Fig. 7B, n = 10 per group). The portion of lung containing metastasized tumor after the iv injection of B16F10 cells

SC

comprised 68.8% and 28.1% of the total lung volume in the F109 and DHP107 groups,

M AN U

respectively. Thus, the DHP107 group showed a significant reduction in tumor burden of the lung compared with that of the F109 group (P < 0.01). A representative mouse lung from each group is shown in Fig. 7B. Moreover, all mice in the F109 group died by day 35;

TE D

however, six mice in the DHP107 group were still alive at 45 d after melanoma cell injection. The survival rate of the DHP107 and F109 groups at 35 d after the tumor cell injection was

EP

67% and 0%, respectively.

AC C

3.7. Paclitaxel concentration in the plasma and tumor tissues after oral DHP107 administration in the mice with melanoma

The antitumor activity of DHP107 was tested in breast and melanoma mouse cancer models. We investigated paclitaxel distribution to the tumor tissue. The concentration of paclitaxel in tumor tissues was measured after the oral administration of DHP107 containing 24

ACCEPTED MANUSCRIPT 1% (w/v) paclitaxel at a dose of 50 mg/kg in the mouse melanoma model. Taxol® as a positive control was also infused intravenously at a dose of 10 mg/kg (Fig. 8, n = 5 per time

RI PT

point). The pharmacokinetic parameters of paclitaxel in the plasma and tumor tissues are summarized in Table 2. Paclitaxel was eliminated more slowly from tumor tissues than from the plasma in both the groups. After the oral administration of DHP107, paclitaxel was well

SC

absorbed and could be detected in the plasma from the first blood-sampling time-point (30

M AN U

min). Paclitaxel was rapidly and effectively distributed to tumor tissues in both the groups. The AUC of tumor tissues was higher than that of the plasma in both the groups. The tumor tissue-to-plasma (T/P) ratio in the Taxol® and DHP107 groups was 4.1 and 18.6, respectively.

EP

4. Discussion

TE D

The T/P ratio in the DHP107 group was 4.5 times higher than that in the Taxol® group.

AC C

We developed DHP107, a new oral formulation of paclitaxel currently being evaluated in clinical studies, to improve the oral BA of paclitaxel without the need for excipients, which could be harmful to hypersensitive patients [10-12]. In this study, to optimize paclitaxel content in the DHP107 formulation, we examined the pharmacokinetic, antitumor activity, and paclitaxel distribution to tumor of DHP107 administered as various formulations and doses. 25

ACCEPTED MANUSCRIPT

We performed an analysis of in vitro release of DHP107 and model compounds from the vehicle formulation (F109; Fig. S1), which facilitates the understanding of the characteristics

RI PT

of the formulation [3, 5]. As the DHP107 formulation is mucoadhesive and can affect paclitaxel absorption from the subsequent orally administered DHP107 for over 48 h, as shown in our previous repeated dosing studies [18], the in vitro release analysis was

SC

performed for 48 h. We selected model compounds with various logP values to compare the

M AN U

release pattern of the drugs with hydrophobicities different from that of F109 used in this study. The DHP107 formulation can entrap hydrophobic drugs and regulate their release. We confirmed that DHP107 can form micronized particles after peristalsis, as the mechanical

TE D

power of the intestinal tract is far less than that exerted by sonication. We observed paclitaxel crystallization from DHP107 after mixing with simulated digestive fluids by polarized

EP

microscopy. As paclitaxel is highly hydrophobic and has poor aqueous solubility, it is easily crystallized in water [1, 4, 22, 31]. Further, because the DHP107 formulation is composed of

AC C

lipids, which have a negative charge due to fatty acids, the physical stability of the formulation may be affected by pH [32]. To develop a highly stable formulation, we had to evaluate the stability of DHP107 formulated with various paclitaxel contents in both simulated gastric juice and bile. When the DHP107 formulations containing 0%–2% (w/v) paclitaxel were dispersed in the simulated gastric juice and bile, their dispersion was stable up to 1% (w/v) paclitaxel in vitro. 26

ACCEPTED MANUSCRIPT

To examine the effect of paclitaxel content in the DHP107 formulations on paclitaxel absorption and to identify the formulation that allows the best absorption of paclitaxel in vivo,

RI PT

DHP107 containing 0.5% and 1% (w/v) paclitaxel was administered to mice at various doses. The AUC of the 1% (w/v) paclitaxel formulation was higher than that of the 0.5% (w/v) paclitaxel formulation at all doses tested. For example, at 25 mg/kg dose, the AUC of the 1%

SC

(w/v) paclitaxel formulation was 9.6 times higher than that of the 0.5% (w/v) paclitaxel

M AN U

formulation (Table 1). This indicates that paclitaxel absorption was increased when the content of paclitaxel in the formulation was increased up to 1% (w/v) at the same dose. In our previous studies, we found that the DHP107 formulation forms the mucoadhesive sponge

TE D

phase in aqueous environments, such as digestive juice in gastrointestinal tract, after oral administration [10, 11, 18]. We suggest that paclitaxel in the DHP107 formulation because of

EP

the sponge phase, which adheres to mucosal cells in the gastrointestinal tract, is absorbed for a long time slowly. The DHP107 formulation containing 1% (w/v) paclitaxel might not be

AC C

disrupted in the gastrointestinal fluid and might maintain higher content of paclitaxel in sponge phase near the absorption site than that containing 0.5% (w/v) paclitaxel. Moreover, this might be because paclitaxel absorption after the administration of formulations containing low paclitaxel contents was more affected by P-gp efflux and first-pass CYP metabolism than that after the administration of formulations containing high paclitaxel contents [28-30]. Paclitaxel is already a well-known substrate and an inducer of P-gp and 27

ACCEPTED MANUSCRIPT

CYP; therefore, the inhibition of paclitaxel absorption after DHP107 administration due to the induction of P-gp and CYP has been studied [18]. We also verified the effect of P-gp and

RI PT

CYP on paclitaxel absorption. The results revealed that paclitaxel absorption was significantly enhanced after the co-administration of DHP107 with a P-gp and CYP inhibitor, cyclosporin A, when compared with the administration of DHP107 alone at all doses as

SC

expected (Fig. S2). DHP107 containing 1% (w/v) paclitaxel is suggested to be better than that

improves patient convenience.

M AN U

containing 0.5% (w/v) paclitaxel at the same dose, because the smaller administration volume

Further, the above results showed that an increase in paclitaxel content in DHP107 can

TE D

also increase paclitaxel absorption. Formulations with different paclitaxel contents ranging from 0.5% to 3% (w/v) were prepared to evaluate the effect of paclitaxel content in DHP107

EP

on paclitaxel absorption when equal volume of DHP107 was administered orally to mice. The AUC of the 1% (w/v) paclitaxel formulation (50 mg/kg) was higher than that of the 0.5%

AC C

(w/v) paclitaxel formulation (25 mg/kg); however, the AUC of the 1% (50 mg/kg) and 2% (w/v) paclitaxel formulations (100 mg/kg) was similar, despite the increased dose of DHP107. A comparison of the AUC of formulations containing 0.5%, 1%, and 2% (w/v) paclitaxel at 100 mg/kg revealed that the AUC of 1% (w/v) paclitaxel formulation was the highest. The maximum BA was also observed when paclitaxel content in DHP107 was 1% (w/v). The Tmax for the 1.5% (w/v) paclitaxel formulation was delayed by 1.5 h. In our previous study, we 28

ACCEPTED MANUSCRIPT

observed that the DHP107 formulation is micronized and micellized by mixing with bile in the gastrointestinal tract [11]. Paclitaxel in the bile fluid might remain stable in the

RI PT

gastrointestinal tract and absorbed for extended periods of time. However, the Tmax of the formulations containing 1.5%–3% (w/v) paclitaxel was shorter than that of the formulations containing 0.5% and 1% (w/v) paclitaxel. This indicates that paclitaxel might not be

SC

entrapped stably in formulations containing 1.5%–3% (w/v) because of paclitaxel

M AN U

crystallization. This was also verified by the results of polarized microscopy (Figs. 2 and 3). Paclitaxel crystallization in the DHP107 formulations containing 1.5% and 2% (w/v) paclitaxel was observed, indicating that paclitaxel precipitation can occur in the

TE D

gastrointestinal tract when taken orally. Therefore, paclitaxel absorption after the administration of DHP107 formulations containing paclitaxel content ≥ 1.5% (w/v) was

EP

decreased in vivo. Based on these results, we considered the DHP107 formulation containing 1% (w/v) paclitaxel as the best. The results showed that the DHP107 formulation containing

AC C

1% (w/v) paclitaxel had the best oral BA partly due to the formation of precipitates when the formulations containing ≥ 1.5% (w/v) paclitaxel are diluted with aqueous fluids. The stability of formulation droplets formed in aqueous solution was important to optimize the formulation as well as the solubility of paclitaxel in the DHP107 formulation. The solubility of paclitaxel in F109 was approximately 3% (w/v) in our previous study [11]. However, crystallized paclitaxel was observed with paclitaxel content of ≥ 1.5% (w/v) in the DHP107 formulation 29

ACCEPTED MANUSCRIPT

when the formulation was mixed with aqueous solution. If the formulation can entrap more than 1% (w/v) paclitaxel in aqueous solution without paclitaxel crystallization, we believe

RI PT

that paclitaxel absorption can be improved. In addition, based on the results of previous studies, we found that paclitaxel absorption is increased when the mucoadhesive ability of the formulation is enhanced [11, 18]. It is not clear whether the mucoadhesive property of the

SC

formulations is dependent on paclitaxel content in the formulation. The mucoadhesiveness of

M AN U

the DHP107 formulation might be affected by paclitaxel in the high paclitaxel content formulation.

Based on the above results, DHP107 containing 1% (w/v) paclitaxel was used in the

TE D

antitumor activity assay. The antitumor activity of DHP107 against various transplanted tumors in mice has been reported [10, 11, 26, 33]. However, the antitumor activity in

EP

melanoma and metastatic cancer model was first confirmed in this study. The oral formulation of DHP107 was administered to mice at doses 4–5 times higher than that of

AC C

Taxol® because the previously reported BA of DHP107 was approximately 20% when the AUC of oral DHP107 was compared with that of iv Taxol® infusion in mice [10]. Tumor growth in mice in the DHP107 group was similarly reduced when compared with that in mice in the Taxol® group in breast cancer and melanoma mouse models. The body weight and temperature of mice treated with oral DHP107 did not change during the experimental period and were similar to those negative control groups; however, the body weight and temperature 30

ACCEPTED MANUSCRIPT of mice treated with iv Taxol® were significantly reduced during the treatment period in all breast cancer models. The body temperature of mice in the Taxol® group might have

RI PT

decreased because of the vehicle used for Taxol® (cremophor EL/ethanol). The side effects of Taxol® formulation are well known. Chemotherapy-related fatigue and debilitation along with reduced food intake, body weight loss, and hypothermia are reported during paclitaxel

SC

treatment [34]. However, chemotherapy-related fatigue could be diminished by changing the

M AN U

formulation of paclitaxel. Ethanol is reported to produce a dose-dependent reduction in colonic temperature when orally administered to rats at doses of 2 and 4 g/kg [35]. In this study, 0.8 g/kg of ethanol was infused intravenously to mice when Taxol® was administered

TE D

to mice at a dose of 12.5 mg/kg. As the level of immunity drops when the body temperature falls and Taxol® frequently lowers white blood cell count [13, 36, 37], the patients with

EP

cancer are at a risk of developing infections when they are treated with Taxol®. Hypothermic status after chemotherapy often is associated with the complication of infections. Most

AC C

DHP107 treatments increase the survival rate with fewer toxic effects than Taxol® in murine cancer models. These effects of DHP107 on mice tumor models are consistent with the previously reported antitumor activity and toxicity of DHP107 in other tumor models [10, 11, 26]. The results of quantification of paclitaxel in the plasma and tumor tissues after oral DHP107 administration in the mouse melanoma model revealed the tumor tissue-to-plasma 31

ACCEPTED MANUSCRIPT ratio of DHP107 was higher than that of Taxol®. The distribution of paclitaxel to various normal tissues after the oral administration of DHP107 has been evaluated in previous studies

RI PT

[10, 12]. However, the distribution of paclitaxel to tumor tissue after DHP107 administration was examined for the first time in this study. Paclitaxel from orally administered DHP107 was better distributed to tumor tissues than that from intravenously infused Taxol®. The tissue

SC

concentration of paclitaxel after the oral administration of DHP107 was significantly higher

M AN U

than that after the iv infusion of Taxol® in various tissues studied [10, 12]. This may be due to saturable paclitaxel tissue distribution and saturable elimination from the plasma after the iv administration of Taxol®. Paclitaxel shows nonlinear pharmacokinetics [12, 15, 28, 29]. The

TE D

plasma AUC increased more than proportionally to the increase in the dose of Taxol® because of the dose-dependent clearance of paclitaxel and cremophor EL [30]. As previously stated,

EP

the nonlinear increase in the plasma paclitaxel concentration does not lead to a proportional increase in the level of paclitaxel in tissues, indicating saturable distribution [28]. Therefore,

AC C

the high initial concentration of paclitaxel following the iv infusion of Taxol® may not be efficiently eliminated and distributed to tissues. Thus, the high concentration of paclitaxel might be eliminated from the blood more slowly and therefore remain in the blood for a longer time in the Taxol® group than that in the DHP107 group. Thus, the distribution of paclitaxel to the tumor and other tissues in the oral DHP107 group might be higher than that in the iv Taxol® group. The low plasma paclitaxel concentration and high tumor paclitaxel 32

ACCEPTED MANUSCRIPT

concentration after DHP107 administration is advantageous because high drug concentrations in the plasma might cause hematological toxicity; however, it is beneficial to maintain high

RI PT

drug levels in target tissues. Paclitaxel was entrapped to the maximum level in the DHP107 formulation containing 1% (w/v) paclitaxel without forming crystal precipitates after the addition of simulated digestive

SC

juice in vitro (Figs. 2 and 3). Furthermore, paclitaxel absorption from the formulation was the

M AN U

best among other formulations (Figs. 4 and 5 and Table 1), with antitumor activity in mice (Figs. 6 and 7). We also found that paclitaxel distribution to tumor was more efficient (higher tumor tissue-to-plasma ratio) after the oral administration of DHP107 than intravenous

TE D

Taxol® administration (Fig. 8 and Table 2). In the previously reported clinical trial data, DHP107 was also systemically absorbed well and showed saturated absorption at high doses

EP

without intolerable toxicity [13]. Therefore, the optimized DHP107 formulation could be

AC C

applied to humans for better paclitaxel absorption.

5. Conclusions

Paclitaxel absorption was increased after the oral administration of DHP107 to mice when the content of paclitaxel in the DHP107 formulation was increased up to 1% (w/v). However, the DHP107 formulations with paclitaxel content higher than 1.5% (w/v) were 33

ACCEPTED MANUSCRIPT

unstable because paclitaxel crystallized in the aqueous solution. Therefore, paclitaxel absorption after the administration of these DHP107 formulations was decreased. We

RI PT

optimized paclitaxel content in the DHP107 formulation to 1% (w/v), and then evaluated the antitumor activity, survival rate, and distribution to tumor of the optimized DHP107 formulation in various mouse cancer models. The antitumor activity of oral DHP107 was

SC

similar to that of iv Taxol®. However, DHP107 improved the survival rate of mice, caused

M AN U

low toxicity, and maintained normal body weight and temperature in comparison with those of Taxol®, and the concentration of paclitaxel in the tumor tissues of mice in the DHP107 group was higher than that in the Taxol® group. The findings of this study indicate that

TE D

DHP107 enhances paclitaxel distribution to tumor tissues by improving its absorption. Overall, DHP107 and Taxol® have comparable antitumor activity, but DHP107 has fewer

EP

toxic effects, and it might be beneficial to use DHP107 for the treatment of patients hypersensitive to Taxol®. In the future, we plan to subject the optimized DHP107 formulation

AC C

to clinical trials.

Acknowledgments

This study was supported by an intramural grant from the Korea Institute of Science and Technology [grant numbers 2E21590 and 2E22310]. 34

ACCEPTED MANUSCRIPT

RI PT

Conflict of interest

We declare no potential conflicts of interest with respect to the research, authorship,

AC C

EP

TE D

M AN U

SC

and/or publication of this article.

35

ACCEPTED MANUSCRIPT

References [1] J. Iqbal, F. Sarti, G. Perera, A. Bernkop-Schnurch, Development and in vivo evaluation of an oral drug delivery system for paclitaxel, Biomaterials 32(1) (2011) 170–175. [2] M. Nakajima, K. Hayashi, Y. Egi, K. Katayama, Y. Amano, M. Uehata, M. Ohtsuki, A. Fujii, K.

RI PT

Oshita, H. Kataoka, K. Chiba, N. Goto, T. Kondo, Effect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanoma, Cancer Chemother. Pharmacol. 52(4) (2003) 319–324.

[3] L. Zhao, S.S. Feng, Enhanced oral bioavailability of paclitaxel formulated in vitamin E-TPGS emulsified nanoparticles of biodegradable polymers: in vitro and in vivo studies, J. Pharm. Sci. 99(8) (2010) 3552–3560.

SC

[4] E. Lee, J. Lee, I.H. Lee, M. Yu, H. Kim, S.Y. Chae, S. Jon, Conjugated chitosan as a novel platform for oral delivery of paclitaxel, J. Med. Chem. 51(20) (2008) 6442–6449.

[5] D. Pandita, A. Ahuja, V. Lather, B. Benjamin, T. Dutta, T. Velpandian, R.K. Khar, Development of

M AN U

lipid-based nanoparticles for enhancing the oral bioavailability of paclitaxel, AAPS PharmSciTech. 12(2) (2011) 712–722.

[6] J.P. Flores, M.W. Saif, Novel oral taxane therapies: recent phase I results, Clin. Investig. (Lond) 3(4) (2013) 333–341.

[7] R. Mo, X. Jin, N. Li, C. Ju, M. Sun, C. Zhang, Q. Ping, The mechanism of enhancement on oral absorption of paclitaxel by N-octyl-O-sulfate chitosan micelles, Biomaterials 32(20) (2011) 4609–

TE D

4620.

[8] J. Han, H. Chung, J.H. Lee, J.E. Suh, G.S. Lee, J.C. Kim, B.H. Kang, Single and two-week repeated oral dose toxicity study of DHP2, a hydrophobic drug delivery vehicle in mice, Toxicol. Res. 20(2) (2004) 123–129.

[9] S.J. Lee, S.W. Kim, H. Chung, Y.T. Park, Y.W. Choi, Y.H. Cho, M.S. Yoon, Bioadhesive drug

EP

delivery system using glyceryl monooleate for the intravesical administration of paclitaxel, Chemotherapy 51(6) (2005) 311–318.

AC C

[10] J.W. Hong, I.H. Lee, Y.H. Kwak, Y.T. Park, H.C. Sung, I.C. Kwon, H. Chung, Efficacy and tissue distribution of DHP107, an oral paclitaxel formulation, Mol. Cancer Ther. 6(12 Pt 1) (2007) 3239– 3247.

[11] I.H. Lee, J.W. Hong, Y. Jang, Y.T. Park, H. Chung, Development, optimization and absorption mechanism of DHP107, oral paclitaxel formulation for single-agent anticancer therapy, in: B. Thomas (Ed.), New Advances in the Basic and Clinical Gastroenterology, InTech, Croatia, 2012, pp. 357–374. [12] B.S. Shin, H.J. Kim, S.H. Hong, J.B. Lee, S.W. Hwang, M.H. Lee, S.D. Yoo, Enhanced absorption and tissue distribution of paclitaxel following oral administration of DHP 107, a novel mucoadhesive lipid dosage form, Cancer Chemother. Pharmacol. 64(1) (2009) 87–94. [13] Y.S. Hong, K.P. Kim, H.S. Lim, K.S. Bae, M.H. Ryu, J.L. Lee, H.M. Chang, Y.K. Kang, H. Kim, T.W. Kim, A phase I study of DHP107, a mucoadhesive lipid form of oral paclitaxel, in patients with 36

ACCEPTED MANUSCRIPT advanced solid tumors: crossover comparisons with intravenous paclitaxel, Invest. New Drugs 31(3) (2013) 616–622. [14] M.H. Ryu, B.Y. Ryoo, T.W. Kim, S.B. Kim, H.S. Lim, K.S. Bae, S.R. Park, Y.W. Jo, H.J. Cho, Y.K. Kang, A phase I/II study of DHP107, a novel oral paclitaxel formulation, in patients with advanced solid tumors (phase I) and gastric cancer (phase II), J. Clin. Oncol. 32(3) (2014) abstr 101.

RI PT

[15] H.S. Lim, K.S. Bae, J.A. Jung, Y.H. Noh, A.K. Hwang, Y.W. Jo, Y.S. Hong, K. Kim, J.L. Lee, S.J. Park, J.E. Kim, Y.K. Kang, T.W. Kim, Predicting the efficacy of an oral paclitaxel formulation (DHP107) through modeling and simulation, Clin. Ther. 37(2) (2015) 402–417.

[16] Y.K. Kang, M.H. Ryu, S.H. Park, P.S. R., K.J. G., J.W. Kim, S.H. Cho, Y.I. Park, S.Y. Rha, M.J. Kang, J.Y. Cho, S.Y. Kang, Y.S. Hong, B.Y. Ryoo, B.H. Nam, Y.W. Jo, K.E. Yoon, S.C. Oh, Efficacy

SC

and safety findings from DREAM: A phase III study of DHP107 (oral paclitaxel) vs iv paclitaxel in patients with gastric cancer after failure of first-line chemotherapy, J. Clin. Oncol. 34(2016) suppl; abstr 4016.

M AN U

[17] J. Verbraecken, P. Van de Heyning, W. De Backer, L. Van Gaal, Body surface area in normalweight, overweight, and obese adults. A comparison study, Metabolism 55(4) (2006) 515–524. [18] Y. Jang, H.J. Chung, J.W. Hong, C.-W. Yun, H. Chung, Absorption mechanism of DHP107, an oral paclitaxel formulation that forms a hydrated lipidic sponge phase, Acta Pharmacol. Sin. 38(1) (2017) 133–145.

[19] H. Chung, S.Y. Jeong, I.C. Kwon, Cubic liquid-crystalline particles as protein and insoluble drug

TE D

delivery systems, in: M.L. Lynch, P.T. Spicer (Eds.), Bicontinuous Liquid Crystals, CRC Press, Taylor and Francis group, Boca Raton, FL, 2005, pp. 353–385. [20] N.T. Annan, A.D. Borza, L.T. Hansen, Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions, Food Res. Int. 41(2) (2008) 184–193.

EP

[21] T. Yang, F.D. Cui, M.K. Choi, J.W. Cho, S.J. Chung, C.K. Shim, D.D. Kim, Enhanced solubility and stability of PEGylated liposomal paclitaxel: in vitro and in vivo evaluation, Int. J. Pharm. 338(1–2)

AC C

(2007) 317–326.

[22] S.C. Kim, D.W. Kim, Y.H. Shim, J.S. Bang, H.S. Oh, S. Wan Kim, M.H. Seo, In vivo evaluation of polymeric micellar paclitaxel formulation: toxicity and efficacy, J. Control. Release 72(1–3) (2001) 191–202.

[23] J.M. Pawelek, K.B. Low, D. Bermudes, Tumor-targeted Salmonella as a novel anticancer vector, Cancer Res. 57(20) (1997) 4537–4544. [24] Y. Saegusa, H. Tabata, Usefulness of infrared thermometry in determining body temperature in mice, J. Vet. Med. Sci. 65(12) (2003) 1365–1367. [25] W. Zhang, Y. Shi, Y. Chen, J. Hao, X. Sha, X. Fang, The potential of pluronic polymeric micelles encapsulated with paclitaxel for the treatment of melanoma using subcutaneous and pulmonary metastatic mice models, Biomaterials 32(25) (2011) 5934–5944. 37

ACCEPTED MANUSCRIPT [26] H.S. Hahn, K.H. Lee, I.H. Lee, J.H. Lee, C.S. Whang, Y.W. Jo, T.J. Kim, Metronomic oral paclitaxel shows anti-tumor effects in an orthotopic mouse model of ovarian cancer, J. Gynecol. Oncol. 25(2) (2014) 130–135. [27] A. Shikanov, S. Shikanov, B. Vaisman, J. Golenser, A.J. Domb, Paclitaxel tumor biodistribution and efficacy after intratumoral injection of a biodegradable extended release implant, Int. J. Pharm.

RI PT

358(1-2) (2008) 114–120. [28] D.S. Sonnichsen, C.A. Hurwitz, C.B. Pratt, J.J. Shuster, M.V. Relling, Saturable

pharmacokinetics and paclitaxel pharmacodynamics in children with solid tumors, J. Clin. Oncol. 12(3) (1994) 532–538.

[29] A. Sparreboom, O. van Tellingen, W.J. Nooijen, J.H. Beijnen, Nonlinear pharmacokinetics of

SC

paclitaxel in mice results from the pharmaceutical vehicle Cremophor EL, Cancer Res. 56(9) (1996) 2112–2115.

[30] A. Sparreboom, O. van Tellingen, W.J. Nooijen, J.H. Beijnen, Tissue distribution, metabolism

M AN U

and excretion of paclitaxel in mice, Anti-Cancer Drug. 7(1) (1996) 78–86.

[31] A.O. Nornoo, H. Zheng, L.B. Lopes, B. Johnson-Restrepo, K. Kannan, R. Reed, Oral microemulsions of paclitaxel: in situ and pharmacokinetic studies, Eur. J. Pharm. Biopharm. 71(2) (2009) 310–317.

[32] H. Hauser, W. Guyer, K. Howell, Lateral distribution of negatively charged lipids in lecithin membranes. Clustering of fatty acids, Biochemistry 18(15) (1979) 3285–3291.

TE D

[33] D.B. Kim, J. Jang, Y.H. Cho, M.S. Yoon, H.S. Chung, Y.T. Park, Y.W. Choi, S.W. Kim, Anticancer efficacy and toxicity of oral GMO-paclitaxel in a hormone refractory prostate cancer model, Korean J. Urol. 47(2) (2006) 143–149.

[34] M.A. Ray, R.A. Trammell, S. Verhulst, S. Ran, L.A. Toth, Development of a mouse model for assessing fatigue during chemotherapy, Comparative medicine 61(2) (2011) 119–130.

EP

[35] R. Myers, Alcohol's effect on body temperature: hypothermia, hyperthermia or poikilothermia?, Brain res. bull. 7(2) (1981) 209–220.

AC C

[36] A.K. Oncken, R. Kirby, E. Rudloff, CRITICAL CARE-Hypothermia in Critically III Dogs and Cats, Compend. Contin. Educ. Vet. 23(6) (2001) 506–520. [37] S. Ganta, H. Devalapally, M. Amiji, Curcumin enhances oral bioavailability and anti-tumor therapeutic efficacy of paclitaxel upon administration in nanoemulsion formulation, J. Pharm. Sci. 99(11) (2010) 4630–4641.

38

ACCEPTED MANUSCRIPT

Table 1 Plasma pharmacokinetic parameters of paclitaxel after the oral administration of DHP107

RI PT

containing 0.5% and 1% (w/v) paclitaxel at various volumes and DHP107 containing various

Dose

Volume

Tmax

Cmax

AUC0-24h

BA

in DHP107 (%, w/v)

(mg/kg)

(mL/kg)

(h)

(µg/mL)

(µg·h/mL)

(%)

25

5

2.0

0.3

0.5

2.1

50

10

2.0

1.5

3.8

8.4

75

15

2.0

6.1

20.3

29.8

100

20

2.0

5.3

19.7

21.6

125

25

2.0

5.3

16.1

14.2

25

2.5

2.0

3.5

4.8

21.2

50

5

2.0

4.2

13.0

28.6

75

7.5

2.0

12.3

23.7

34.7

100

10

2.0

18.7

36.2

39.8

125

12.5

2.0

18.8

42.5

37.3

150

15

2.0

22.5

51.0

37.3

25

5

2.0

0.3

0.5

2.1

50

5

2.0

4.2

13.0

28.6

75

5

0.5

10.8

12.2

17.9

100

5

0.5

9.1

12.8

14.0

125

5

0.5

3.2

3.8

3.4

150

5

0.5

6.3

5.4

3.9

0.5 1 1.5 2 2.5 3

M AN U

TE D

1

AC C

0.5

SC

Paclitaxel Formulation

EP

paclitaxel content at various doses in the same volume (n = 5 per time point).

ACCEPTED MANUSCRIPT

Table 2 Plasma and tumor pharmacokinetic parameters of paclitaxel after the iv injection of Taxol®

Taxol® iv (10 mg/kg)

DHP107 po (50 mg/kg)

Parameter

Plasma

Tumor

Plasma

Tumor

Tmax (h)

-

0.5

2.0

4.0

Cmax (µg/mL or µg/g)

-

12.3

5.7

16.2

AUC0-12h (µg·h/mL or µg·h/g)

28.8

118.3

289.6

T/P ratio*

4.1

SC

RI PT

and the oral administration of DHP107 (n = 5 per time point).

AC C

EP

TE D

tumor tissue-to-plasma ratio

18.6

M AN U

*

15.6

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 1. Droplet-size distribution (intensity-weighted) determined for a mixture of DHP107 in

AC C

EP

TE D

water.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

Fig. 2. Paclitaxel crystallization from DHP107 in 0%, 0.5%, 1%, 1.5%, and 2% (w/v) paclitaxel formulations by diluting to 1.5, 5, and 20 times (v/v) in simulated gastric juice

AC C

EP

monitored by optical microscopy (left) and polarized light microscopy (right).

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

Fig. 3. Paclitaxel crystallization from DHP107 in 0%, 0.5%, 1%, 1.5%, and 2% (w/v) paclitaxel formulations by diluting to 1.5, 5, and 20 times (v/v) in simulated bile monitored

AC C

EP

by optical microscopy (left) and polarized light microscopy (right).

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

EP

Fig. 4. Paclitaxel concentration in the plasma was analyzed at several time points after the

AC C

oral DHP107 administration of 0.5% (w/v) paclitaxel formulation at doses of 25–125 mg/kg and 1% (w/v) paclitaxel formulation at doses of 25–150 mg/kg to BALB/c mice (n = 5 per time point). Upper panels are paclitaxel concentration by time and lower panels are AUC and bioavailability (BA) by administration doses. The BA was dose normalized by the intravenous Taxol® at a dose of 10 mg/kg.

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

Fig. 5. Paclitaxel concentration in the plasma analyzed at several time points after the oral administration of DHP107 containing 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% (w/v) paclitaxel to BALB/c mice at doses of 25–150 mg/kg at an equal volume of the formulation (5 mL/kg) (n = 5 per time point). Left panel indicates paclitaxel concentration by time and the right panel

TE D

indicates AUC and bioavailability (BA) by administration doses. The BA was dose

AC C

EP

normalized by the intravenous Taxol® at a dose of 10 mg/kg.

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

Fig. 6. Antitumor activity of intravenous (iv) saline (closed circle), oral paclitaxel free formulation (F109; open circle), iv Taxol® (closed down triangle) and Abraxane® (open up triangle), and oral DHP107 [1% (w/v) paclitaxel formulation, 25 mg/kg; closed square and 50 mg/kg; open square] in BALB/c and nude mice implanted subcutaneously with EMT-6, MDA-MB-231, and MX-1 cell lines (n = 10 per group). The tumor size, body weight and temperature, and survival rate were measured during the experiments.

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 7. (A) Antitumor activity of intravenous (iv) Taxol® at a dose of 10 mg/kg and 5 mL/kg

EP

of oral F109 and DHP107 at a dose of 50 mg/kg in C57BL6 mice subcutaneously inoculated

AC C

with melanoma cell lines. The tumor size and survival rate of mice were measured during the experiments (n = 10 per group). (B) Antitumor activity of oral F109 (5 mL/kg) and DHP107 (50 mg/kg) in a mouse pulmonary metastatic melanoma model. The proportion (percent) of lungs exhibiting tumor metastasis and lung modulation was measured on day 30 after tumor implantation (left; *P < 0.01) and the survival rate of mice were measured during the experiments (right; n = 10 per group).

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 8. Paclitaxel concentration in the plasma and tumor tissue after the intravenous Taxol®

AC C

EP

time point).

TE D

(10 mg/kg) and oral DHP107 (50 mg/kg) administration to mice with melanoma (n = 5 per