Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Summary

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Abstract

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN.

To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

Introduction

Chemotherapy is a primary treatment modality for many cancers yet it is often associated with severe adverse effects and limited efficacy due to drug resistance and other factors^1,2,3. To improve the outcome of chemotherapy, drug combination regimens have been applied in the clinic based on considerations such as non-overlapping toxicities, different mechanisms of drug action, and non-cross drug resistance^4,5,6. In clinical trials, a better tumor response rate was often observed usin....

Protocol

All animal experiments were approved by the Animal Care Committee of University Health Network at the Ontario Cancer Institute and conducted in accordance with the Canadian Council on Animal Care Guidelines.

1. Biological Sample Preparation

1. Collect the whole blood, major organs, and breast tumor at predetermined time-points after intravenous (i.v.) administration of drug-containing formulations (e.g., DMPLN, liposomal DOX)
   1. Inject a breast tumor-bearing mouse i.v. with a prepared drug-containing formulation.
   2. Anesthetize the mouse at designated time-points (e.g., 15 min) by....

Results

Two anticancer drugs, DOX and MMC, as well as the DOX metabolite, DOXol, were simultaneously detected without any biological interference under the same applied gradient HPLC condition using 4-MU as the I.S. for both the fluorescence and UV detectors. DOX, MMC, DOXol and 4-MU were well-separated from each other with retention times of 5.7 min for MMC, 10.4 min for DOXol, 10.9 min for 4-MU, and 11.1 min for DOX (Figure 2). Each drug in whole blood and various .......

Discussion

Compared to other chromatographic methods that enable the detection of a single drug species at a time, the present HPLC protocol is able to simultaneously quantitate three drug compounds (DOX, MMC, and DOXol) in the same biological matrix without the need to change the mobile phase. This preparation and analysis method has been successfully applied to determine the pharmacokinetics and bio-distribution of two nanoparticle-based drug delivery systems (i.e., liposomal DOX and DMPLN)²². Sin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Doxorubicin	Polymed Theraeutics	111023	Anticancer drug
Mitomycin C	Polymed Theraeutics	060814	Anticancer drug
Doxorubicinol (DOXol)	Toronto Research Chemicals	D558020	Metabolite of DOX
4-Methylumbelliferone sodium salt	Sigma-Aldrich	M1508	Internal standard
Myristic Acid	Sigma-Aldrich	544-63-8	Materials for poly-lipid hybrid nanoparticles
Polyoxyethylene (100) Stearate	Spectrum	M1402	Materials for poly-lipid hybrid nanoparticles
Polyoxyethylene (40) Stearate	Sigma-Aldrich	P3440	Materials for poly-lipid hybrid nanoparticles
Pluronic F68 (PF68)	BASF Corp.	9003-11-6	Materials for poly-lipid hybrid nanoparticles
Ultrasonication (UP100H)	Hielscher, Ultrasound Technology	NA	Nanoparticle preparation
Water Bath (ISOTEMP 3016HS)	Fisher Scientific	NA	Nanoparticle preparation
Liposomal Doxorubicin  (Caelyx)	Janssen	Purchased from the pharmacy Princess Margaret Hospital	Clinically-approved nanoparticle formulation
HPLC-graded Methanol	Caledon Chemicals	6701-7-40	HPLC mobile phase composition
HPLC-graded H2O	Caledon Chemicals	8801-7-40	HPLC mobile phase composition
HPLC-graded Acetonitrile	Caledon Chemicals	1401-7-40	HPLC mobile phase composition
Trifluoroacetic Acid	Sigma-Aldrich	302031	HPLC mobile phase composition
0.45 μm Nylon Membrane Filter Paper	Whatman	WHA7404004	HPLC mobile phase preparation
1cc Plastic Syringes	Becton, Dickinson and Company	2606-309659	Treatment injection
5cc Plastic Syringes	Becton, Dickinson and Company	2608-309646	Tissue collections
30G 1/2 Needles	Becton, Dickinson and Company	305106	Treatment injection
25G 5/8 Needles	Becton, Dickinson and Company	305122	Tissue collections
Sterile 0.9% Saline	Univeristy of Toronto House Brand	1011	Tissue perfusion
13 ml Rounded-bottom conical tube	SARSTEDT	62.515.006	Prolyprolene, tissue homogenization
Alpha Minimum Essential Medium (MEM)	Gibco	12571063	Cell medium
1 x Phosphate Buffer Saline	Gibco	10010023	Tissue homogenization
Triton X-100	Sigma-Aldrich	X100-100 ML	Tissue homogenization
Formic acid	Caledon Chemicals	1/5/3840	Adjust pH for extraction solvent
Sodium heparin sprayed plastic tubes	Becton, Dickinson and Company	367878	Blood collection
Analytical Weigh Balance	Sartorius	CPA225D	NA
pH meters	Fisher Scientific	13-637-671	accumet BASIC
Vortex Mixter	Fisher Scientific	02-215-365	Vortexing samples at desired speed
1.5 ml  Microcentrifuge Tube	Fisherbrand	2043-05408129	Prolyprolene
Model 1000 homogenizer	Fisher Scientific	08-451-672	Tissue homogenization
Centrifuge 5702R	Eppendorf	5702R	Extraction preparation
Heated Evaporator System	Glas-Col	NA	Sample reconstitution
HPLC Screw Thread Vials	DIKMA	5320	HPLC sample injection
HPLC Screw Caps with PTFE White Silicone Septa	DIKMA	5325	HPLC sample injection
HPLC Polypropylene Insert	Agilent Technologies	5182-0549	Maximum volume 250 μl, HPLC sample injection
Xbridge C18 Column	Waters Corporation	186003117	Drug analysis
Gradient pump	Waters Corporation	W600	Drug analysis
Auto-sampler	Waters Corporation	W2707	Drug analysis
Photodiode array detector	Waters Corporation	W2998	Drug analysis
Multi λ fluoresence detector	Waters Corporation	W2475	Drug analysis
EMPOWER 2	Waters Corporation	NA	Data analysis software
Scientist	Micromath	NA	Pharmacokinetic analysis
Female Balb/c Mice	Jackson Laboratory	001026	In vivo
EMT6/WT Breast Cancer Cells	Provided by Dr. Ian Tannock; Ontario Cancer Institute	NA	In vivo