Determination of In Vivo Behavior of Mitomycin C-Loaded O/W Soybean Oil Microemulsion and Mitomycin C Solution Via Gamma Camera Imaging

Preparation of oil-in-water microemulsion

For microemulsion formulation, soybean oil was used as the oil phase. Lecithin and Tween 80 at a ratio of 1:2 (S) were surfactants and ethanol was the cosurfactant (coS). Lecithin was dissolved in predetermined amount of ethanol by stirring with a vortex at 1000 rpm. Then, oil and Tween 80 were added and mixed. Finally, distilled water was added drop by drop until the turbidity was observed, while stirring with a magnetic stirrer (IKA Labortechnik). Ideal S/coS weight ratios and micromulsion existence areas were determined with the aid of phase diagrams drawn using a computer program developed in the computer center at the Ege University Faculty of Pharmacy. ¹⁹ The HLB value of the S/coS mixture was 11.33, which is suitable for O/W microemulsion formulation. In the final, MMC was added to the microemulsion formulation (1 mg/1g). ²⁰ MMC-loaded microemulsion is abbreviated as MMC-M.

Preparation ^99mTc labeled MMC solution and ^99mTc labeled MMC-M

MMC was radiolabeled with radioactive technetium (^99m Tc) at the Radiopharmacy Department of the Pharmacy Faculty of Ege University as follows: to a 5-mL vial, 1 mL of the MMC solution (1 mg MMC in 1 mL normal saline), 25 μL of freshly prepared solution of stannous chloride dihydrate (1 mg/mL) in 0.1N HCl, and 74 MBq (2 mCi) Na^99mTcO₄ were added. The mixture was vortexed for 30 seconds and filtered through a membrane filter (0.22 μm) directly into a sterile vial. The solution was incubated in a closed container for 10 minutes at room temperature. The radiochemical purity was evaluated by instant thin layer chromatography (ITLC).

^99mTc labeled MMC-M was prepared as follows: 1 mL of radiolabeled MMC mixture was incorporated into 0.92 mg oily phase (surfactant and oil). Then, 0.095 mL distilled water was added and stirred with a magnetic stirrer.

Radiochemical analysis

Radiochemical analysis was performed on ITLC-SG strips with the acid citrate dextrose buffer as a mobile phase. The ^99mTc labeled MMC remained at the point of spotting, whereas hydrolyzed ^99mTc and free ^99mTc impurities moved with the solvent front (Rf: 0.8–1.0).

Gamma camera imaging of in vivo MMC distribution

To understand the whole body localization of therapeutic dose of MMC-M, gamma scintigraphy was used. The in vivo study conducted was approved by the Ege University Local Ethique Committee (Protocol no: 2009/2). The ^99mTc labeled MMC-M and ^99mTc labeled MMC solution as control were evaluated in New Zealand white male rabbits (2 kg) by recording static images at different time points. Three rabbits were used to observe in vivo behavior of MMC-M and one as the control for MMC solution. The radiolabeled formulations were injected intravenously through the right marginal ear vein of each rabbit using volumes of 0.120 mL and activities of approximately 0.120 mCi. After delivery of formulations, static images were obtained by a gamma camera (Toshiba digital GCA 602 A) equipped with a low-energy general-purpose parallel-hole collimator, which was used for static image acquisition at 15th, 30th, 60th, 90th minutes. Energy windows were set at 140 keV (20% width).

Microemulsion preparation and its properties

The droplet size of microemulsions with and without MMC was determined as 57.67±3.16 nm and 34.65±5.86 nm, respectively. The pH values of MMC unloaded microemulsions were between 6.80–6.95. These pH and the viscosity value (32.4 cPs) were appropriate for i.v. application. Incorporation of MMC into the M system did not significantly affect the pH value of the microemulsion formulation. The addition of MMC slightly increases the electrical conductivity because of its amphiphylic character. ²⁰ As shown in the previous data, the addition of an amphiphylic drug increases the conductivity value of a microemulsion system. ^17,20,21

According to previous reported data on the hemolitic activity, microemulsion formulations are safe and appropriate for i.v. application. ^{22 –24} Previously, we showed that the microemulsion has the ability to reduce the hemolytic potential of T80, which is used as the hydrophilic surfactant for microemulsion production. Besides, cytotoxicity tests were carried out in Calu1 and A-549 cell lines with the microemulsion formulation. It was observed that, microemulsion showed no significant cytotoxic effect at all applied doses on Calu1 cells. For A549 cells, the viability was over 81% up to 210 μM dose. ²⁰

Gamma camera imaging of in vivo behavior of MMC

The radiolabeling efficiency of ^99mTc labeled MMC was found to be greater than 90%. Gamma camera images of the rabbits after the i.v. administration of ^99mTc labeled MMC-M and ^99mTc labeled MMC solution are shown in Fig. 1. Radioactivity in the bladder observed in 30 minutes after injection of the MMC solution shows that urinary excretion of the latter starts very rapidly (Fig. 1A). On the other hand, following administration of MMC-loaded microemulsion, no radioactivity appeared in the bladder during 90 minutes (Fig. 1B). The unabated radioactivity in the liver during the experiment shows that this formulation is stably localized in the liver.

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FIG. 1.

Gamma camera images of rabbits administered with radiolabeled drug. (A) Rabbit administered with MMC solution as control; (B) Rabbit administered with MMC-microemulsion. L, liver; S, spleen; B, bladder; K, kidneys; A, administration site (the right marginal ear vein).

Radiopharmaceuticals, which are used for imaging either the biliary tract or phagocytic cells also provide liver imaging. Due to the bile circulation, these radiopharmaceuticals usually appear in the intestine approximately in 1 hour after administration. In the present study, no activity was observed in the intestine during imaging experiments, only liver hepatocytes, which are of phagocytic character were observed.

Also, radioactivity is clearly seen in the liver in the images obtained after both the MMC solution and MMC-M, while later only the MMC solution is eliminated via urine. The liver is an organ composed of hepatocytes, which have phagocytic property for certain size of particles. Targeting of the liver by ^99mTc labeled MMC-M is to be expected, considering that MMC-M is a colloidal carrier system containing about 50 nm lipid droplets. Following the administration of ^99mTc labeled MMC solution, radioactivity was seen in the bladder alongside the liver. Although no visual change was observed in the system after radiolabeling, and also MMC was below its solubility maximum, it may have formed colloidal molecules with ^99mTc in the solution. It could be interesting to see if such a formation took place, to explain the high radioactivity in the liver after MMC solution administration.

Another possible explanation for the high radioactivity in the liver after administration of the drug solution is that MMC has previously been shown to be metabolized by xanthine oxidase and microsomal enzymes such as NADPH-cytochrome P450 and cytochrome b5, which are abundant in the liver, ^{2 –4} while approximately 10% of the drug is excreted in the urine. ⁵ As to the octanol/water partition coefficient definition, MMC was determined as 76% in the water phase and 24% in the octanol phase. Also, it has been reported that MMC tends to be located at the lipid/water interface, thus causing the drug-loaded microemulsion droplets to be larger in size than the ones of unloaded microemulsion. ²⁰ Even though the external phase in the MMC-M system is water, no excretion was seen via urine for 90 minutes after administration. This finding confirms that a majority of MMC in the system is located at the interface of the droplets. In conclusion, the microemulsion system ensures a longer drug presence in the liver.

It is definitely difficult to foresee the biological behavior of the drug in case of liver failure. Given that the solution of MMC may be excreted more actively via urine it may be preferable to treat with the MMC solution in cases of liver failure, considering that MMC-M may not be eliminated from the body. So, another set of experiments, including both healthy animals and animals with liver failure model could give valuable information about the elimination and the tolerability of the developed system.

Nevertheless, the results of gamma camera imaging and our previous in vitro release studies ²⁰ indicate that the MMC-M system is more appropriate than the MMC solution to treat liver cancers because of its better targeting and its longer stable location in the target organ.