Patent Application
20080145440
The entire disclosure of Japanese Patent Application No. 2006-341244, filed Dec. 19, 2006, and Han Hee CHO et. al, “Drug Delivery System Using Complex of Anticancer Agent (Cis-platin) and Salmon-Derived DNA”, Official Journal of the Japan Society of Drug Delivery System, Vol. 21, No. 3 (May 2006), p. 365 is expressly incorporated by reference herein.
1. Field of the Invention
The present invention relates to a microparticle preparation that encapsulates a pharmaceutical agent and a medical composition that contains the microparticle preparation.
2. Description of Related Art
Cisplatin (cis-dichlorodiammineplatinum (II) or CDDP) has been widely used in treatments of a malignant tumor such as a testicular tumor, bladder cancer, ovary cancer, uterine cervix cancer and ureteropelvic junction tumor. The cisplatin is an effective anticancer agent, but can cause severe side-effects such as a neural toxicity, kidney toxicity etc. Accordingly, cisplatin has been administered in a restrictive manner. In order to reduce the side effects of cisplatin while maintaining an anticancer effect, it is important to selectively deliver cisplatin only to a tumor site and to maintain a high concentration of cisplatin. In this manner, cisplatin is administered intracavemously, intraperitoneally and intratumorally.
However, cisplatin in solution is rapidly absorbed into systemic circulation, so that the high concentration of the pharmacological agent can be hardly maintained at a target site.
Consequently, there has been an attempt to use a drug delivery system (DDS) such as a liposome, polymer micelle or a microparticle so as to maintain the high concentration of cisplatin at the target site. A number of researches have been carried out on encapsulating cisplatin by use of a biodegradable polymer. As such examples, refer to Document 1: JP-A-2006-312015 and Document 2: JP-A-2006-248978.
However, in the technology of the polymer-coated composition disclosed in Document 1 and the liposome preparation in Document 2, since a large amount of cisplatin is released in an early-phase, cisplatin is not continuously released at a constant concentration.
An object of the present invention is to provide a microparticle preparation that can reduce the early-phase release amount, maintain a release of a pharmaceutical agent over a long period of time and enhance therapeutic efficacy for patients, and a medical composition containing the microparticle preparation.
A microparticle preparation according to an aspect of the present invention contains double-helical DNA, in which a pharmaceutical agent is intercalated into the double-helical DNA.
The double-helical DNA is generally formed by a pair of polynucleotide chains having a right-handed helix. In sugar-phosphate backbones of the pair of polynucleotide chains, a pair of planar bases having structural complementarity protrude toward a center portion of the helix perpendicularly to an axis of the helix, such that the bases are hydrogen-bonded with each other. In a case of a B structure, there is a gap (width and height: approximately 1.1 nm and 0.34 nm respectively) between base pairs of the chains of the DNA. A small molecule having a planar structure can be inserted into the gap, which is called “intercalation (intercalate)”. Such a phenomenon may be promoted by an electric charge and hydrophobic property of the small molecule.
As a material for the double-helical DNA according to the present invention, for example, a fish milt may be advantageously used. Since a fish milt nucleus contains a lot of DNA and has been a discarded material, the milt is an advantageous material for mass-producing the double-helical DNA at low cost. The fish is salmon, herring, trout, cod and the like. After skin, strings and vessels are removed from the milt, the milt is deprived of oil content and refined to obtain the double-helical DNA according to the present invention.
According to the aspect of the present invention, since the microparticle preparation contains a pharmaceutical agent that is intercalated into the double-helical DNA, the microparticle preparation exhibits an excellent slow-release property. Accordingly, for example, a pharmaceutical agent having a severe toxicity can be slowly delivered to a target site.
Particularly, a use of the double-helical DNA derivatized by a ligand having target-cell specificity is advantageous because a specific target site can be selectively cured.
In the microparticle preparation according to the aspect of the present invention, it is advantageous that the pharmaceutical agent is an anticancer agent, and specifically advantageous that the anticancer agent consists of a platinum compound selected from a group consisting of cisplatin, carboplatin, ormaplatin, oxialiplatin and lobaplatin. Particularly, when the microparticle preparation is applied to cisplatin (hereinafter, “CDDP”) that is an anticancer agent having an extremely severe toxicity, the micro preparation can minimize the side effects and continuously provide an anticancer effect to on cancer cells for a long period of time.
As such double-helical DNA, salmon DNA may be advantageously used, which is a biodegradable and nontoxic natural high molecule.
According to the aspect of the present invention, the microparticle preparation can be used as an easily injectable preparation. When the microparticle preparation is provided to medical industries, the microparticle preparation can reduce the side effects entailed by anticancer agent administration, alleviate pains that terminal cancer patients suffer from, and contribute to reduction of medical care costs.
According to the aspect of the present invention, the microparticle preparation may be supported by a pharmaceutically-permissible carrier, so that the microparticle preparation can be used as a medical composition for treating a tumor.
The present invention will be described below with reference to examples, but it should be noted that the present invention is not limited to the contents of the examples.
After the fish (salmon) milt underwent a water-rinsing and caustic-soda treatment, the milt underwent a protease treatment and a solid-liquid separation with ethanol to obtain double-helical DNA.
A microparticle containing CDDP was manufactured by Solvent Evaporation Method using an O/O emulsion (oil in oil emulsion). Specifically, the microparticte was manufactured by the following method:
500 mg of the double-helical DNA (molecular weight: one million and more) was dissolved in 50 ml of distillated water, which was added with 5 ml of 1 percent-by-mass CDDP solution whose solvent was N,N-dimethylformamide (manufactured by Wako Pure Chemical Industries, Ltd.), and was stirred. Next, the CDDP-DNA mixed solution was added into liquid paraffin containing solbitan monooleat (span 80) of a temperature of 28 degrees Celsius. Being stirred, the CDDP-DNA mixed solution and the liquid paraffin were heated up to 40 degrees Celsius in increments of 2 degrees per ten minutes, and the CDDP-DNA mixed solution and the liquid paraffin were reacted with each other for forty eight hours. The paraffin solution underwent a centrifugal separation at 10000 rpm for ten minutes, and the microparticle was obtained. After being rinsed in n-hexane five times and then in 2-propyl alcohol one time, the microparticle was freeze-dried.
The double-helical DNA prepared in the above-described method (1) was a white and water-soluble material. Containing the CDDP, the microparticle obtained by the Solvent Evaporation Method became a yellow and insoluble material (hereinafter, the microparticle may be referred to as “CDDP-DNA MS”). Such a change suggests that the CDDP was intercalated into a double-helical structure of the DNA such that the CDDP was chemically bonded with the DNA to form a cross-linking. Accordingly, it is considered that the CDDP was stably incorporated into the microparticle.
In order to measure CDDP content in the obtained microparticle, 10 mg of the microparticle was added to 10 ml of nuclease-tris-HCl buffer solution (3 unit/ml, sigma) containing a DNA-degrading enzyme and stirred until the microparticle was completely dissolved. In addition, 10 mg of the CDDP was dissolved in 10 ml of tris-HCl buffer solution, and a solution having a concentration of 0.5 to 2 μg/ml was used as a standard solution. The CDDP content was measured three times per a sample with an atomic absorption spectrometer (Hitachi Model Z-8000).
An atomic absorption spectrum analysis was performed under the following conditions:
Pt lamp: 1=265.9 nm
Slit width: 1.9 A
Carrier gas: argon (1 l/min)
measurement mode: BGC
amount of infusion: 10 μl.
As a result, the CDDP content in the CDDP-DNA MS was 4 percent-by-mass (theoretical figure: 9 percent-by-mass) and an uptake efficiency was 45 percent.
The CDDP-DNA MS (10 mg) was added to 10 ml of tris-HCl buffer solution (pH 7.4) containing 0.01 percent-by-mass Tween 80. Being oscillated, the buffer solution was incubated at a temperature of 37 degrees Celsius respectively for two hours, one day, three days, five days and seven days. 5 ml of the buffer solution was extracted at each point, and a Pt amount in the solution was measured by measuring absorbency under 265.95 nm with an atomic absorption spectrometer. The measurement was performed independently three times.
Using mouse-derived colon cancer cells (colon-26), a toxicity of the microparticle to cancer cells was experimented. Specifically, the experiment was conducted according to the following method:
The colon-26 cells were disseminated on a 96 well-plate at 1′104 concentration and cultured for twenty four hours. Then, the cells were treated with free CDDP (free CDDP: 1 μg/ml, 5 μg/ml, 10 μg/ml), DNA only (5 mg/ml) and the CDDP-DNA MS (CDDP-MS: 1 mg/ml, 10 mg/ml) and cultured for forty eight hours. Cellular toxicity was examined by measuring absorbency under 450 nm with a CCK-8 kit (Dojindo, Kumamoto, Japan), the CCK-8 kit measuring formazan substitution of tetrazolium based on an intercellular enzyme.
Cell growth was slightly depressed in the colon cancer cells of colon-26 treated with the DNA only. However, since no effect was made on the cell-growth in an experiment of toxicity to fibroblast L-929, the cell-growth depression in colon-26 is considered not to have been induced by the toxicity.
The cell group having undergone the 10 μg/ml free CDDP treatment showed the cytostatic effect of approximately 50 percent. In the cells treated with the CDDP-DNA MS at concentrations of 1 mg/ml and 10 mg/ml respectively, the cell growth was significantly depressed as compared with a control (standard sample). However, the concentration difference of the CDDP-DNA MS showed no significant difference in the cell-growth depression effect. It is now clear that the CDDP released from the CDDP-DNA MS retains activity in depressing the cell-growth.
As an experimental animal, six-week-old BALB/c male mice (weight 25 g, Shimizu Laboratory Supplies Co., Ltd.) was kept under an environment where a constant temperature and humidity was maintained (temperature: 24 degrees Celsius +/−2 degrees, humidity: 55+/−10%). The mice were divided into control group, a CDDP-DNA MS group and a free CDDP group, each including five mice. During an experiment, the mice were allowed to access to feed and water without restriction. The CDDP-DNA MS, free CDDP and DNA only were respectively suspended in normal saline containing 0.01 percent by mass Tween 80, and each solution was adjusted.
Normal saline was set as the control. The free CDDP was prepared to have concentrations respectively of 5 mg/kg, 10 mg/kg and 20 mg/kg, and the mice in the free CDDP group were intraperitoneally administered with the free CDDP one time for each concentration. The CDDP-DNA MS was prepared to have CDDP concentrations respectively of 20 mg/kg and 40 mg/kg, and the mice in the CDDP-DNA MS group were intraperitoneally administered with the CDDP-DNA MS one time for each concentration. Then, mice conditions were observed, whether or not the mice were alive was checked, and weight of the mouse was measured. The mice were sacrificed in 30 days after the administration. Blood was taken from hearts of the mice and a hematological assessment and biochemical examination of blood were conducted. Further, after dissection, internal organs were macroscopically observed.
The mice were sacrificed on a twentieth day after the agent administration, and
Based on the above results, it is understood that the CDDP-DNA MS administration entails much less toxicity than the free CDDP administration.
As an experimental animal, a six-week-old BALB/c male mouse (weight 25 g, Shimizu Laboratory Supplies Co., Ltd.) was selected. Based on weight measured on a date of cancer implanting, the mice were divided by stratified randomization method into a control group, a CDDP-DNA MS group and a free CDDP group such that each group included seven mice. In the experiment, the same agent treatment was performed as in the toxicity experiment. Specifically, the experiment was conducted in the following methods:
Mouse-derived colon cancer (colon-26 adenocarcinoma) cells (1′106) were intraperitoneally implanted in the mice to provide cancer-bearing mice. In six days after the cancer implanting, agents (the free CDDP and CDDP-DNA MS) were intraperitoneally administered. Weight changes and survival rate were observed from the day of the administration. In
After the agent administration, the free CDDP group showed weight reduction and appearance changes due to CDDP toxicity. In the free CDDP group in which 20 mg/kg of CDDP was administered, a minimum weight was observed on a fifth day after the administration and a half number of the group eventually died. The death is considered to be caused by the CDDP toxicity.
On the other hand, neither weight reduction nor appearance change was observed in the CDDP-DNA MS group. In the free CDDP group, luster of the mouse hair was lost after the CDDP administration.
The appearance changes due to cancer formation were observed from a thirtieth day after the administration. In the free CDDP group, the mice started to die of cancer after a fortieth day. The concentration difference between the free CDDP 5 mg/kg and 10 mg/kg showed no significant difference in the anticancer effect On the other hand, in the CDDP-DNA MS group, it was obvious from an appearance observation that a tumor formation was depressed, and the CDDP-DNA MS showed a significant difference in life prolongation effect as compared with the control and free CDDP groups.
From the results, it is understood that the administration of the CDDP-DNA MS provides an excellent anticancer effect.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-341244 | Dec 2006 | JP | national |
