Methods and apparatus for extracting doxorubicin from blood and measuring doxorubicin in blood

Abstract

Doxorubicin is extracted from blood using anionic material, such as a resin comprising sulfonated polystyrene divinylbenzene beads, and polyethersulfone membrane, or both. After exposing the resin and/or membrane to blood in order to remove doxorubicin therefrom, the doxorubicin maybe extracted from the resin and/or membrane by exposing the material to an extraction solution, sonicating the extraction solution to enhance release of the doxorubicin, and repeating the exposure and sonication in order to remove substantially all of doxorubicin from the resin.

Claims

1. A method for measuring doxorubicin present in a patient's blood, said method comprising: mixing a sample of the patient's blood with a silver nitrate solution; adding a volume of methanol to the mixture; further mixing the blood, silver nitrate, and methanol together for at least 10 seconds; letting the mixed blood, silver nitrate, and methanol stand for at least 30 minutes to precipitate particles and form a supernatant; separating the supernatant via chromatography, wherein the separated supernatant contains doxorubicin originally present in the blood sample; and analyzing the separated supernatant via spectroscopy, wherein the UV absorbance of doxorubicin is measured.

2. The method of claim 1, including the step of analyzing the separated supernatant via spectroscopy at 254 nm.

3. The method of claim 1, including the step of analyzing the separated supernatant via spectroscopy at 480 nm.

4. The method of claim 1, wherein a chromatography buffer comprises acetonitrile and NaH.sub.2PO.sub.4.

5. The method of claim 1, wherein the total mixed volume of the sample of the patient's blood with the silver nitrate solution is below 200 μl.

6. The method of claim 1, wherein the volume of methanol added to the mixture is less than or equal to 1 ml.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Extraction solutions suitable for use in the present invention may be prepared as follows: a. Solution 1 i. Mixing 100 ml of 20% weight by volume KCl solution with 100 ml of organic solution (acetone, ethanol, or acetonitrile) ii. After excess salt has precipitated, take off the extraction solution supernatant and add 1% weight by volume of detergent (CHAPS, SDS, Triton X-100, or CTAB). iii. After 1 hour of stirring, spin down the detergent extraction solutions, to remove excess precipitates, then filter supernatant using a solvent filter. b. Solution 2 i. Mixing 120 ml of 20% weight by volume KCl solution with 80 ml of organic solution (acetone, ethanol, or acetonitrile) making the ending concentration 60% KCL and 40% organic. ii. After excess salt has precipitated take off the extraction solution supernatant add 1% weight by volume of detergent (CHAPS, SDS, Triton X-100, or CTAB).

(2) iii. After 1 hour of stirring spin down the detergent extraction solutions, to remove excess precipitates, then filter supernatant using a solvent filter. c. Solution 3 i. Mixing 80 ml ml of 20% weight by volume KCL solution with 120 ml of organic solution (acetone, ethanol, or acetonitrile) making the ending concentration 40% KCL and 60% organic. ii. After excess salt has precipitated take off the extraction solution supernatant add 1% weight by volume of detergent (CHAPS, SDS, Triton X-100, or CTAB). iii. After 1 hour of stirring spin down the detergent extraction solutions, to remove excess precipitates, then filter supernatant using a solvent filter. d. Solution 4 i. Mixing 90 ml of 20% weight by volume KCL solution with 110 ml of organic solution (acetone, ethanol, or acetonitrile) making the ending concentration 45% KCL and 55% organic. ii. After excess salt has precipitated take off the extraction solution supernatant add 1% weight by volume of detergent (CHAPS, SDS, Triton X-100, or CTAB). iii. After 1 hour of stirring spin down the detergent extraction solutions, to remove excess precipitates, then filter supernatant using a solvent filter.

(3) Doxorubicin may be removed from blood in vivo or in vitro by exposure of the blood to an ion exchange resin. For example, doxorubicin may be removed from blood by exposure to cationic resin comprising a mixture of sulfonated polystyrene divinylbenzene beads and/or polyethersulfone sulfonate membrane.

(4) In vivo removal of doxorubicin from a patient may be accomplished by incorporating the cationic sulfonated polystyrene divinylbenzene resin or beads, and/or polyethersulfone sulfonate membrane into the filtration catheters described in PCT Publication WO 2014/100201, previously incorporated herein by reference. The filtration device may then be positioned with a patient's blood vessel, as described in the PCT Publication and left in place for a time sufficient to remove doxorubicin until the resin is partially or fully loaded and/or the doxorubicin is partially or completely removed from the blood.

(5) Once the cationic sulfonated polystyrene divinylbenzene and polyethersulfone sulfonate resin bead mixture of the present invention is partially or fully loaded with doxorubicin, the doxorubicin may be extracted from a portion of the bead mixture as follows: a. Add 0.19 grams of loaded resin/membrane to a 50 ml falcon tube. b. Then add 50 ml of extraction solution to the container. c. To extract doxorubicin from the ion exchange material we place the extraction solution with the loaded beads under ultrasonic mixing by using an ultrasonic bath or an ultrasonic probe for 30 minutes. d. After 30 minutes take a 1 ml sample/aliquot from the bead containing extraction solution. e. Then decant the drug filled supernatant after the beads are allowed to settle out of solution. f. Add 50 ml of extra extraction solution to the container holding the beads. g. Now ultrasonically mix the new bead containing extraction solution using an ultrasonic bath or an ultrasonic probe for 30 minutes to extract.

(6) Doxorubicin extracted from the beads may be analyzed as follows: a. After 1 hour under ultrasonic mixing/reloading of the extraction solution all of the doxorubicin has eluted off of the ion exchange material. b. Samples analyzed using a Molecular Devices Spectramax M2 Multi-Mode or, in the alternative, high pressure liquid chromatography (HPLC) absorbance and fluorescence microplate reader measuring excitation at 480 nm and emission at 550 nm. c. Doxorubicin concentrations are compared to standard concentration curves.

(7) Doxorubicin in blood may be analyzed by performing the following steps: Mix 100 μl of blood with 25 μl of AgNO3 (30% volume solution in water) Mix vigorously for 15 seconds Add 875 μl of Methanol Mix vigorously for 15 minutes Leave for 60 minutes Remove the solid particulates via centrifugation Analyze via chromatography

(8) This method requires fewer steps than the prior method described earlier, uses smaller sample and reagent sizes, and results in a single peak that provides more accurate results.

(9) One or more business methods may incorporate the methods described above. As an example of a business method, hospital or clinic customers may perform extractions for a patient, and then send the device (such as a catheter) used in the extraction to a service provider. The service provider may then perform an analysis of the device, and/or of the filtration materials as described above, in order to quantify the amount of therapeutic removed from the blood of the patient.

(10) Following analysis via chromatography in one of the methods described above, the amount of doxorubicin extracted from a patient's circulatory system is determined. The amount of doxorubicin extracted may be entered into a formula in order to determine the effective dosage to the patient. And the effective dosage to the patient may be recorded in order to track a patient's cumulative lifetime dosage.

(11) For example, prior to a patient's first treatment, the prescribed dosage (Dosage.sup.PRE) is recorded, and the drug administered to the patient. A filtering device is employed as described above in order to remove chemotherapeutic agent from the bloodstream after it has passed through the target tissue. The amount of agent extracted from the filter material is calculated according to the methods described above, and quantified as the extracted dosage (Dosage.sup.EXT). The extracted dosage is subtracted from the prescribed dosage, in order to determine a first effective dosage to the patient (Dosage.sup.EFF 1):
Dosage.sup.PRE−Dosage.sup.EXT=Dosage.sup.EFF 1
The first effective dosage is recorded and maintained in the patient's medical chart. In subsequent treatments, the effective dosage is analyzed and recorded, and assigned a subsequent number. Each successively numbered effective dosage is added to the previous effective dosage, thereby tracking the total lifetime dosage (Dosage.sup.LIFE).
Dosage.sup.EFF 1+Dosage.sup.EFF 2+Dosage.sup.EFF 3=Dosage.sup.LIFE

(12) A kit comprising individually packaged devices and reagents as described above may be assembled and sold commercially. A kit, for example, may include: 1: Catheter having a reservoir containing sulfonated polystyrene beads; 2: 3 vials KCl solution: 100 ml; 120 ml; 90 ml 3: 3 vials of Ethanol solution: 100 ml; 80 ml; 110 ml 4: Detergent 5: Instructions for use: catheter, extraction methods and formula

(13) The foregoing examples are not intended to limit the scope of the invention. All modifications, equivalents and alternatives are within the scope of the invention. As an example, therapeutic agents other than Doxorubicin may be filtered from a patient's blood, the filtered agent extracted and quantified, and the lifetime dosage of the agent tracked.