CN114323894A - Method for analyzing medicine in tissue - Google Patents
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- 238000004445 quantitative analysis Methods 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 4
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- 239000013062 quality control Sample Substances 0.000 claims description 35
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- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 13
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 13
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims description 13
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims description 13
- 239000008103 glucose Substances 0.000 claims description 10
- 238000003908 quality control method Methods 0.000 claims description 10
- 230000008014 freezing Effects 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 9
- 238000011161 development Methods 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000012417 linear regression Methods 0.000 claims description 5
- 230000002500 effect on skin Effects 0.000 claims description 4
- 238000000211 autoradiogram Methods 0.000 claims description 3
- 238000011002 quantification Methods 0.000 claims description 3
- 238000007493 shaping process Methods 0.000 claims description 3
- 238000000376 autoradiography Methods 0.000 claims description 2
- 238000012937 correction Methods 0.000 claims description 2
- 238000007405 data analysis Methods 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims 1
- 230000008520 organization Effects 0.000 claims 1
- 210000001519 tissue Anatomy 0.000 abstract description 32
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- 210000004207 dermis Anatomy 0.000 abstract description 6
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- 206010033675 panniculitis Diseases 0.000 abstract description 6
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- 241000282887 Suidae Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
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- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
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Abstract
The invention provides a method for analyzing a drug in a tissue, which adopts a quantitative whole body radioactive self-developing technology (QWBA) and a tissue embedding slice to carry out quantitative analysis and detection on the drug distribution condition in an isolated tissue. The invention avoids transverse slicing after longitudinal slicing, saves experimental time, accurately positions each layer structure of the skin and visually observes the distribution condition of the medicine in the horny layer, the epidermal layer, the dermis layer and the subcutaneous tissue of the skin. Similar effects are also obtained for other tissues.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a method for analyzing a medicine in a tissue.
Background
Transdermal drug delivery refers to a form of drug delivery, usually called transdermal drug delivery system or transdermal therapeutic system, in which the drug is released from a specially designed device and absorbed through the intact skin into the systemic blood system. The main characteristics are as follows:
firstly, the inactivation of the medicine in the gastrointestinal tract and the first pass effect of the liver are avoided;
the blood concentration is stable and can be kept within the effective concentration range for a long time;
thirdly, the irritation of the medicine to the gastrointestinal tract is reduced;
and fourthly, the safety is improved, and the medicine can be stopped at any time.
Structure of the skin: the skin is composed of epidermis, dermis and subcutaneous adipose tissue. The dermis and subcutaneous tissue offer little resistance to penetration of the drug and the capillaries are developed, from which the drug is absorbed into the systemic circulation.
After the conventional skin smearing medicine is applied, the skin tissues of all layers are difficult to separate, all the skin tissues need to be longitudinally sliced and then transversely sliced to select the required part (such as a specific selected dermis layer), the time is consumed, and the sampling cannot be accurately positioned to the required cortex layer. After the skin is smeared and administered, the skin tissue on the longitudinal section is easily polluted by the medicine during longitudinal slicing, and at the moment, the medicine is detected by selecting the homogenate of the required part by using the transverse slice, so that whether the detection result has a polluted part or not and the degree of pollution cannot be judged, and the data result is inaccurate.
In addition to analyzing drug distribution after dermal administration, there is also a need to analyze drug distribution in other stratified tissues, especially those with tumors.
Disclosure of Invention
In order to save the experimental time, accurately position and visually observe the distribution condition of the medicine in the tissue, the invention provides a method for analyzing the medicine in the tissue. The method adopts quantitative whole body radioactive autography technology (QWBA) and tissue embedded slice to carry out quantitative analysis and detection on the distribution condition of the medicine in the isolated tissue.
In some embodiments, the method comprises the following steps:
s1, taking the target tissue acted by the radioactive labeled drug, and freezing and shaping;
s2, embedding the target tissue sample;
s3 sample for standard curveThe preparation of (1): will be provided with14Preparing the C-glucose plasma to a set concentration, adding the prepared C-glucose plasma into a hole wrapped by a sodium carboxymethylcellulose embedding block, freezing and embedding to obtain a sample for the standard curve;
s4, preparation of quality control samples: will be provided with14Preparing the C-glucose to a set concentration by using plasma, adding the prepared C-glucose into a blank hole wrapped by a sodium carboxymethylcellulose embedding block where a target tissue sample is located, freezing and embedding to obtain the quality control sample;
s5, slicing and dehydrating;
s6, exposure and development: after exposure, scanning by a laser scanning imager to obtain a radioactive self-development image;
s7, data analysis: the radioactive concentration in the autoradiogram image obtained in step S6 was quantified using image analysis software.
Further, the sodium carboxymethyl cellulose in the sodium carboxymethyl cellulose embedded block is 2.2-3% of sodium carboxymethyl cellulose (w/v). Preferably, it is 2.5% sodium carboxymethylcellulose (w/v).
In some embodiments, the step S7 includes the steps of:
s7.1, measuring the radioactive concentration of the sample for the standard curve by using a liquid scintillation counter to obtain a theoretical value of the sample for the standard curve; and (3) using the image analysis software to select the unit area gray value of the sample for the standard curve and the theoretical value of the sample for the standard curve for pairing and quantification to obtain a standard curve and a linear regression equation: y is a × x; y represents a unit area gray scale value, x represents a correction density, and a represents a coefficient;
s7.2, measuring the radioactive concentration of the quality control sample by using a liquid scintillation counter to obtain a theoretical value of the quality control sample; using the image analysis software to select the gray value of the quality control sample in unit area, and calculating according to the linear regression equation obtained in the step S7.1 to obtain the corrected concentration of the quality control sample;
s7.3, the acceptance standard of the standard curve and the quality control sample comprises 1) to 3): 1) if the deviation between the corrected concentration of a single quality control sample and the theoretical value of the quality control sample is within an acceptable range, the quality control sample is qualified; 2) at least 2/3 of the quality control sample is acceptable; 3) the determination coefficient of the standard curve is more than or equal to 0.9; the acceptable range is ± 15%; quantitative analysis of the target tissue sample with the image analysis software is performed only if 1) -3) of the strips satisfy the acceptance criteria. In some embodiments, the step S7 further includes the step S7.4 of excluding radioactive residues from the region outside the non-target tissue when the analysis region is circled by the image analysis software.
In some embodiments, a is 1.35030.
In some embodiments, the target tissue is skin; step S1 is to take the skin after the radiolabeled drug is applied by dermal administration, and freeze-set. Further, the step S7 includes a step S7.4 of excluding radioactive residues in the non-stratum corneum region outside the section when the analysis region is circled by the image analysis software. Further, the skin sample obtained in step S1 is put into dry ice for freeze-setting.
In some embodiments, the step S2 of embedding the target tissue sample includes placing the frozen and shaped target tissue obtained in the step S1 in an embedding container with a longitudinal section facing upward. Further, in the step S2 of embedding the target tissue sample, the frozen and shaped target tissue obtained in the step S1 is vertically placed in an embedding container with a longitudinal section facing upward.
In some embodiments, the quantitative range of the standard curve in step S3 is 0.5-8000 nCi/g. Further, the quantitative range of the standard curve in the step S3 is 1-8000 nCi/g.
In some embodiments, the concentration of the quality control sample in step S4 comprises 3 gradients, which are: 2-4 times (preferably 3 times) the lowest concentration of the sample for standard curve in said step 3, 10% ± 2% of the highest concentration of the sample for standard curve in said step 3 and 75% ± 5% of the highest concentration of the sample for standard curve in said step 3.
Preferably, the concentration of the quality control sample in step S4 includes 3 gradients, which are: 3 times the lowest concentration of the sample for the standard curve in step 3, 10% of the highest concentration of the sample for the standard curve in step 3, and 75% of the highest concentration of the sample for the standard curve in step 3.
In some embodiments, the section of the sodium carboxymethyl cellulose embedded block in which the target tissue sample is located in step S5 is a section performed parallel to a longitudinal section of the target tissue.
In some embodiments, the image analysis software in step S7 is AIDA image analysis software.
The invention has the following beneficial effects:
1. the application of QWBA avoids transverse slicing after longitudinal slicing, saves experimental time, accurately positions each layer structure of skin, and visually observes the distribution of the medicine in the horny layer, epidermal layer, dermal layer and subcutaneous tissue of the skin. Similar effects are also obtained for other tissues.
2. The distribution condition of the medicine can be visually observed, the medicine is prevented from polluting the sample during collection, the concentration of the medicine in the sample is prevented from being high, and data distortion is avoided. Namely, when the analysis area is circled by using image analysis software in the step S7.4, the radioactive residues in the non-stratum corneum area on the outer circle of the section can be removed, so that the false high concentration of the sample drug caused by drug pollution on the sample during collection is effectively avoided, and data distortion is avoided.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a standard curve of example 1.
Fig. 2 is an autoradiogram of the drug in a longitudinal section of skin. Where M01 is the animal number, here minipig (minipig) number 01.
Detailed Description
In order to make the technical means, the characteristics, the purposes and the functions of the invention easy to understand, the invention is further described with reference to the specific drawings. However, the present invention is not limited to the following embodiments.
It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention.
The following examples are further described by taking skin drug delivery to detect drug distribution in skin as an example, and other tissues can be treated similarly with reference to skin, and are not described herein again. The quality control sample and the sample to be detected need to be located in the same embedded block, and the plasma of the same kind as the sample to be detected needs to be used as a solvent (for example, the sample to be detected is the skin of a pig, and the solvent of the quality control sample needs to be the plasma of the pig), so as to monitor whether the processing process is accurate and the stability of the instrument analysis.
Example 1
1, administration and sampling: using a radiolabeled compound (14C-compound) were applied to the skin of pigs and dried skin tissue was excised at the indicated time points with a 7mm diameter dermatome to a depth of 7-8 mm. Taking out the skin, placing the skin into a culture dish, and immediately placing the skin into dry ice for freezing and shaping.
2, sample embedding: 2.5% sodium carboxymethylcellulose (w/v) was poured into a rectangular frame, covered only with the bottom plate, and pre-frozen in liquid nitrogen to a semi-frozen state. The skin was removed with forceps with the longitudinal cut facing up, placed vertically into semi-solidified 2.5% sodium carboxymethylcellulose (w/v), added enough 2.5% sodium carboxymethylcellulose (w/v) to cover the skin filling the frame, placed in liquid nitrogen to freeze completely, and then transferred to a freezer at-20 ℃ for overnight freezing.
3 preparation of standard curve: using rat plasma14C-glucose stock solution (from Perkin Elmar) was diluted to the indicated concentration to obtain a sample for a standard curve and added to pre-perforated 2.5% carboxymethyl celluloseAnd (5) freezing and embedding the sodium ascorbate (w/v). The quantitative range of the standard curve is about 1-8000 nCi/g. And (5) measuring the radiation concentration of the standard curve by using a sample through a liquid scintillation counter to obtain a theoretical value. In this example, 10 samples for the calibration curve were set, and the theoretical values thereof were: 0.940, 4.18, 40.8, 82.4, 423, 839, 1711, 3877, 5922, 7994 nCi/g.
4 Quality Control (QC) sample preparation: dilution with Bama miniature pig plasma14And C-glucose is used for preparing QC samples to the specified concentration, (the concentration of the QC samples is divided into three types of low, medium and high, namely 3 times of the lowest concentration of a standard curve, 10% of the highest concentration of the standard curve and 75% of the highest concentration of the standard curve) and the radiation concentration of the standard samples used for preparing QC is measured by a liquid scintillation counter. Three holes with the diameter of 10mm are drilled in parallel in the blank of the sample embedding block by using an electric drill, and then the quality control samples with low, medium and high concentrations are filled in the holes respectively.
5 standard curve and section of specimen embedded block: the embedded blocks of the samples were sectioned using a Leica CM3600 cryomicrotome (Leica Biosystems, Germany) to a slice thickness of about 30 μm. Sections were transferred to tape with section collection tape and dehydrated in a cryomicrotome (-20 ℃) for at least 24 hours.
6, exposure and development: since the development plate (phosphor screen) is recycled, it is necessary to uniformly irradiate the development plate in a screen cleaner for at least 30 minutes before exposure and development to remove the residual latent image or background. The dehydrated dried sample tape (including the quality control sample) and the standard curve sample collection tape were fixed on a developing plate, and the developing plate was pressed tightly in an exposure box and exposed for about 4 days. Autoradiographic images were obtained in a dark room, scanned using an Amersham Typhoon RGB imager.
The concentration of radioactivity in autoradiographic tissue was quantified using AIDA image analysis software (Raytest GmbH, Germany).
7.1 the standard curve data measured by the liquid scintillation counter are as follows: 0.940, 4.18, 40.8, 82.4, 423, 839, 1711, 3877, 5922, 7994nCi/g, which is the theoretical value of the radioactive concentration, is abbreviated as the theoretical value in the present invention. Selection criteria using AIDA softwareThe curve is matched and quantified by the unit area gray value of the sample and the theoretical value measured by the liquid scintillation counter, and a standard curve is made. From the calibration curve (FIG. 1), the calibration concentrations of the samples for calibration curve were 0.980, 3.59, 38.9, 77.3, 472, 845, 1851, 3823, 5927, 7983 nCi/g. The linear regression equation of the standard curve is that y is 1.35030 xx, and y represents the unit area gray scale value and has the unit PSL/mm2(ii) a x represents the corrected concentration in nCi/g; determining the coefficient (r)2) Is 0.9998, and meets the standard curve range.
7.2 the data of the quality control sample measured by the liquid scintillation counter (liquid scintillation counter for short) are as follows: 6007. 883, 3.19nCi/g, as theoretical values. And (3) selecting the unit area grey value of the quality control sample by using AIDA software, and calculating the corrected concentration of the quality control sample to be 6152, 1004 and 3.04nCi/g according to the standard curve.
7.3 Standard Curve and acceptance Standard of QC samples
And analyzing the quality control sample in the autoradiography image obtained by scanning the section. When the corrected concentration of the section of the quality control sample reaches the following standard, the standard curve and the quality control sample can be judged to be qualified: 1) if the deviation of the corrected concentration of a single quality control sample and the theoretical value (measured value of the liquid scintillation meter) is within the acceptable range (+ -15%), the quality control sample is qualified; 2) at least 2/3 of quality control samples; 3) coefficient of determination (r) of standard curve2) Not less than 0.9. Only if the three standards are completely met, the standard curve and the quality control sample are qualified, and the method can be used for carrying out quantitative analysis on the tissue sample to be detected. If the above standard is not met, a section which is operated in parallel with the target tissue sample is taken again and then analyzed again.
7.4 Using AIDA software to coil selected volumes of stratum corneum, epidermis, dermis, subcutaneous tissue and report radioactive concentrations. The concentrations of the cuticle, epidermis, dermis and subcutaneous tissues of the pigskin 4h after the administration are 2311, 1008, 1.64 and 0.35nCi/g respectively. From the radioactivity image (fig. 2), the distribution of the relevant radioactivity in the stratum corneum and epidermis layers can be visually observed. The outer ring of the image observation has a small amount of radioactive residues, so that the liquid medicine is contaminated when the skin taking device takes the skin downwards. The part of the area is not selected during image selection, so that sample pollution is effectively avoided.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. A method for analyzing medicine in tissue features that the quantitative analysis and detection of medicine distribution in vitro tissue are performed by quantitative whole-body autoradiography and tissue embedded slice.
2. The method of analyzing a drug in tissue of claim 1, comprising the steps of:
s1, taking the target tissue acted by the radioactive labeled drug, and freezing and shaping;
s2, embedding the target tissue sample;
s3, preparation of sample for standard curve: will be provided with14Preparing the C-glucose plasma to a set concentration, adding the prepared C-glucose plasma into a hole wrapped by a sodium carboxymethylcellulose embedding block, freezing and embedding to obtain a sample for the standard curve;
s4, preparation of quality control samples: will be provided with14Preparing the C-glucose to a set concentration by using plasma, adding the prepared C-glucose into a blank hole wrapped by a sodium carboxymethylcellulose embedding block where a target tissue sample is located, freezing and embedding to obtain the quality control sample;
s5, slicing and dehydrating;
s6, exposure and development: after exposure, scanning by a laser scanning imager to obtain a radioactive self-development image;
s7, data analysis: the radioactive concentration in the autoradiogram image obtained in step S6 was quantified using image analysis software.
3. The method for analyzing a drug in a tissue according to claim 2, wherein the step S7 includes the steps of:
s7.1, measuring the radioactive concentration of the sample for the standard curve by using a liquid scintillation counter to obtain a theoretical value of the sample for the standard curve; and (3) using the image analysis software to select the unit area gray value of the sample for the standard curve and the theoretical value of the sample for the standard curve for pairing and quantification to obtain a standard curve and a linear regression equation: y is a × x; y represents a unit area gray scale value, x represents a correction density, and a represents a coefficient;
s7.2, measuring the radioactive concentration of the quality control sample by using a liquid scintillation counter to obtain a theoretical value of the quality control sample; using the image analysis software to select the gray value of the quality control sample in unit area, and calculating according to the linear regression equation obtained in the step S7.1 to obtain the corrected concentration of the quality control sample;
s7.3, the acceptance standard of the standard curve and the quality control sample comprises 1) to 3): 1) if the deviation between the corrected concentration of a single quality control sample and the theoretical value of the quality control sample is within an acceptable range, the quality control sample is qualified; 2) at least 2/3 of the quality control sample is acceptable; 3) the determination coefficient of the standard curve is more than or equal to 0.9; the acceptable range is ± 15%; quantitative analysis of the target tissue sample with the image analysis software is performed only if 1) -3) of the strips satisfy the acceptance criteria.
4. The method for analyzing a drug in a tissue according to claim 3, wherein said step S7 further comprises the step S7.4 of excluding radioactive residues from a region outside the non-target tissue outside the section when the analysis region is circled by said image analysis software.
5. The method of drug analysis in tissue of claim 2, wherein the target tissue is skin; step S1 is to take the skin after the radiolabeled drug is applied by dermal administration, and freeze-set.
6. The method for analyzing a drug in a tissue according to claim 2, wherein the embedding of the target tissue sample in the step S2 is performed by placing the frozen and fixed target tissue obtained in the step S1 in an embedding container with a longitudinal section thereof facing upward.
7. The method of claim 2, wherein the quantification of the standard curve in step S3 is in the range of 0.5 to 8000 nCi/g.
8. The method for analyzing a drug in a tissue according to claim 2, wherein the concentration of the quality control sample in step S4 comprises 3 gradients, which are: 3 times the lowest concentration of the sample for the standard curve in step 3, 10% of the highest concentration of the sample for the standard curve in step 3, and 75% of the highest concentration of the sample for the standard curve in step 3.
9. The method for analyzing a drug in a tissue according to claim 2, wherein the section of the embedded mass of sodium carboxymethyl cellulose in which the target tissue sample is located in step S5 is a section parallel to a longitudinal section of the target tissue.
10. The method for analyzing a drug in an organization according to claim 2, wherein the image analysis software in the step S7 is AIDA image analysis software.
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