WO2012090909A1 - 脂溶性の測定方法 - Google Patents
脂溶性の測定方法 Download PDFInfo
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- WO2012090909A1 WO2012090909A1 PCT/JP2011/080018 JP2011080018W WO2012090909A1 WO 2012090909 A1 WO2012090909 A1 WO 2012090909A1 JP 2011080018 W JP2011080018 W JP 2011080018W WO 2012090909 A1 WO2012090909 A1 WO 2012090909A1
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- the present invention relates to a technique for measuring fat solubility of a drug.
- the present invention relates to a high-throughput drug lipid solubility measurement technique.
- fat solubility is an important molecular property that affects oral absorption, membrane permeability, solubility, volume of distribution, metabolic stability, and the like.
- Distribution parameters such as logP (partition coefficient) and logD (distribution coefficient) are widely used as parameters indicating fat solubility, and a flask shaking method is generally used as an actual measurement method.
- the flask shaking method is a method in which a test substance and two kinds of solvents are placed in a flask and shaken, and then the amount of the test substance contained in the organic phase and the aqueous phase is quantified to obtain a distribution coefficient.
- Distribution coefficient measurement methods are also defined in JIS standards (JIS Z7260-107, OECD Test Guideline 107).
- the HPLC method exists as a simpler system.
- the HPLC method is a method of indirectly measuring the partition coefficient of a test substance by utilizing the correlation between the retention time and the distribution coefficient of reversed-phase chromatography.
- the distribution coefficient of the test substance is determined using the correlation.
- the flask shaking method can accurately evaluate the fat solubility, the operation is complicated and it is not suitable for multi-sample treatment.
- the HPLC method can process many samples compared to the flask shaking method, but it cannot be applied to a compound having low solubility in water or a compound that reacts with a column carrier, and has a known distribution coefficient. If the chemical structures of the substance and test substance are significantly different, the partition coefficient cannot be measured accurately.
- Patent Document 1 As a method for measuring the fat solubility of a test substance quickly and in large quantities, Patent Document 1, Non-Patent Documents 1 and 2 propose a method for measuring fat solubility using a multiwell plate.
- fat solubility is measured by quantifying three types of the test substance contained in the organic phase and the test substance contained in the aqueous phase: only the organic phase, only the aqueous phase, and both phases. A method of adopting either value is described.
- the flask shaking method can evaluate the fat solubility relatively accurately, but the operation is complicated, and after distributing the sample, the two phases are taken out separately by the layer separation operation and quantified by HPLC or the like. Since multi-step manual operation is required, measurement takes time and is not suitable for multi-sample simultaneous processing.
- the amount of solvent is reduced, and the process is automated using a multiwell plate such as 96-well, thereby measuring the fat solubility by flask shaking method.
- a multiwell plate such as 96-well
- the amount of the solvent is reduced, the measurement accuracy of fat solubility is likely to be lowered, and it is difficult to perform high-throughput fat solubility measurement for a low fat-soluble substance having a log D of less than 1 or a high fat-soluble substance having a log D of more than 3. there were.
- an object of the present invention is to provide a method for quickly and accurately measuring fat solubility of a wide range of substances.
- the inventor of the present invention measured the fat solubility using two or more types of solvent systems in which the ratio of the hydrophobic solvent to the hydrophilic solvent was changed. The inventors have found that the solubility can be measured accurately and quickly, and have completed the present invention.
- a method for measuring fat solubility of a test substance comprising: (1) mixing a predetermined amount of a test substance in a first solvent system comprising a hydrophobic solvent and a hydrophilic solvent in a first ratio; 2) mixing a predetermined amount of a test substance with a second solvent system comprising the same hydrophobic solvent and hydrophilic solvent as the first solvent system in a second ratio; and (3) the first solvent system. Measuring the test substance contained in the hydrophobic phase or hydrophilic phase of the first solvent system after reaching the distribution equilibrium; and (4) the first solvent after the second solvent system reaches the distribution equilibrium.
- An apparatus for measuring fat solubility of a test substance a dispenser for dispensing a hydrophobic solvent and a hydrophilic solvent into a container at a first ratio and a second ratio, and a test substance
- a computer for calculating the fat solubility of the test substance from the ratio of the neutral solvent and the measurement result of the test substance.
- the fat solubility of a substance can be measured quickly and accurately.
- FIG. 1 is a graph comparing log D (Example) measured according to the present invention and log D (Comparative Example) measured by a flask shaking method.
- FIG. 2 is a graph comparing log D (Example) measured by the present invention and log D (Comparative Example) measured by the flask shaking method for 14 commercially available compounds.
- the present invention is a method for measuring the fat solubility of a test substance, wherein (1) a predetermined amount of a test substance is mixed in a first solvent system comprising a hydrophobic solvent and a hydrophilic solvent in a first ratio. A first step; (2) a step of mixing a predetermined amount of a test substance in a second solvent system comprising a hydrophobic solvent and a hydrophilic solvent in a second ratio; and (3) a first solvent system. Measuring the test substance contained in the hydrophobic phase or hydrophilic phase of the first solvent system after reaching the distribution equilibrium; and (4) the first solvent after the second solvent system reaches the distribution equilibrium. A step of measuring a test substance contained in a phase different from the phase measured for the system, and (5) a step of calculating the fat solubility of the test substance based on the results obtained in the third and fourth steps, including.
- the substance whose fat solubility is measured is any chemical or biological substance, and may be, for example, an organic compound, protein, peptide, or nucleic acid.
- the substance for measuring fat solubility according to the present invention include polycyclic aromatic or aliphatic hydrocarbon, halogen-containing aromatic or aliphatic hydrocarbon, nitrogen and oxygen-containing aromatic or aliphatic hydrocarbon, and fat-soluble.
- Vitamins and the like can be mentioned, and among them, compounds that are acceptable as active ingredients of pharmaceuticals, in particular, low molecular compounds are preferable, compounds having a molecular weight of 1000 or less are more preferable, and nitrogen and oxygen-containing aromatic or aliphatic hydrocarbons Further, a compound having a molecular weight of 1000 or less is more preferable.
- the substance whose fat solubility is measured may be solid or liquid, and may be dissolved in a suitable solvent such as DMSO (dimethyl sulfoxide).
- DMSO dimethyl sulfoxide
- the fat solubility of the substance to be measured according to the present invention is not particularly limited, and according to the present invention, it is possible to measure from a low fat soluble substance having a log D of ⁇ 1 or less to a highly fat soluble substance having a log D of 5 or more. .
- even a highly lipid-soluble substance having a log D of more than 3 and a low fat-soluble substance having a log D of less than 1 can accurately measure the fat solubility.
- the test substance is a compound having a log D of ⁇ 2 to 6, another embodiment is a compound of ⁇ 2 to 5, and another embodiment is a compound of ⁇ 1 to 5.
- fat solubility is a parameter indicating the hydrophobicity of a substance, and specifically includes partition coefficients such as logD and logP.
- partition coefficients such as logD and logP.
- the concentration ratio of the substance distributed between the hydrophilic phase and the hydrophobic phase becomes constant regardless of the amount of the test substance. That is, when the concentration of the test substance in the hydrophilic phase is Cw and the concentration of the test substance in the hydrophobic phase is Co, the ratio (Co / Cw) between them is constant, and the common logarithm is called a distribution coefficient.
- logP partition coefficient
- log D distributed coefficient
- log D is a parameter that takes into account changes in fat solubility due to dissociation of molecules in different pH environments.
- the fat solubility is evaluated by log D. It is common.
- log D measured using a buffer adjusted to pH 7.4 is called log D 7.4, and is widely used for evaluating the fat solubility of substances.
- the lipophilicity of a substance is measured using a hydrophobic solvent and a hydrophilic solvent.
- the hydrophobic solvent and the hydrophilic solvent are not miscible or hardly miscible with each other.
- the hydrophobic solvent in the present invention is a so-called nonpolar solvent and is not miscible or hardly miscible with a polar solvent such as water.
- the dielectric constant of a hydrophobic solvent is lower than that of a hydrophilic solvent such as water.
- hydrophobic solvents examples include organic solvents such as 1-octanol (octane-1-ol) and aliphatic hydrocarbons (cyclohexane, dodecane, hexadecane or halogenated hydrocarbons). Particularly, 1-octanol is hydrophobic. It is preferable to use as an ionic solvent.
- organic solvents such as 1-octanol (octane-1-ol) and aliphatic hydrocarbons (cyclohexane, dodecane, hexadecane or halogenated hydrocarbons).
- 1-octanol is hydrophobic. It is preferable to use as an ionic solvent.
- the hydrophilic solvent in the present invention is a so-called polar solvent, which means a solvent having molecules that are electronically charged, and is not miscible or hardly miscible with a hydrophobic solvent.
- the hydrophilic solvent is, for example, an aqueous solution of a buffer salt having a high buffering ability within a certain pH range (that is, a buffer solution, specifically, a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, And 3-N-tris (hydroxymethyl) methylamino-2-hydroxypropanesulfonic acid buffer, etc.).
- the pH range may be in the range of 0 to 14, and a buffer adjusted to have a pH of about 7.4, particularly a phosphate buffer buffered at pH 7.4 is used as the hydrophilic solvent. It is preferable.
- the concentration of the buffer solution can be appropriately selected within a range that does not affect the measurement in terms of buffer capacity and salt precipitation by mixing with a hydrophobic solvent.
- phosphate buffer buffered at pH 7.4 The concentration of the solution (in terms of phosphoric acid) is preferably 0.5 to 20 mM, more preferably 1 to 10 mM, and even more preferably 5 to 10 mM.
- the “ratio of the hydrophobic solvent and the hydrophilic solvent” in the present specification means “the volume ratio of the hydrophobic phase and the hydrophilic phase formed by mixing the hydrophobic solvent and the hydrophilic solvent”.
- “A first solvent system comprising a hydrophobic solvent and a hydrophilic solvent in a first ratio” means that the volume ratio of the hydrophobic phase to the hydrophilic phase formed by mixing the hydrophobic solvent and the hydrophilic solvent is Means a first solvent system with a ratio of 1. Therefore, considering the change in the total volume due to the mixing of the hydrophobic solvent and the hydrophilic solvent and the time required to reach the distribution equilibrium, the hydrophobic solvent and the hydrophilic solvent are sufficiently mixed in advance to saturate each other. It is preferable to prepare a mixed liquid and use the hydrophobic phase as a hydrophobic solvent and the hydrophilic phase as a hydrophilic solvent.
- a predetermined amount of a substance is mixed with a first solvent system comprising a hydrophobic solvent and a hydrophilic solvent in a first ratio, and the same hydrophobic solvent and hydrophilicity as the first solvent system are further mixed.
- a predetermined amount of material is mixed in a second solvent system comprising a solvent in a second ratio.
- the hydrophilic phase can be sampled without separating the hydrophobic phase, and the fat solubility can be measured. Layer separation operations can be avoided.
- the amount of the test substance to be mixed with the first solvent system and the amount of the test substance to be mixed with the second solvent system may be known, and both may be the same or different.
- the first ratio and the second ratio of the hydrophobic solvent and the hydrophilic solvent are not particularly limited as long as they are different.
- the first ratio (volume ratio) of the hydrophobic solvent and the hydrophilic solvent can be 50:50 to 1:99, and 20:80 to 1:99. Is preferably 10:90 to 1:99, more preferably 1:14 to 1:49.
- the ratio of the hydrophobic solvent can be made higher than that in the first solvent system.
- the second ratio (volume ratio) of the hydrophobic solvent to the hydrophilic solvent is 90:10 to 1:99, more preferably 80:20 to 20:80.
- the second ratio (volume ratio) of the hydrophobic solvent and the hydrophilic solvent may be 99: 1 to 50:50, and the hydrophobic solvent may be increased.
- it is preferable to set the second ratio (volume ratio) to 80:20 to 20:80, particularly 55:45 to 45:55, because the measurement accuracy near logD 0 is improved.
- the total amount of the hydrophobic solvent and the hydrophilic solvent is not particularly limited, but is preferably 50 ⁇ L to 10 mL, more preferably 100 ⁇ L to 5 mL, even more preferably 500 ⁇ L to 2 mL, and even more preferably 500 ⁇ L to 500 mL from the viewpoint of performing high-throughput analysis. 1.5 mL is even more preferable, and 800 ⁇ L to 1.2 mL is most preferable.
- the first ratio is 10:90 to 1:99 (more preferably 1:14 to 1:49), with a second ratio of 80:20 to 20:80 (more preferably 55:45 to 45:55), during shaking in the first solvent system and the second solvent system
- the test substance concentration is preferably 2.5 to 20 ⁇ M.
- the test substance contained in the hydrophobic phase or the hydrophilic phase of the first solvent system is measured, and further, the second solvent system reaches the distribution equilibrium. Then, the test substance contained in a phase different from the phase measured for the first solvent system is measured.
- the measurement accuracy can be improved by making the phase for measuring the test substance different between the first solvent system and the second solvent system.
- the test substance in the hydrophilic phase is measured for the first solvent system, and the test substance in the hydrophobic phase is measured for the second solvent system.
- the first ratio is 10:90 to 1:99 (more preferably 1:14 to 1:49)
- the second When the ratio of the solvent is 80:20 to 20:80 (more preferably 55:45 to 45:55) and the total amount of the hydrophilic solvent and the hydrophobic solvent is 500 ⁇ L to 2 mL, high-throughput and high-precision analysis is performed. Preferred above.
- the test substance in the hydrophobic phase and / or the hydrophilic phase is measured after reaching the partition equilibrium.
- distribution equilibrium is reached after a lapse of a certain time from the addition of the test substance, but the time to reach distribution equilibrium may be shortened by stirring and shaking.
- the time to reach the distribution equilibrium depends on the amount of the solvent and the like, but is, for example, 1 minute to 24 hours, preferably 10 minutes to 12 hours, more preferably 30 minutes to 3 hours.
- a solvent and a test substance can be put into a container by a known method and taken out from the container. That is, a known dispenser (dispenser), a sampler (sampler), or the like can be used, and these operations can be automated.
- a known dispenser dispenser
- a sampler sampler
- the method of the present invention is particularly suitable for high-throughput analysis because it can be easily automated using standard equipment.
- the method for measuring a test substance is not particularly limited, and a known method can be used. Specifically, measurement with an absorbance detector such as UV, liquid chromatography (LC), gas chromatography (GC), capillary electrophoresis (CE), etc., and measurement with a combination of these preferred detectors For example, but not limited to.
- the detector for liquid chromatography (LC) include an absorbance detector such as UV, a mass spectrometer (MS), and / or a conductivity detector. For high-throughput and high-precision analysis, LC / MS measurement may be preferable.
- a hydrophilic phase test substance and a hydrophobic phase test substance in a plurality of solvent systems having different phase ratios are measured, and the fat solubility of the test substance is calculated using the following formula I based on the result.
- (A ⁇ C 1 ⁇ D + b ⁇ C 1 ) / X (c ⁇ C 2 + d ⁇ C 2 / D) / Y (Formula I)
- D distribution ratio
- X amount of test substance added to the first solvent system
- Y amount of test substance added to the second solvent system
- C 1 concentration of test substance in the hydrophilic phase in the first solvent system
- C 2 second Concentration of test substance in the hydrophobic phase in the solvent system
- a amount of hydrophobic solvent in the first solvent system (volume of the hydrophobic phase)
- b Amount of hydrophilic solvent in the first solvent system (volume of hydrophilic phase)
- c amount of hydrophobic solvent in the second solvent system (volume of hydrophobic phase)
- d
- concentrations C 1 and C 2 in the above formula values correlating with the concentrations may be used instead. Specifically, it is a liquid chromatography or LC / MS peak area value.
- the distribution ratio D is calculated using the above equation, and log D or log P is obtained by calculating the common logarithm as necessary.
- the present invention relates to an apparatus for measuring the fat solubility of a test substance. That is, the present invention is an apparatus for measuring the fat solubility of a test substance, and dispenser for dispensing a hydrophobic solvent and a hydrophilic solvent into a container at a first ratio and a second ratio.
- a hydrophobic phase or a hydrophilic phase from a first solvent system containing a first ratio of a hydrophobic solvent and a hydrophilic solvent in a container at a first ratio
- Sampling a phase different from the phase sampled in the first solvent system from a second solvent system comprising a dispenser for diluting with a solvent and a second ratio of hydrophobic and hydrophilic solvents;
- a second solvent system comprising a hydrophobic solvent and a hydrophilic solvent in a second ratio from the second solvent system to the first solvent system.
- a computer is provided for calculating the fat solubility of the test substance from the measurement result.
- the apparatus of the present invention may further include a thermostatic chamber for maintaining the container at a constant temperature, and in order to promote the test substance to reach distribution equilibrium, a shaker for shaking the container, It is preferable to further include a centrifuge for separating the hydrophobic phase and the hydrophilic phase into layers.
- Each part constituting the apparatus of the present invention is preferably automated so that a series of analyzes are automatically performed.
- a well plate such as a multiwell plate as a container.
- the ratio of the solvent system and the container capacity (area) should be selected.
- the amount of the organic phase per 1 mm 2 of the liquid surface area in the well is preferably 0.5 to 2 ⁇ L, more preferably 1 ⁇ L.
- the well volume is preferably 50 ⁇ L to 10 mL, more preferably 100 ⁇ L to 5 mL, and the cross-sectional area of the well on the liquid surface is preferably 10 mm 2 to 500 mm 2. 20 mm 2 to 100 mm 2 is more preferable.
- a material of a container such as a plate, polypropylene or glass is suitable.
- Example 1 log D 7.4 was measured for 38 compounds of the lipid solubility measurement test target according to the present invention.
- the solution amount per well was 1 mL, and a phosphate buffer buffered with 1-octanol as a hydrophobic solvent and pH 7.4 as a hydrophilic solvent was used.
- Measurement is 96 deep well plate (Gleiner, model number 780270: inner dimensions of the upper part of the well are 8.2 mm in length and 8.2 mm in width, the inner dimension of the lower part of the well is 7.37 mm in both length and width, and the well depth is 41 mm. , Volume 2 mL, made of polypropylene). Operations such as dispensing, shaking, and dilution were automated by a dispensing robot (Hamilton, MicroLabSTARlet), and the movement of the plate from apparatus to apparatus was performed manually. The specific experimental procedure is as follows.
- phosphate buffer pH 7.4 Dulbecco reagent (for 1 L of PBS solution manufactured by SIGMA: 8 g of sodium chloride, 1.15 g of sodium monohydrogen phosphate, 0.2 g of potassium chloride, anhydrous phosphoric acid) The volume was adjusted to 1 L (containing 0.2 g of potassium dihydrogen), and then stirred, and unnecessary substances were filtered off. The pH of the solution was measured and adjusted to pH 7.4 with 1N HCl.
- (2) Preparation of saturated 1-octanol and saturated phosphate buffer Add sufficient amounts of 1-octanol (Kanto Chemical: for partition coefficient measurement) and phosphate buffer prepared in (1) to the separatory funnel.
- the lower phase (aqueous phase) was extracted from the first well, and a sample diluted 10-fold with methanol was prepared.
- the upper phase (1-octanol phase) was extracted from the second well, and a sample diluted 10-fold with methanol and a sample diluted 50-fold were prepared.
- Nitrogen gas was used as the cone gas, the flow rate was measured at 50 L / h, and the desolvation gas flow rate was measured at 750 L / h.
- the source temperature was set to 130 ° C and the solvent removal temperature was set to 350 ° C.
- the capillary voltage was set to 3.5 kV. (6)
- the lower phase (no dilution) sample of the first well and the lower phase of the first well are diluted 10-fold with methanol, and the upper phase of the second well is diluted with methanol.
- samples with a large peak area (samples with a low dilution rate) of each sample obtained by LC / MS measurement were selected for the upper phase and the lower phase, respectively.
- C 2 / C 1 was calculated from the area value and the dilution rate, and the log D was automatically calculated by substituting it into the formula (II).
- the peak area of a sample with a low dilution rate is remarkably large and the LC / MS quantification is considered to be in a low range (in this test condition, the peak area value is 750,000 or more)
- the dilution rate A large sample was selected and its peak area was used for LogD calculation.
- sample to be measured (partially half of microspatel) is placed in a 10 mL screw centrifuge tube and saturated phosphoric acid when LogD ⁇ 0, using physical property prediction software represented by ACD / Labs (Fujitsu). About 6 mL of buffer solution was added, and when Log D ⁇ 0, about 6 mL of saturated 1-octanol was added. Ultrasonic irradiation was performed for several tens of seconds to disperse the sample to obtain a mixed solution.
- the screw centrifuge tube was vigorously shaken for 10 minutes and then centrifuged at 3000 rpm for 10 minutes.
- the gradient conditions were as follows: 0-0.5 minutes: solution B 10-50%, 0.5-5 minutes: 50-100%, 5-7 minutes: 100%.
- the flow rate was 0.3 mL / min.
- HPLC used LC1100 made from Agilent which has a binary pump.
- As the column stationary phase a reverse phase column (Imtakt Cadenza CD-C18 2.0ID ⁇ 50 mm) having an octadecyl group was used.
- the column temperature was 40 ° C.
- MS used 1946D made by Agilent.
- the Drying Gas Flow was set to 12.0 L / min, the Drying Gas Temp was set to 350 ° C., and the Capillary Voltage was set to 3000V.
- the detection mode was ESI +, and the detected ions were optimized by the sample to acquire data. Typical ions were [M + H] + and [MH] +.
- the fragmentor voltage was measured by optimizing the sample. Optimization was performed in the range of 40 to 200 V, and measurements were typically made at 80 V and 100 V.
- (6) In the LogD calculation, samples with a large peak area (samples with low dilution rate) obtained by LC / MS measurement are selected for the upper phase and the lower phase, respectively, and the peak area is calculated for LogD. Used (area of 1-octanol phase ⁇ dilution ratio / area value of aqueous phase ⁇ dilution ratio distribution ratio, LogD was calculated by taking the common logarithm of this distribution ratio).
- the contamination of the hydrophobic phase at the time of sampling is suppressed,
- the concentration of the test substance contained in the hydrophilic phase could be improved, and as a result, the fat solubility could be measured with high accuracy.
- the fat solubility of a substance can be measured quickly and accurately.
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Abstract
Description
1. 被験物質の脂溶性を測定する方法であって、(1)疎水性溶媒と親水性溶媒を第1の比率で含んでなる第1の溶媒系に所定量の被験物質を混合する工程と、(2)第1の溶媒系と同じ疎水性溶媒と親水性溶媒を第2の比率で含んでなる第2の溶媒系に所定量の被験物質を混合する工程と、(3)第1の溶媒系が分配平衡に達した後、第1の溶媒系の疎水相または親水相に含まれる被験物質を測定する工程と、(4)第2の溶媒系が分配平衡に達した後、第1の溶媒系について測定した相とは別の相に含まれる被験物質を測定する工程と、(5)第3および第4の工程で得られた二つの値に基づいて被験物質の脂溶性を算出する工程と、を含む上記方法。
2. 疎水性溶媒と親水性溶媒との第1の比率が50:50~1:99(容積比)である、上記1に記載の方法。
3. 疎水性溶媒と親水性溶媒との第2の比率が80:20~20:80(容積比)である、上記1または2に記載の方法。
4. 疎水性溶媒が1-オクタノールであり、親水性溶媒が緩衝液である、上記1~3のいずれかに記載の方法。
5. 第1から第5の工程を自動で行う、上記1~4のいずれかに記載の方法。
6. logD測定値が-2~6の被験物質に用いるための、上記1~5のいずれかに記載の方法。
7. 以下の式:
(a×C1×D+b×C1)/X=(c×C2+d×C2/D)/Y (式I)
式中、D=分配比、
X=第1の溶媒系に添加した被験物質の量
Y=第2の溶媒系に添加した被験物質の量
C1=第1の溶媒系における親水相中の被験物質の濃度
C2=第2の溶媒系における疎水相中の被験物質の濃度
a=第1の溶媒系における疎水性溶媒の量(疎水相の容積)
b=第1の溶媒系における親水性溶媒の量(親水相の容積)
c=第2の溶媒系における疎水性溶媒の量(疎水相の容積)
d=第2の溶媒系における親水性溶媒の量(親水相の容積)
に基づいて被験物質の脂溶性を算出する、上記1~6のいずれかに記載の方法。
8. 被験物質の脂溶性を測定するための装置であって、疎水性溶媒と親水性溶媒とを第1の比率および第2の比率で容器に分注するための分注機と、被験物質を分注するための分注機と、疎水性溶媒と親水性溶媒とを第1の比率で含む第1の溶媒系から疎水相または親水相をサンプリングし、疎水性溶媒と親水性溶媒とを第2の比率で含む第2の溶媒系から第1の溶媒系でサンプリングした相とは別の相をサンプリングするための分注機と、被験物質を測定するための分析機と、疎水性溶媒と親水性溶媒の比率および被験物質の測定結果から被験物質の脂溶性を算出するため計算機とを含む、上記装置。
9. 以下の式:
(a×C1×D+b×C1)/X=(c×C2+d×C2/D)/Y (式I)
式中、D=分配比、
X=第1の溶媒系に添加した被験物質の量
Y=第2の溶媒系に添加した被験物質の量
C1=第1の溶媒系における親水相中の被験物質の濃度
C2=第2の溶媒系における疎水相中の被験物質の濃度
a=第1の溶媒系における疎水性溶媒の量(疎水相の容積)
b=第1の溶媒系における親水性溶媒の量(親水相の容積)
c=第2の溶媒系における疎水性溶媒の量(疎水相の容積)
d=第2の溶媒系における親水性溶媒の量(親水相の容積)
に基づいて被験物質の脂溶性を算出する、上記8に記載の装置。
(a×C1×D+b×C1)/X=(c×C2+d×C2/D)/Y (式I)
式中、D=分配比、
X=第1の溶媒系に添加した被験物質の量
Y=第2の溶媒系に添加した被験物質の量
C1=第1の溶媒系における親水相中の被験物質の濃度
C2=第2の溶媒系における疎水相中の被験物質の濃度
a=第1の溶媒系における疎水性溶媒の量(疎水相の容積)
b=第1の溶媒系における親水性溶媒の量(親水相の容積)
c=第2の溶媒系における疎水性溶媒の量(疎水相の容積)
d=第2の溶媒系における親水性溶媒の量(親水相の容積)
A.本発明による脂溶性の測定
試験対象の38化合物について本発明によりlogD7.4を測定した。1ウェルあたりの溶液量を1mLとし、疎水性溶媒として1-オクタノール、親水性溶媒としてpH7.4で緩衝化したリン酸緩衝液を用いた。また、第1の溶媒系および第2の溶媒系に添加する被験物質の量は同じとした(式IのX=Y)。第1の溶媒系は、1-オクタノール:リン酸緩衝液=5:95(容積比)とし、リン酸緩衝液相(以下、水相)に分配された被験物質を液体クロマトグラフィーおよび質量分析(LC/MS)によって測定した。第2の溶媒系は、1-オクタノール:リン酸緩衝液=50:50(容積比)とし、1-オクタノール相(疎水相)に分配された被験物質を同様に測定した。
5×C1×D+95×C1=50×C2+50×C2/D
D2+(19-10C2/C1)D-10C2/C1=0 (式II)
(1) リン酸緩衝液(pH7.4)の調製
ダルベッコ試薬(SIGMA製PBS溶液1L用:塩化ナトリウム8g、無水リン酸一水素ナトリウム1.15g、塩化カリウム0.2g、無水リン酸二水素カリウム0.2gを含む)を1Lにメスアップし、その後、撹拌し、不要物をろ別した。溶液のpHを測定し、1N HClでpH7.4に調整した。
(2) 飽和1-オクタノールと飽和リン酸緩衝液の調製
1-オクタノール(関東化学:分配係数測定用)と(1)で調製したリン酸緩衝液とをそれぞれ十分な量ずつ分液漏斗に加え、十分に混合した後、室温で一晩静置した。上層と下層を取り分けて、リン酸緩衝液飽和1-オクタノール(以下、飽和1-オクタノール)と1-オクタノール飽和リン酸緩衝液(以下、飽和リン酸緩衝液)を得た。それぞれの溶液は室温で保存した。
(3) 測定化合物のDMSO溶液
測定化合物をDMSOに添加し、10mMの溶液を調製した。
(1) 測定化合物の10mM DMSO溶液を96ディープウェルプレートに10μL分注し、飽和1-オクタノール490μLをさらに分注した。吸引吐出による撹拌を10回行い、測定化合物のオクタノール標準液を調製した。
(2) オクタノール標準液50μLを2つのウェルに分注し、1つ目のウェルには、飽和リン酸緩衝液950μLを分注し(疎水性溶媒:親水性溶媒=5:95)、2つ目のウェルには、飽和リン酸緩衝液500μLと飽和1-オクタノール450μLを分注した(疎水性溶媒:親水性溶媒=50:50)。
(3) マルチウェルプレートに圧力硬化シールを接着後、振盪器にて25℃で3時間振盪した。振盪後、3000rpmで5分間遠心処理した。
(4) 1つ目のウェルから下相(水相)を抜き取り、メタノールで10倍希釈したサンプルを調製した。2つ目のウェルから上相(1-オクタノール相)を抜き取り、メタノールで10倍希釈したサンプルと、50倍希釈したサンプルを調製した。1つ目のウェルの下相(希釈なし)と下相の10倍希釈サンプル、2つ目のウェルの上相の10倍希釈サンプルと50倍希釈サンプルの計4サンプルについて、LC/MSにより測定化合物を測定した。
上記4サンプルをLC/MSのオートサンプラーにセットし、自動的に測定した。
(1) 2Lのメスフラスコに適当量の超純水(ミリポア社の超純水製造装置により調製)を加えた後、ホールピペットを用いてギ酸(関東化学)2mLを滴下した。その後、超純水でメスアップし、転倒混和を行った。移動相用の瓶に移し、超音波処理を10分間行い脱気して、0.1%ギ酸水溶液を調製した。
(2) 1Lのメスフラスコに適当量のアセトニトリル(和光純薬製)を加えた後、ホールピペットを用いてギ酸(関東化学)1mLを滴下した。その後、アセトニトリルでメスアップし、転倒混和を行った。移動相用の瓶に移し、超音波処理を10分間行い脱気して、0.1%ギ酸アセトニトリル溶液を調製した。
(3) HPLCの移動相として、0.1%ギ酸水溶液(A液)および0.1%ギ酸アセトニトリル溶液(B液)を用いた。グラジエント条件は、0~0.3分:B液10%、0.3~1.5分:B液10~95%、1.5~2.25分:B液95%、2.25~3.0分:B液10%とした。流速は0.6mL/分とした。
(4) HPLCは、バイナリーポンプを有するWaters社製Acquityを使用した。カラム固定相として、オクタデシル基を有する逆相カラム(Acquity BEH C18、1.7μm、2.0×30mm)を用いた。カラム温度は50℃で実施した。
(5) MSはWaters社製ZQ又はSQDを使用した。検出モードはESI+で,検出イオンは[M+H]+でデータを取得した。コーン電圧は25Vで測定した。コーンガスとして窒素ガスを用い,流量は50L/hで測定し,脱溶媒ガスの流量は750L/hで測定した。またソース温度は130℃に,脱溶媒温度は350℃に設定した。キャピラリー電圧は3.5kVに設定した。
(6) LogDの計算では、1つ目のウェルの下相(希釈なし)サンプルと1つ目のウェルの下相をメタノールで10倍希釈したサンプル、2つ目のウェルの上相をメタノールで10倍希釈したサンプルと50倍希釈したサンプルについて、LC/MS測定で得られた各サンプルのピーク面積が大きいサンプル(希釈率の低いサンプル)を、上相と下相でそれぞれ選択し、そのピーク面積値と希釈率からC2/C1を算出し、それを式(II)に代入にしてlogDを自動算出した。ただし、希釈率の低いサンプルでのピーク面積が著しく大きく、LC/MSの定量性が低い範囲にあると考えられる場合(本試験条件においては、ピーク面積値が750000以上の場合)は、希釈率の大きいサンプルを選択して、そのピーク面積をLogD算出に用いた。
B.フラスコ振盪法による脂溶性の測定
以下の手順によりフラスコ振盪法により脂溶性を測定した。
Aと同様にして、溶液を調製した。
試料を適量(ミクロスパーテルに半分くらい)とり、10mLのネジ口遠沈管に入れ、ACD/Labs(富士通)に代表される物性値予測ソフトにより、LogD<0の場合は飽和リン酸緩衝液を約6mL加え、LogD≧0の場合は飽和1-オクタノールを約6mL加えた。超音波照射を数10秒実施し、試料を分散させ、混合液とした。
上記8サンプルをLC/MSのオートサンプラーにセットし、自動的に測定した。
(1) 2Lのメスフラスコに10mMとなるように酢酸アンモニウム(和光純薬製)約1.5gを秤量し、超純水(ミリポア社の超純水製造装置により調製)でメスアップし、転倒混和を行った。移動相用の瓶に移し、超音波処理を10分間行い脱気して、10mM酢酸アンモニウム水溶液を調製した。
(2) 2Lのメスフラスコに10mMとなるように酢酸アンモニウム(和光純薬製)約1.5gを秤量し、メタノール(和光純薬製)でメスアップし、転倒混和を行った。移動相用の瓶に移し、超音波処理を10分間行い脱気して、10mM酢酸アンモニウムアセトニトリル溶液を調製した。
(3) HPLCの移動相として、10mM酢酸アンモニウム水溶液(A液)および10mM酢酸アンモニウムメタノール溶液(B液)を用いた。グラジエント条件は、0―0.5分:B液10~50%、0.5―5分:50~100%、5―7分:100%とした。流速は0.3mL/分とした。
(4) HPLCは、バイナリーポンプを有するAgilent社製LC1100を使用した。カラム固定相として、オクタデシル基を有する逆相カラム(Imtakt Cadenza CD―C18 2.0ID×50mm)を用いた。カラム温度は40℃で実施した。
(5) MSはAgilent社製1946Dを使用した。Drying Gas Flowは12.0L/分、Drying Gas Tempを350℃、Capillary Voltageを3000Vに設定した。検出モードはESI+で、検出イオンはサンプルにより最適化を行いデータを取得した。代表的なイオンは[M+H]+や[M-H]+であった。フラグメンター電圧はサンプルにより最適化を行い測定した。40~200Vの範囲で最適化を行い代表的には80Vや100Vで測定した。
(6) LogDの計算では、LC/MS測定で得られた各サンプルのピーク面積が大きいサンプル(希釈率の低いサンプル)を、上相と下相でそれぞれ選択し、そのピーク面積をLogD算出に用いた(1-オクタノール相の面積×希釈倍率/水相の面積値×希釈倍率=分配比、この分配比の常用対数をとることでLogDを算出)。ただし、希釈率の低いサンプルでのピーク面積が著しく大きく、LC/MSの定量性が低い範囲にあると考えられる場合は、希釈率の大きいサンプルを選択して、そのピーク面積をLogD算出に用いた。
市販の14化合物の他、表1に化合物1~24として示した24化合物についても測定を行った。表1に測定したlogDの値を示す。96ウェルのマルチウェルプレートを用いて上記測定を行うことにより、38化合物のlogD7.4を約13時間で測定することができ、本発明によって極めてハイスループットな分析が可能になった。
Claims (9)
- (1) 疎水性溶媒と親水性溶媒を第1の比率で含んでなる第1の溶媒系に所定量の被験物質を混合する工程と、
(2) 第1の溶媒系と同じ疎水性溶媒と親水性溶媒を第2の比率で含んでなる第2の溶媒系に所定量の被験物質を混合する工程と、
(3) 第1の溶媒系が分配平衡に達した後、第1の溶媒系の疎水相または親水相に含まれる被験物質を測定する工程と、
(4) 第2の溶媒系が分配平衡に達した後、第1の溶媒系について測定した相とは別の相に含まれる被験物質を測定する工程と、
(5) 第3および第4の工程で得られた二つの値に基づいて被験物質の脂溶性を算出する工程と、
を含む、被験物質の脂溶性を測定する方法。 - 疎水性溶媒と親水性溶媒との第1の比率が50:50~1:99(容積比)である、請求項1に記載の方法。
- 疎水性溶媒と親水性溶媒との第2の比率が80:20~20:80(容積比)である、請求項1または2に記載の方法。
- 疎水性溶媒が1-オクタノールであり、親水性溶媒が緩衝液である、請求項1~3のいずれかに記載の方法。
- 第1から第5の工程を自動で行う、請求項1~4のいずれかに記載の方法。
- logD測定値が-2~6の被験物質に用いるための、請求項1~5のいずれかに記載の方法。
- 以下の式:
(a×C1×D+b×C1)/X=(c×C2+d×C2/D)/Y (式I)
式中、D=分配比、
X=第1の溶媒系に添加した被験物質の量
Y=第2の溶媒系に添加した被験物質の量
C1=第1の溶媒系における親水相中の被験物質の濃度
C2=第2の溶媒系における疎水相中の被験物質の濃度
a=第1の溶媒系における疎水性溶媒の量(疎水相の容積)
b=第1の溶媒系における親水性溶媒の量(親水相の容積)
c=第2の溶媒系における疎水性溶媒の量(疎水相の容積)
d=第2の溶媒系における親水性溶媒の量(親水相の容積)
に基づいて被験物質の脂溶性を算出する、請求項1~6のいずれかに記載の方法。 - 疎水性溶媒と親水性溶媒とを第1の比率および第2の比率で容器に分注するための分注機と、
被験物質を分注するための分注機と、
疎水性溶媒と親水性溶媒とを第1の比率で含む第1の溶媒系から疎水性溶媒相または親水性溶媒相をサンプリングし、疎水性溶媒と親水性溶媒とを第2の比率で含む第2の溶媒系から第1の溶媒系でサンプリングした相とは別の相をサンプリングするための分注機と、
被験物質を測定するための分析機と、
疎水性溶媒と親水性溶媒の比率および被験物質の測定結果から被験物質の脂溶性を算出するため計算機と、
を含む、被験物質の脂溶性を測定するための装置。 - 以下の式:
(a×C1×D+b×C1)/X=(c×C2+d×C2/D)/Y (式I)
式中、D=分配比、
X=第1の溶媒系に添加した被験物質の量
Y=第2の溶媒系に添加した被験物質の量
C1=第1の溶媒系における親水相中の被験物質の濃度
C2=第2の溶媒系における疎水相中の被験物質の濃度
a=第1の溶媒系における疎水性溶媒の量(疎水相の容積)
b=第1の溶媒系における親水性溶媒の量(親水相の容積)
c=第2の溶媒系における疎水性溶媒の量(疎水相の容積)
d=第2の溶媒系における親水性溶媒の量(親水相の容積)
に基づいて被験物質の脂溶性を算出する、請求項8に記載の装置。
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JP2015017813A (ja) * | 2013-07-09 | 2015-01-29 | 第一三共株式会社 | 自動分注機を用いた分配係数測定の完全自動化方法 |
WO2014014926A3 (en) * | 2012-07-19 | 2015-07-16 | Merck Sharp & Dohme Corp. | Magnetic particle separator |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH11344485A (ja) * | 1998-03-30 | 1999-12-14 | Eisai Co Ltd | 脂溶性薬物の溶出試験法 |
JP2006267105A (ja) * | 2005-03-21 | 2006-10-05 | F Hoffmann La Roche Ag | 担体媒介分配システム(camdis) |
-
2011
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---|---|---|---|---|
JPH11344485A (ja) * | 1998-03-30 | 1999-12-14 | Eisai Co Ltd | 脂溶性薬物の溶出試験法 |
JP2006267105A (ja) * | 2005-03-21 | 2006-10-05 | F Hoffmann La Roche Ag | 担体媒介分配システム(camdis) |
Non-Patent Citations (3)
Title |
---|
ALELYUNAS Y.W. ET AL.: "A high throughput dried DMSO LogD lipophilicity measurement based on 96-well shake-flask and atmospheric pressure photoionization masss pectrometry detection", J CHROMATOGR A, vol. 1217, no. 12, March 2010 (2010-03-01), pages 1950 - 1955, XP026921330 * |
RYO MIZOGUCHI: "High Throughput LogD Sokutei System no Kochiku", JOURNAL OF PHARMACEUTICAL SCIENCE AND TECHNOLOGY, vol. 69, 30 April 2009 (2009-04-30), JAPAN, pages 211, XP008170366 * |
STOPHER D. ET AL.: "An improved method for the determination of distribution coefficients", J PHARM PHARMACOL, vol. 42, no. 2, February 1990 (1990-02-01), pages 144, XP055119462 * |
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WO2014014926A3 (en) * | 2012-07-19 | 2015-07-16 | Merck Sharp & Dohme Corp. | Magnetic particle separator |
JP2015017813A (ja) * | 2013-07-09 | 2015-01-29 | 第一三共株式会社 | 自動分注機を用いた分配係数測定の完全自動化方法 |
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