JP4902964B2 - Drug metabolism inhibitors and their use - Google Patents

Description

  The present invention relates to a substance that inhibits drug metabolism and improves the absorption rate of the drug, and use thereof.

Many drugs (pharmaceuticals) are known to undergo metabolism by enzymes such as cytochrome P450 3A (CYP3A) in the small intestine. Metabolism in this small intestine reduces the absorption rate of drugs in the small intestine, and is cited as the cause of low bioavailability and large interindividual variability.
On the other hand, as a method for improving drug absorption, administering a composition comprising an amount of lipid effective to delay the passage of the drug through the small intestine for a time sufficient for absorption of the drug, It is known to use fatty acids such as acids (Patent Document 1).
It is also known that a polyunsaturated fatty acid is contained in a drug as a drug absorption accelerator for transmucosal such as nasal mucosa (Patent Document 2 and Patent Document 3).
However, it is not known that polyunsaturated fatty acids such as DHA have an action of inhibiting drug metabolism in the small intestine.

Japanese National Patent Publication No. 11-505258 JP 2000-128805 A JP 2001-240558 A

  An object of the present invention is to provide a means for inhibiting the metabolism of a drug in the small intestine to improve the absorption rate of the drug and obtain high bioavailability.

The present inventor has found that polyunsaturated fatty acids such as DHA can control (suppress) the metabolism of drugs in the small intestine, and the function of inhibiting the metabolism of polyunsaturated fatty acids improves the absorption rate of drugs in the small intestine as a result. And found that it contributes to high bioavailability, leading to the present invention. In addition, the present inventor unexpectedly found that even when a polyunsaturated fatty acid such as DHA is orally administered, it reaches the small intestine without being decomposed, and that its metabolic inhibitory function can be applied to a drug for oral administration. I found it.
Furthermore, the present inventor has discovered that polyunsaturated fatty acids such as DHA suppress drug metabolism for a certain period of time, but lose their ability to inhibit after a certain period of time. It has also been found that the effective time can be controlled in the case of a pharmaceutical used together with an active ingredient. In addition, polyunsaturated fatty acids suppress drug metabolism for a certain period of time, so effective bioavailability can be achieved by matching the time to reach the absorption site of the drug and the metabolism suppression time. . Such a function of inhibiting metabolism for a certain period of time due to the unsaturated polyhydric fatty acid can exhibit particularly remarkable utility in a pharmaceutical for oral administration.
Conventionally, it is well known that grapefruit juice improves the absorption of orally administered drugs by inhibiting the metabolism of cytochrome P450 3A (CYP3A). However, the inhibition of CYP3A metabolism by grapefruit juice is due to complete inactivation of the enzyme activity of CYP3A, which means that everything that should be metabolized by CYP3A is inherently absorbed, and the metabolism inhibition becomes too strong. Whereas there is a risk, the metabolic inhibitory ability by polyunsaturated fatty acids such as DHA of the present invention is effective for a certain period of time as described above, so there is no such risk.

That is, the present invention relates to the following.
(1) A drug metabolism inhibitor comprising a polyunsaturated fatty acid.
(2) The metabolic inhibitor according to (1) above, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).
(3) The metabolic inhibitor according to (1) or (2) above, wherein the drug is for oral administration.
(4) The metabolic inhibitor according to any one of (1) to (3), which inhibits metabolism by cytochrome P450 3A (CYP3A) in the small intestine.
(5) The metabolic inhibitor according to any one of (1) to (4) above, wherein the active ingredient of the drug is cyclosporine or saquinavir.
(6) A pharmaceutical composition comprising an active ingredient of a drug and a polyunsaturated fatty acid as a metabolic inhibitor.
(7) The pharmaceutical composition according to the above (6), wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).
(8) The composition according to the above (6) or (7), wherein the pharmaceutical composition is for oral administration.
(9) A pharmaceutical composition comprising cyclosporine or saquinavir and a polyunsaturated fatty acid as a metabolic inhibitor.
(10) The pharmaceutical composition according to the above (9), wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).
(11) Use of polyunsaturated fatty acids as drug metabolism inhibitors.
(12) Use of docosahexaenoic acid (DHA) as a drug metabolism inhibitor.

  According to the present invention, metabolism of a drug in the small intestine can be suppressed, the absorption rate of the drug in the small intestine can be improved, and high bioavailability can be obtained. Furthermore, according to the present invention, even a drug having a low absorption rate in the small intestine is likely to be used as a medicine, so that there is a great merit in drug development.

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the following description.
The metabolic inhibitor of the present invention is a polyunsaturated fatty acid such as docosahexaenoic acid (DHA), linolenic acid, γ-linolenic acid, arachidonic acid, and retionic acid, with DHA being most preferred.

  The active ingredient of the drug used in the present invention is generally easily metabolized by cytochrome P450 3A (CYP3A) or the like in the small intestine, or is completely metabolized by cytochrome P450 3A (CYP3A) in a small amount. Anything can be used as long as it is not effective. For example, cyclosporine such as cyclosporin A (CyA), saquinavir known as an anti-AIDS drug, and the like can be mentioned.

  Examples of the form of the preparation of the present invention include solid preparations such as powders, powders, granules, tablets and capsules, liquid preparations such as solutions, suspensions and emulsions, lyophilized preparations, and the like. It can be prepared by means.

In Examples 1 to 5 below, cyclosporin A (CyA) was selected as an active ingredient, and it was examined whether or not the absorption rate of CyA in the small intestine was enhanced by DHA.
The reagents used in the examples are as follows.

<Reagent>
Cyclosporine A (CyA): Noval Dispharma Co., Ltd. Corn oil (Wako)
cis-4,7,10,13,16,19-docosahexaenoic acid (DHA): Sigma D-2534
Polyethylene glycol 200: Wako
Anesthetic Enflurane: Dynapot Cyclosporine Measurement Kit TDXFLX ^TM : Abbott
P-glycoprotein inhibitor PSC833: Novaldispharma Co., Ltd. Testosterone: Wako208-08341
6β-Hydroxy Testosterone: Sigma H-2898
17α-methyltestosterone: Sigma M-7252
Human liver microsomes: Gentest452161
Albumin: Bovine SigmaA-4503

<Influence of DHA on cyclosporine absorption rate in small intestine>
CyA was administered to rats, and the influence of DHA on blood cyclosporine concentration was examined.
(Reagent preparation)
For oral administration, CyA dissolves in corn oil. DHA was added to corn oil (final concentration 5, 10, 20 wt / vol%).
In the case of intravenous administration, CyA was dissolved in ethanol and then dissolved in polyethylene glycol 200 (final ethanol concentration 10 vol / vol%).

(Surgery)
220-250 g Wister male rats (Japanese medical science animals) were fasted for 24 hours, and the left hind leg femoral artery and femoral vein were cannulated under enflurane anesthesia (introduction 5%, maintenance 2%). After waking up, CyA was administered and the rats were fixed in the Bolman cage.

(Method of administration)
In the case of oral administration, CyA was dissolved in corn oil and administered with an oral sonde. The dose was 5 mg / mL of CyA solution administered at 5 mg per kg of rat. When DHA was added, it was added to corn oil at a final concentration of 5, 10, 20 wt / vol%.
In the case of intravenous administration, 1 ml / kg corn oil (which may contain DHA) was orally administered 3 hours before administration, and intravenous administration of a 10 vol / vol% ethanol solution of CyA was performed. When DHA was added, it was added to corn oil at a final concentration of 5, 10, 20 wt / vol%.

(Blood collection)
After administration of CyA, heparin blood was collected over time. In the case of oral administration, 1, 2, 3, 4, 6, 9, 12, 24 hours after administration, and in the case of intravenous administration, 0.167, 0.5, 1, 2, 4, 8, 12 after administration. 24 hours later, blood was collected.

(Blood CyA titration method)
The amount of CyA in the blood was measured with a cyclosporine measurement kit TDXFLX ^™ (Abbott).

(result)
Table 1 and FIG. 1 show the blood CyA concentration (mean value: mean) of rats (number of individuals 6 to 7) orally administered with CyA dissolved in corn oil (base material). In Table 1, the standard deviation (SD) is also shown. The concentration of CyA in rats after oral administration of CyA was reported in the literature (Biochemical Pharmacology 63 (4): 777-783 (2002) Yokogawa K, Shimada T, Higashi Y, Itoh Y, Masue T, Ishizaki J, Asahi M , Miyamoto K., Modulation of mdr1a and CYP3A gene expression in the intestine and liver as possible cause of changes in the cyclosporin A disposition kinetics by dexamethasone). In the group in which DHA was added to the base material, the blood CyA concentration increased according to the amount of DHA added from the second hour of administration, and in the group to which 10 and 20 wt / vol% DHA was added, it significantly increased.
Further, as is apparent from FIG. 1, the increase in blood CyA concentration due to the addition of DHA is transient.
The blood CyA concentration of rats (number of individuals 3 to 4) to which CyA was intravenously administered showed the same result as that in the previously reported literature. No effect was observed when DHA was orally administered in advance (Table 2 and FIG. 2).
From this result, it was found that in the case of oral administration of CyA, the addition of DHA significantly increased the blood CyA concentration, whereas in the case of intravenous administration, addition of DHA had no effect. It was.

<Effects of pre-administration of DHA>
In this example, the effect on the blood concentration of CyA when DHA was administered in advance was examined.
The same test as in Example 1 was conducted except that 20 wt / vol% DHA was administered to rats 3 hours before CyA was orally administered.
The results are shown in FIG. There is almost no difference in the blood concentration of CyA when DHA is administered in advance and when it is not administered (control).
From this result, it was found that when DHA was administered in advance, CyA orally administered did not have the same effect as DHA co-administration. This indicates that the effects of DHA are not as persistent as grapefruit juice and are less dangerous.

<Influence of DHA on dynamic parameters>
CyA was administered to rats in the same manner as in Example 1, and pharmacokinetic parameters after administration were measured.
In the case of oral administration, the value of AUC (area under the blood concentration time curve) increases with the addition of DHA, and the group to which 10 and 20% by weight of DHA is added becomes higher according to the added dose. It was significant. The values of systemic clearance Cltot / F and volume of distribution Vdss / F decreased with the addition of DHA, and were significant in the groups to which 10 and 20% by weight of DHA was added. There was no difference in half-life T1 / 2 (Figure 4). The AUC was processed by using the software Win Nonlin (Pharsight Corp) according to the straight trapezoidal method.
In the case of intravenous administration, there was no significant difference in AUC, Cltot / F, Vdss / F, and T1 / 2 even when DHA was orally administered in advance (FIG. 5).
From this result, it is understood that in the case of oral administration of CyA, the addition of DHA has an advantageous effect on the pharmacokinetic parameters, whereas in the case of intravenous administration, the addition of DHA has no effect. It was.

  From the results of Examples 1 and 3, it was suggested that DHA affects the absorption process of CyA in the small intestine and does not affect the metabolism of CYP3A4 in the liver. Therefore, in order to examine this, next, it was confirmed whether DHA had an effect on the metabolism of CyA by CYP3A4 using human and rat liver microsomes, and Caco-2 expressing P-glycoprotein. It was confirmed whether DHA affects the permeability of CyA using cells.

<Influence of DHA on metabolism>
(Liver microsome metabolism experiment)
Testosterone (15, 30, 100, 300 μM) was metabolized in human tubes and rat liver microsomes (Gentest) in a test tube at 37 ° C. for 10 minutes, and the metabolite (6-β-hydroxylated testosterone) was separated and eluted by HPLC. . DHA (10, 20, 30 μM) was added during the metabolic reaction.
By using SAAMII (Saam Institude software) in the following equation, Vmax, Km, and Ki were obtained.
The HPLC conditions are as follows.
Liquid A water: acetonitrile: methanol = 58: 2: 40
Liquid B Water: acetonitrile: methanol = 28: 32: 40
Gradient elution is performed using A and B solutions.
Internal standard: 17α-methyltestosterone Column: Super-ODS (TOSOH)
Temperature: 42 ° C
Flow rate: 1 mL / min

The results are shown in Table 3.
From this result, it was found that the Ki (inhibition constant) of DHA on testosterone metabolism by the liver was small and the inhibitory effect was high.

  Further, the reciprocal of the concentration of testosterone as a substrate and the reciprocal of the purification rate of 6β-hydroxylated testosterone as a metabolite were plotted (Line-Weaver Burk Plot), and the inhibition mode was examined. As a result, DHA competitively inhibited the testosterone metabolism using human microsomes depending on the concentration (FIG. 6).

<Influence of DHA on permeability of cyclosporine>
A permeability test (transport assay using Caco-2 cells) was performed using Caco-2 cells expressing P-glycoprotein.
(cell)
The Caco-2 cells used are ATCC-derived cells obtained from Hisamitsu Pharmaceutical Co., Ltd., and are subcultured at least once a week. For the passage, Caco-2 cells cultured in a culture dish (IWAKI 3020-100) having a diameter of 100 mm were washed twice with PBS (−), reacted with trypsin-EDTA, peeled off from the dish and collected. The cells were counted after concentrating the cells by centrifugation. Finally, we were seeded a new dish one per 10 ⁶ cells. For transport assay, 63,000 Caco-2 cells were seeded per well in Transwell (costar 3402, pore size 3.0 μm, diameter 12 mm). The culture medium was changed once every two to three days after one week of culture, and a transport assay was performed around three weeks of culture.

(Transport assay using Caco-2 cells)
[ ³ H] -CyA transport assay was performed using Caco-2 cells cultured for 3 weeks. A portion of the buffer was collected from transwell at 30, 60, 90 and 120 minutes after the start of transport, and the radioactivity was measured with a liquid scintillation counter.
The transport assay conditions are as follows.
Transwell (costar3402): Pore size 3.0μm, diameter 12mm
Transport buffer: Hanks balanced solt solution (pH 7.4)
In the presence of 0.1% BSA
CyA concentration: 50 nM (3 Ci / mmol)
PSC833 concentration: 10 μM
DHA concentration: 100 μM

Whether DHA affects the permeability of CyA using Caco-2 cells expressing P-glycoprotein was examined by placing PSC833, a P-glycoprotein inhibitor, as a positive control. As a result, the transportation from the apical side to the basal side and the transportation from the basal side to the apical side were recognized linearly according to time. In contrast to these transports, PSC833 inhibited transport from the basal side to the apical side. The reason why PSC833 did not inhibit the transport from the apical side to the basal side is unknown. Therefore, DHA was evaluated for transport from the basal side to the apical side. As a result, DHA 100 μM did not affect the transport from the basal side to the apical side. (Table 4 and Fig. 7)

From the results of Examples 4 and 5 above, it was suggested that the improvement of CyA bioavailability by DHA was the result of inhibiting CyP3A4 in the small intestine rather than via P-glycoprotein.
From the above, the possibility of a drug delivery system (DDS) using DHA for increasing the drug absorption rate has been clarified.
Considering that DHA is not toxic to living organisms, the use of DHA is clinically effective.

<Influence on metabolism of polyunsaturated fatty acids>
Metabolism inhibiting ability was tested about polyunsaturated fatty acid including other than DHA. As a control, a saturated fatty acid and a monovalent unsaturated fatty acid were also tested.
Testosterone was metabolized using rat liver microsomes in the same manner as in Example 4, and the metabolite (6-β hydroxylated testosterone) concentration was measured. Metabolism was carried out for 10 minutes at a fatty acid concentration of 50 μM, a testosterone concentration of 15 μM, and a temperature of 37 ° C.
Table 5 shows the 6-β hydroxylated testosterone (6βOHT) concentration when each fatty acid was used as an inhibitor as a ratio to the 6-β hydroxylated testosterone (6βOHT) concentration when no fatty acid was added.
From the results of Table 5, saturated fatty acids do not have the ability to inhibit metabolism, but unsaturated fatty acids have the ability to inhibit metabolism, and even unsaturated fatty acids such as linolenic acid, γ-linolenic acid, arachidonic acid, retionic acid, DHA, etc. It can be seen that the ability to inhibit metabolism is further increased when the polyunsaturated fatty acid is used. Furthermore, it was found from Table 5 that DHA exhibits the highest metabolic inhibition ability among polyunsaturated fatty acids.

<Influence of DHA on the absorption rate of saquinavir in the small intestine>
The following examples show the results of testing for saquinavir (HIV protease inhibitor) instead of CyA.
In the same manner as in Example 1, saquinavir was administered to rats, and the influence of DHA on the blood saquinavir concentration was examined.
FIG. 8 shows the blood concentration of saquinavir in rats (number of individuals 4-7) orally administered with 50 mg / kg of saquinavir. In the group to which DHA was added, the blood saquinavir concentration increased according to the amount of DHA added from the second hour after administration, and significantly increased in the group to which 10% wt / vol% DHA was added. As is clear from FIG. 8, the increase in blood saquinavir concentration due to the addition of DHA was transient.
On the other hand, FIG. 9 shows the blood concentration of saquinavir in rats (number of individuals 5) intravenously administered with 10 mg / kg of saquinavir. No effect was observed when DHA was orally administered in advance.
From this result, as for Cyquinavir, as in CyA, the blood concentration was significantly increased by the addition of DHA in the case of oral administration, whereas the effect of adding DHA in the case of intravenous administration was also affected. I found that there was no.

<Effects of pre-administration of DHA>
In this example, the effect on the blood concentration of saquinavir when DHA was administered in advance was examined.
The same test as in Example 1 was performed except that 10 wt / vol% DHA was administered to 3 rats 3 hours before oral administration of saquinavir (50 mg / kg).
The results are shown in FIG. FIG. 10 shows the average value and standard deviation of three rats. There is almost no difference in the blood concentration of saquinavir (SQV) when DHA is administered in advance and when it is not administered (control).
From this result, it was found that when DHA was administered in advance, even when saquinavir was orally administered, there was no effect as with DHA co-administration. This indicates that, as in Example 2, the effects of DHA are not as persistent as grapefruit juice and are less dangerous.

<Influence of DHA on kinetic parameters (Saquinavir)>
Saquinavir was administered to rats in the same manner as in Example 1, and pharmacokinetic parameters after administration were measured. The results are shown in Table 6.
In the case of oral administration, the value of AUC increased with the addition of DHA depending on the dose. The values of systemic clearance Cltot / F and distribution volume Vdss / F were decreased according to the dose of DHA added, and there was no difference in half-life T1 / 2.
In the case of intravenous administration, there was no significant difference in AUC, Cltot / F, Vdss / F, and T1 / 2 even when DHA was orally administered in advance.
From this result, it was found that in the case of oral administration of saquinavir, the addition of DHA has an advantageous effect on the pharmacokinetic parameters, whereas in the case of intravenous administration, addition of DHA has no effect. It was.

  From the results of Examples 6 and 7, it was suggested that DHA affects the absorption process of saquinavir in the small intestine and has no effect on the metabolism of saquinavir in the liver, as in the case of CyA. Therefore, in order to examine this, it was next confirmed whether or not DHA affects the metabolism of saquinavir by CYP3A4 using rat liver microsomes.

<Influence of DHA on metabolism (Saquinavir)>
As in Example 3, saquinavir (1.95, 6.3, 13 μM / L) was metabolized in a test tube at 37 ° C. for 10 minutes using rat liver microsomes (Gentest), and the metabolites were separated and eluted by HPLC. . DHA (10, 50, 100, 200, 500 μM) was added during the metabolic reaction. Ketoconazol, known as a representative inhibitor of CYP3A4, was used as a positive control. The results are shown in FIG. In FIG. 11, the vertical axis represents the amount of saquinavir metabolites, and the lower the value, the more inhibited the metabolism. From FIG. 11, it was found that the addition of DHA inhibited saquinavir metabolism in a concentration-dependent manner. 500 μM DHA showed almost the same inhibitory activity as 1 μM ketoconazole, and DHA was found to show a relatively strong CYP3A4 metabolism inhibitory activity.

  According to the DHA of the present invention, the metabolism of a drug in the small intestine can be suppressed, the absorption rate of the drug in the small intestine can be improved, and high bioavailability can be obtained.

The influence of DHA with respect to the blood CyA density | concentration at the time of oral administration is shown. The influence of DHA with respect to the blood CyA density | concentration at the time of intravenous administration is shown. The influence with respect to the blood CyA density | concentration at the time of administering DHA previously is shown. The influence of DHA on the pharmacokinetic parameter after oral administration of CyA is shown. Figure 2 shows the effect of DHA on pharmacokinetic parameters after intravenous administration of CyA. Figure 3 shows the effect of DHA on CYP3A4 metabolism of testosterone using human liver microsomes. The influence of PSC833 and DHA on the transport from the basal side to the apical side of CyA in Caco-2 cells is shown. The influence of DHA with respect to the blood saquinavir concentration at the time of oral administration is shown. The influence of DHA with respect to the blood saquinavir density | concentration at the time of intravenous administration is shown. The influence of DHA with respect to the blood saquinavir density | concentration at the time of administering DHA previously is shown. Fig. 3 shows the influence of DHA on CYP3A4 metabolism of saquinavir using rat liver microsomes.

Claims (7)

A drug metabolism inhibitor comprising docosahexaenoic acid (DHA) . The metabolic inhibitor according to claim 1, wherein the drug is for oral administration. The metabolic inhibitor according to claim 1 or 2 , which inhibits metabolism by cytochrome P450 3A (CYP3A) in the small intestine. The metabolic inhibitor according to any one of claims 1 to 3 , wherein the active ingredient of the drug is cyclosporine or saquinavir. A pharmaceutical composition comprising an active ingredient of a drug and docosahexaenoic acid (DHA) as a metabolic inhibitor. The composition according to claim 5, wherein the pharmaceutical composition is for oral administration. A pharmaceutical composition comprising cyclosporine or saquinavir and docosahexaenoic acid (DHA) as a metabolic inhibitor.