KR19990016002A - Chitochrome P450 substrate containing L7171350 - Google Patents

Abstract

The present invention relates to a substrate for chitochrome P450 having LB71350 of the general formula (I) as a main component.

(I)

Description

Chitochrome P450 substrate containing L7171350

The present invention relates to a composition containing LB71350 of the following structural formula (I), an HIV protease inhibitor, and more particularly to a substrate for chitochrome P450 containing LB71350 as a main component.

(I)

When administered in vivo, the drug undergoes absorption, distribution, metabolism, and excretion. Drugs that are absorbed systemically change their concentration over time in the blood, and the effect is proportional to the blood concentration. A constant relationship is established between dose-blood concentration-drug efficacy, and the degree of blood concentration for the dose of drug to the living body is expressed as bioavailability.

At this time, factors affecting bioavailability include solubility in the gastrointestinal tract, drug absorption rate through the gastrointestinal tract, and first-pass effect in the small intestine and liver. In other words, bioavailability is a function of solubility, drug absorption rate, and first-pass effect. For drugs with low bioavailability, bioavailability can be greatly improved by improving the aforementioned properties. When solubility is low and bioavailability is low, solubility can be improved by using an appropriate salt, another derivative, or an appropriate amount of a dissolution aid. When drug absorption rate is low and bioavailability is low, Derivatives or pro-drugs that can enhance partitioning and permeability can be used to improve the bioavailability of the drug. On the other hand, the first pass effect is a metabolic effect of enzymes in the small intestine and liver, and each drug is affected by a metabolic system unique to each drug. Minimizing the effect can increase bioavailability.

AIDS (Acquired Immunodeficiency Syndrome) Therapeutics are broadly divided into nucleotide derivatives that inhibit reverse transcriptase, non-nucleotide classes, HIV protease inhibitors, TAT protein inhibitors, and other anti-HIV neutralizing antibodies and sugars. Protein formation inhibitors. In the early stages of the development of AIDS therapeutics, reverse transcriptase inhibitors were actively studied, but the toxicity and resistance problems became serious. Recently, many studies are being conducted to suppress HIV protease, which is essential for viral infection, as an effective target of inhibiting the growth of the virus.

However, there are many difficulties in finding an HIV protease inhibitor that has both strong antiviral activity and good oral bioavailability, which are essential conditions to be a useful AIDS therapeutic. In other words, effective long-term bioavailability can be expected when long-term administration for chronic diseases such as AIDS is effective. Unfortunately, most protease inhibitors are characterized by low bioavailability during oral administration. The reason is that the protease inhibitors are a combination of hydrophobic peptides, and the metabolism by the enzyme is so large that the first-pass effect is very high in the absorption process.

Chitochrome P450 (CYP450) is well known as a metabolic enzyme that lowers bioavailability. Among these, CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP1A2, CYP1A1 are microsomal enzymes that play an important role in the metabolism of drugs. Ther. 270 (1994), 414-423). Therefore, if appropriately inhibit the application of metabolic enzymes that lower the bioavailability can greatly improve the bioavailability. For example, the combination of Saquinavir and Norvir can significantly improve plasma concentrations of Saquinavir (Kempf et al., Antimicrob. Agents Chemother. 41 (1997), 654-660). This is because CYP3A4 involved in metabolism was effectively suppressed.

The present invention relates to a reagent for a chitochrome P450 substrate based on the HIV protease inhibitor LB71350.

1A graphically depicts the effect of DMP4520 on in vitro metabolism of LB71350 in dog cytosol fractions.

1B graphically depicts the effect of LB71350 on in vitro metabolism of DMP450 in the cytokine fraction of dogs.

Figure 2a graphically shows the concentration in plasma when LB71350 (30 mg / kg) in mice alone or in combination with DMP 450 (30 mg / kg).

2B graphically shows plasma concentrations of DMP450 (30 mg / kg) in mice alone or in combination with LB71350 (30 mg / kg).

3a graphically shows plasma concentrations in dogs when LB71350 (30 mg / kg) was administered alone or in combination with DMP 450 (30 mg / kg).

3B graphically shows plasma concentrations in dogs when DMP450 (30 mg / kg) was administered alone or in combination with LB71350 (30 mg / kg).

4 is a graph showing the mutual effects on the in vitro metabolism of LB71350 and DMP450 in the cytosol fraction of dogs when co-administration of LB71350 and DMP450.

4A shows the effect of DMP450 on LB71350,

4B shows the effect of LB71350 on DMP450.

The present invention relates to a substrate for metabolic enzyme containing LB71350 represented by the following structural formula (I) as a main component. The invention also relates to LB71350 as a reagent for chitochrome substrates.

Structural Formula 1

LB71350 (Korean Patent Application Nos. 95-37292 and 96-78663) and DMP450 (Biochemistry, vol. 36 , no. 7, pp. 1573-1590 (1997) and Chem. Biology, vol. 3 , no. 4, pp. 301-314 (1996)) are known as HIV protease inhibitors. LB71350 is nonionic and has very high metabolism by enzymes in the small intestine and liver during absorption. DMP450, on the other hand, is ionic and has very low metabolism by enzymes in the small intestine and liver during absorption. In order to minimize the metabolism of LB71350 by enzymes in the small intestine and liver, the present inventors have found that LB71350 acts as a substrate for cytochrome (Ctochorome) P450, an enzyme involved in drug metabolism in the small intestine and liver. The present invention has been completed by finding out.

Pharmaceutical compositions for the administration of HIV protease inhibitors LB71350 and DMP450 to patients include all pharmaceutically acceptable compositions including liquid, semi-liquid, and solid phases. Includes all acceptable methods of administration.

Auxiliary ingredients for making LB71350 and DMP450 compositions into liquid and semi-liquid formulations include (A) a solvent selected from propylene glycol, polyethylene glycol, polyethylene glycol copolymers, or (B) polyoxyethylene (20) sorbitan monolau Latex, polyoxyethylene (20) sorbitan monooleate, polyoxyethyleneglycerol triricinoleate, polyethylene glycol (40) hydrated castor oil, fractionated palm oil, 2- (2-ethoxyethoxy) ethanol and polyethylene polypropylene Selected solvents of glycols or mixtures thereof are possible. In addition, ethanol or propylene glycol can also be used to prepare liquid, semi-liquid formulations. Solid dosage forms may be capsules and tablets, etc., excipients may be used starch, lactose and the like used in the general formulation may include a lubricant such as magnesium stearate. In particular, DMP450 may be prepared in drug delivery formulations such as double or coated tablets, which are previously eluted with rapid release and LB71350 is later eluted with slow release.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Experimental Example 1

LB71350 and DMP450 were administered alone or orally in combination with LB71350 and DMP450 at 30 mg / kg, and the plasma concentrations were determined (DMP450 was administered 30 minutes before co-administration).

Prior to administration, the SD rats (200-300 g body weight) were fasted for 16-20 hours, but water was allowed to drink freely. A blood collection catheter (PE-50) was inserted into the femoral artery and the subject was orally administered with 30 mg / kg of the compound. Blood samples were collected from the subjects at 0, 10, 20, 30, 60, 120, 180, 240, 360, and 480 minutes after administration. Plasma samples were quantified by reverse phase high performance liquid chromatography (HPLC) with ultraviolet absorber followed by protein-treatment. The area under the concentration curve of the compound (hereinafter AUC) was determined by the trapezoidal method. Experiments were conducted in groups of three rats, with the figures expressed as the mean of each group. T _max (time to reach maximum concentration), C _max (maximum concentration value), AUC for both single and combination doses are shown in Table 1, and the plasma concentration curve for time is shown in FIG. 1.

Results of Oral Administration in Rats (Mean ± Standard Deviation) Item Single dose (n = 3) Concomitant administration (n = 3) LB71350 Dose (mg / kg) t _max (min) C _max (μg / ml) AUC (μg.min / ml) 3020 ± 8.74.1 ± 2.1509.3 ± 269.4 30 (DMP450) 200 ± 34.64.6 ± 0.71771 ± 405 DMP450 Dose (mg / kg) t _max (min) C _max (μg / ml) AUC (μg.min / ml) 30 100 ± 34.65.8 ± 1.91505 ± 892 30 (LB71350) 232 ± 1814.2 ± 1.41753 ± 694 AUC (area under concentration curve) is calculated up to 480 minutes

Experimental Example 2

LB71350 (10 mg / kg) and DMP450 (5 mg / kg) were administered to dogs (Beagle Eagle) alone or in combination with LB71350 and DMP450 (5 mg / kg), respectively. DMP450 was administered 30 minutes before).

Prior to dosing, the Eagle Eagle (11-14 kg) was fasted for 16-20 hours, but water was allowed to drink freely. Compounds were orally administered to each subject at a dose of 10 mg / kg (LB71350) or 5 mg / kg (DMP450). Blood was collected from the forelimb vein at 0, 10, 20, 40, 60, 90, 120, 180, 240, 360, 480, 600, 720 minutes after administration, and centrifuged until plasma was isolated and analyzed. Frozen at -20 ° C. Plasma samples were quantified by reverse phase high performance liquid chromatography (HPLC) with an ultraviolet absorber followed by protein treatment. The area under the concentration curve of the compound (hereinafter AUC) was determined by the trapezoidal method. Experiments were conducted in groups of 3-5 dogs, with the figures expressed as the mean of each group. T _max , C _max , and AUC for both single and co-administration are shown in Table 2 and the plasma concentration curves over time.

Results of Oral Administration in Dogs (Mean ± Standard Deviation) Item Alone Concomitant administration LB71350 Dose (mg / kg) t _max (min) C _max (μg / ml) AUC (μg.min / ml) 10 (n = 5) 28 ± 16.40.97 ± 0.4764.5 ± 19.7 5 (DMP450, n = 3) 26.7 ± 11.63.65 ± 0.55908.2 ± 276.7 DMP450 Dose (mg / kg) t _max (min) C _max (μg / ml) AUC (μg.min / ml) 5 (n = 3) 60 ± 0.02.64 ± 0.74971.3 ± 273.0 10 (LB71350, n = 3) 76.7 ± 64.32.88 ± 0.671297.9 ± 432.6 AUC (area under concentration curve) is calculated up to 720 minutes

Experimental Example 3

LB71350 (10 μg / ml) alone or LB71350 (10 μg / ml) and DMP450 (10 μg / ml) were simultaneously reacted with the liver cytosol fraction (s9) in dogs. DMP450 was administered 5 minutes before).

Some of the dog's livers were homogeneously homogenous after being placed in a 2-fold dose of 0.1 M Tris-acetate buffer (pH) 7.4) containing 0.1 M potassium chloride and 1 mM EDTA. Thereafter, the homogenate was centrifuged at 9,000 g for 20 minutes, and then the upper layer (s9) was separated, and the upper layer was further centrifuged at 105,000 g for 1 hour. The thin ferret was then dispersed in 0.1 M tetrasodium pyrophosphate containing 1 mM EDTA and then centrifuged at 105,000 g for 1 hour. Thereafter, the ferret was added to a double dose of 50 mM Tris-acetate buffer (pH 7.4) containing 20% glycerol and 1 mM EDTA to obtain a microsomal fraction (m). Metabolic experiments were carried out in a NADPH activation system containing 6.7 mg / ml s9 or 2 mg / ml microsome protein, and reaction conditions were 37 ° C. and 150 opm. The drug concentration curves of the reaction system by single and combination reactions are shown in FIG. 3.

Experimental Example 4

The liver microsomal fraction (m) of dogs contained the specific substrates of CYP3A4 (nifedipine: 10 ug / ml), CYP2C19 (propranolol: 1000 ug / ml), and CYP2E1 (chlorzoxazone: 1000 ug / ml) of CYP450 enzymes alone or DMP450 ( 10 μg / ml), and the disappearance concentrations of specific substrates and the concentrations of their generated metabolites were determined (DMP450 was administered 5 minutes before the simultaneous reaction).

Liver metabolism experiments were carried out in the same manner as in Experimental Example 3, and specific substrates of the reaction system by specific reactions alone or in combination with DMP450 were shown in Table 3.

Effect of DMP450 on In vitro Metabolism of CYP450 Substrate in Canine Microsomes (n = 2) Substrate Concentration (mg / ml) -Time Profile Minutes Nifedipine (CYP3A4) Propranolol (CYP2C19) Chlor trisonone (CYP2E1) Control + DMP450 Control + DMP450 Control + DMP450 N1 N2 N1 N2 N1 N2 N1 N2 N1 N2 N1 N2 0 10 10 10 10 1000 1000 1000 1000 1000 1000 1000 1000 10 7.98 7.13 9.10 8.92 972 974 978 969 971 971 972 975 20 6.08 5.13 8.21 7.66 972 974 976 973 974 977 978 985 60 2.33 1.21 6.79 6.32 884 878 888 874 890 891 885 898

Generated Metabolite Concentration (μg / ml) -Time Profile Minutes Nifedipine Oxide (CYP3A4) Hydroxy propranolol (CYP2C19) Hydroxy Chlorosazone (CYP2E1) Control + DMP450 Control + DMP450 Control + DMP450 N1 N2 N1 N2 N1 N2 N1 N2 N1 N2 N1 N2 0 0 0 0 0 0 0 0 0 0 0 0 0 10 0.88 0.95 0.37 0.43 0.86 1.02 0.66 0.86 1.25 1.54 0.68 0.96 20 1.14 1.13 0.60 0.67 1.62 1.90 1.26 1.55 2.42 2.97 1.44 1.99 60 0.85 0.40 1.06 1.15 4.45 4.83 3.36 3.72 6.83 8.14 4.98 6.41

Experimental Example 5

LB71350 (10 ug / ml) alone or in CYP450 enzymes was used for CYP3A4 (ketoconazole), CYP2C9 (sulphaphenazole), CYP2C19 (tranylcypramine), CYP2D6 (quinidine), CYP2E1 (4-methylpyrazole) and CYP1A2. (furafylline) and specific inhibitors of CYP1A1 (α-naphthoflavone) (40 μg / ml) were reacted at the same time, and then the loss concentration of LB71350 was determined (specific inhibitors were administered 5 minutes before the simultaneous reaction).

Hepatic metabolism experiments were carried out in the same manner as in Experimental Example 3, and LB71350 drug concentrations in the reaction system by LB71350 alone or in combination with specific inhibitors are shown in Table 4.

Effect of CYP Inhibitor on In Vitro Metabolism of LB17350 Substrate in Canine Microsomes (n = 2) LB71350 Concentration (μg / ml) -Time Profile Minutes LB71350 sole + CYP1A1 + CYP1A2 + CYP2E1 N1 N2 N1 N2 N1 N2 N1 N2 0 10 10 10 40 10 10 10 10 10 9.27 8.86 9.91 70 9.30 9.21 9.77 9.54 30 7.95 7.06 9.78 70 8.39 8.28 9.44 9.30 60 6.17 4.72 9.57 9.74 7.14 6.68 9.12 8.92

(continue)

Minutes LB71350 sole + CYP1A1 + CYP1A2 + CYP2E1 N1 N2 N1 N2 N1 N2 N1 N2 0 10 10 10 10 10 10 10 10 10 9.82 9.73 9.82 9.94 9.72 9.88 9.54 9.60 30 9.53 9.54 9.41 9.31 9.42 9.98 8.51 8.22 60 9.36 9.26 9.14 8.93 9.09 9.04 6.68

Experimental Example 6

LB 71350 (10 μg / ml) alone or LB 71350 (10 μg / ml) and DMP 450 (10 μg / ml) were reacted to the liver cytosol fraction (s9) in dogs, and the disappearance of the drug was determined. In simultaneous reaction, LB 71350 and DMP 450 were administered simultaneously).

Hepatic metabolism experiments were performed in the same manner as in Experimental Example 3, and the drug concentration curves of the reaction system by single and combination reactions are shown in FIG. 4.

As can be seen, LB71350 was metabolized very quickly in the liver cytosol fraction (s9) of the dog, while DMP450 was very stable in the liver cytosol fraction (s9) of the dog (FIG. 1). LB71350 was mainly metabolized by CYP3A4 in hepatic microsomal fraction (m), but metabolism by CYP1A1, CYP2E1, CYP2D6, and CYP2C9 was also confirmed (Table 4), and DMP450 selectively inhibited CYP3A4 (Table 3). As a result of co-administration of LB71350 and DMP450 in mice and dogs, plasma concentrations of LB71350 in mice and dogs were greatly improved by DMP450 (FIGS. 1 and 2). By co-administration of DMP450, the AUC of LB71350 increased about 3.5-fold in rats, about 14-fold in dogs, and _Cmax increased about 3.8-fold in dogs (Table 1 and Table 2). Hepatic metabolism of LB71350, measured by loss half-life in liver metabolism experiments, was reduced to about one eighth by DMP450 (FIG. 3), which is consistent with the results of the bioreaction. This is because DMP450 strongly inhibited the CYP3A4 enzyme, resulting in a significant decrease in LB71350 metabolism. In addition, liver metabolism (loss half-life) of LB71350 is reduced by about 1 minute when DMP450 is administered simultaneously (Fig. 4), even when LB71350 and DMP 450 are orally administered simultaneously. Both show an increase. On the other hand, DMP450 was not affected by LB71350 in both hepatic metabolism and oral administration.

Claims (2)

A composition which is a substrate for chitochrome P450 containing LB71350 as a main component represented by the following structural formula (I): (I) The method of claim 2, The composition wherein the chitochrome P450 is CYPA4, CYP1A1, CYP2E1, CYP2D6, or CYP2C9.