CN105796496A - Fenofibrate-containing nano-liposome as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a fenofibrate nano liposome, a preparation method and application thereof. Fenofibrate nano-liposomes of the present invention include fenofibrate and nano-liposomes; the nano-liposomes are composed of phospholipids and cholesterol; the mass ratio of the cholesterol to the phospholipids is 1:3-15; The mass ratio of the fenofibrate to the phospholipid is 1:4-40. Its preparation method comprises the following steps: (1) dissolving phospholipids, cholesterol, and fenofibrate in a solvent, mixing, and then removing the solvent to obtain a mixture; (2) buffering the mixture obtained in step (1) The solution is hydrated to obtain a nanoliposome system; (3) the nanoliposome system obtained in step (2) is subjected to ultrasonic treatment with a probe, and then filtered to obtain a fenofibrate nanoliposome. The fenofibrate nano liposome of the invention is applied to the preparation of medicines for treating non-alcoholic fatty liver. The fenofibrate nano liposome of the invention can improve the oral bioavailability of the fenofibrate.

Description

A kind of fenofibrate nano liposome and its preparation method and application

technical field

The invention relates to a fenofibrate nano liposome and a preparation method and application thereof, belonging to the technical field of medicine.

Background technique

Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by excessive fat deposition (imaging or histological evidence) in hepatocytes caused by causes other than alcohol and other clear liver injury factors. NAFLD is the most common chronic liver disease in the West; in my country, with the increase in the number of obese people, the incidence of NAFLD is also on the rise. Patients with NAFLD often suffer from metabolic diseases such as obesity, diabetes, and dyslipidemia. Many studies have shown that excessive fat deposition in non-adipose tissue is toxic. According to the "second hit" hypothesis, excessive lipids act as a "first hit", which can cause "second hits" such as oxidative stress, mitochondrial dysfunction, and inflammation, so that NAFLD can progress to hepatitis, liver fibrosis, and even liver cancer. At present, there is no clear plan and drug for the treatment of NAFLD clinically. The general treatment mainly includes lifestyle changes, weight loss, and the use of lipid-lowering drugs (statins), hypoglycemic drugs (thiazolidinediones, metformin, etc.), anti-diabetic drugs, etc. Oxidants (acetylcysteine, vitamin E, etc.), however, the effects of these treatment options are not ideal. Therefore, it is urgent to find effective drugs to prevent and treat NAFLD clinically.

Fenofibrate is a third-generation phenoxyacetic acid lipid-regulating drug, which is clinically used to treat hyperlipidemia. In addition, it can affect processes such as inflammation, oxidation, and apoptosis. In some animal experiments and human experiments, it also has a certain effect on fatty liver. In terms of the mechanism of action, it is relatively clear that fenofibrate can activate PPARα, thereby increasing the hydrolysis of triglycerides, increasing the synthesis of high-density lipoprotein (HDL), and achieving the effect of reducing fat deposition. However, fenofibrate belongs to class II drugs in the Biopharmaceutical Classification System (BCS), that is, a drug with low solubility and high permeability, and this shortcoming of poor solubility in water limits the oral absorption of fenofibrate, and its Oral bioavailability is poor. At present, the existing fenofibrate pharmaceutical preparations include micronized preparations, nanocrystals, solid dispersions, self-emulsifying drug delivery systems, and the like. However, the above new formulation types all have certain limitations. Although the micronized preparation increases the specific surface area of the drug, this increase increases the surface energy of the drug powder, thereby making the powder in an unstable state, easy to aggregate, and poor in fluidity and dispersion. Such as tablets and capsules) are more difficult and have a certain impact on absorption; nanocrystals may cause cytotoxicity and immune reactions due to the particle size being at the nanometer level; Dispersed state and poor stability and the shortcomings of industrialization difficulties; a large number of surfactants in the self-emulsifying drug delivery system has certain toxic and side effects on the gastrointestinal tract. Therefore, finding a suitable drug delivery system to increase the bioavailability of fenofibrate will be of great help to both its basic research and clinical administration in the treatment of fatty liver.

Contents of the invention

The object of the present invention is to provide a kind of fenofibrate nano liposome and its preparation method and application. The fenofibrate nano liposome of the invention can improve the oral bioavailability of the fenofibrate.

Fenofibrate nano-liposome provided by the invention, it comprises fenofibrate and nano-liposome;

The nanoliposome is composed of phospholipids and cholesterol;

The mass ratio of the cholesterol to the phospholipid is 1:3-15;

The mass ratio of the fenofibrate to the phospholipid is 1:4-40.

In the above-mentioned fenofibrate nanoliposomes, the mass ratio of the cholesterol to the phospholipids may be 1:4 to 12, specifically 1:4, 1:9 or 1:4 to 9;

The mass ratio of the fenofibrate to the phospholipid may be 1:5-35, specifically 1:9, 1:30 or 1:5-30.

In the present invention, the phospholipids are any phospholipids that can be used in the field of medicine, including natural phospholipids, fully synthetic phospholipids and semi-synthetic phospholipids.

The present invention also provides the preparation method of the above-mentioned fenofibrate nano-liposome, comprising the following steps: (1) dissolving phospholipids, cholesterol, and fenofibrate in a solvent, mixing, and then removing the solvent to obtain a mixture ;

(2) hydrating the mixture obtained in step (1) with a buffer solution to obtain a nanoliposome system;

(3) The nanoliposome system obtained in step (2) is subjected to probe ultrasonic treatment, and then filtered to obtain fenofibrate nanoliposome.

In the above preparation method, the solvent is at least one of chloroform, methanol, absolute ethanol and methylene chloride;

The volume ratio of the mass of the phospholipid to the chloroform may be 1g:4-27mL, specifically 1g:13.33mL, 1g:16.67mL or 1g:13-17mL.

In the above preparation method, the method of vacuum distillation is used to remove the solvent, and the temperature of the vacuum distillation can be 30-50°C, specifically 37°C or 30-45°C;

The step (1) further includes the step of drying the mixture, specifically by vacuum drying overnight at room temperature (referring to 10-30°C), and the dried mixture is in the form of a film.

In the above-mentioned preparation method, the buffer solution is citric acid-disodium hydrogen phosphate buffer, bicarbonate buffer, phosphate buffer, disodium hydrogen phosphate-citric acid buffer, citrate buffer and at least one of acetate buffers;

The mass ratio of the phospholipid to the buffer solution may be 1g:13-75mL, specifically 1g:22.67mL, 1g:55.56mL or 1g:22.67-55.56mL;

The concentration of the buffer solution may be 0.002-0.02mmol/L, specifically 0.01mmol/L, 0.002-0.01mmol/L, 0.01-0.02mmol/L or 0.005-0.015mmol/L.

In the present invention, the buffer solution specifically adopts PBS buffer, and the specific formula of PBS buffer is _NaCl137mmol /L, _KCl2.7mmol /L, _{Na2HPO410mmol} /L, _KH2PO42mmol /L.

In the above preparation method, the hydration temperature may be 35-40°C, specifically 37°C, 37-40°C or 35-37°C;

The hydration time may be 20-50 minutes, specifically 30 minutes, 20-30 minutes, 30-50 minutes or 20-40 minutes.

In the above-mentioned preparation method, the ultrasonic treatment of the probe is carried out in an ice-water bath; the purpose of the ultrasonic treatment of the probe is to reduce the particle size of the product;

A 0.45 μm microporous membrane is used for the filtration; the purpose of the filtration is to homogenize the particle size of the product.

In the above-mentioned preparation method, the conditions for carrying out the ultrasound of the probe for every 0.5 g of the nanoliposomes are:

The frequency can be 50-100Hz, specifically 70Hz;

The power can be 120~300W, specifically 240W;

The probe ultrasound can be performed 1-6 times, specifically 2 times; each time can be 10-40 minutes, specifically 30 minutes; each time interval is 5-20 minutes, specifically 10 minutes.

The fenofibrate nanoliposome of the present invention is applied to the medicine for treating nonalcoholic fatty liver in the preparation of medicine for treating nonalcoholic fatty liver; the medicine for treating nonalcoholic fatty liver is any pharmaceutically acceptable dosage form or salt form.

The present invention has the following advantages:

The fenofibrate nano liposome of the present invention has good solubility in water; its fluidity and dispersibility are good; it can reduce the degradation of fenofibrate in the gastrointestinal tract; release medicine slowly; it can improve the oral efficacy of fenofibrate Bioavailability; can prepare drugs for treating non-alcoholic fatty liver disease; have liver passive targeting; can reduce adverse reactions of fenofibrate.

Description of drawings

Fig. 1 is the in vitro release diagram of fenofibrate nanoliposomes, wherein the number of parallel experiments n=3.

Fig. 2 is a drug-time curve diagram after one administration of fenofibrate nanoliposome and fenofibrate bulk drug to mice, wherein the number of parallel experiments n=6.

Figure 3A-D shows the oil red O staining results of mouse liver morphological sections in the fenofibrate prevention experiment of NAFLD. A. control group; B. MCD model group; C. fenofibrate raw material drug group; D. fenofibrate nanoliposome group. E is the result of liver lipid extraction; in Figure 3, * means P<0.05, ** means P<0.01, and the number of parallel experiments is n=5.

Figure 4 is the steady-state blood concentration of fenofibrate in the mice in the NAFLD prevention experiment; in Figure 4, * means P<0.05, and the number of parallel experiments is n=5.

Figure 5 is the oil red O staining results of mouse liver morphological sections in the fenofibrate treatment of NAFLD experiment, wherein Figure 5A is the control group; Figure 5B is the MCD model group; Figure 5C is the low-dose fenofibrate raw material group; 5D is a low-dose fenofibrate nanoliposome group; Figure 5E is a high-dose fenofibrate API group; Figure 5F is a high-dose fenofibrate nanoliposome group; Figure 5G is liver lipid extraction Results; * is P<0.05, ** is P<0.01, *** is P<0.001, and the number of parallel experiments is n=5.

Fig. 6 is the steady-state blood concentration of fenofibrate in mice in the experiment of treating NAFLD; in Fig. 6, ** is P<0.01, *** is P<0.001, and the number of parallel experiments is n=5.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the preparation of fenofibrate nano liposome

Accurately weigh fenofibrate, phospholipids and cholesterol, and place them in a 50mL eggplant-shaped bottle. The mass ratio of phospholipids and cholesterol is 9:1, the drug-to-lipid ratio (mass ratio) is 1:10 (the lipid mass is the total mass of phospholipids and cholesterol), and the mass of fenofibrate is 25 mg. Add 3mL of chloroform and shake fully to dissolve the drug and lipid completely. The film was formed by rotary evaporation under reduced pressure at 37° C., and vacuum-dried overnight at room temperature (25° C.), so that the film formed on the bottle wall was evenly dried. Add 6mL of PBS buffer solution (the specific formula is NaCl137mmol/L, KCl2.7mmol/L, _{Na2HPO4 10mmol} /L, _KH2PO4 2mmol/L, PBS buffer solution concentration is 0.01mmol/L ₎ in the eggplant-shaped bottle, The hydration temperature was 37°C, and the hydration time was 30 minutes; the probe was ultrasonicated in an ice-water bath to reduce the particle size, the ultrasonic frequency was 70Hz, and the ultrasonic power was 240W (30min/time, twice in total, with an interval of 10min). Filter with a 0.45 μm microporous membrane to homogenize the particle size to obtain fenofibrate nanoliposomes.

Embodiment 2, the preparation of fenofibrate nano liposome

Accurately weigh fenofibrate, phospholipids and cholesterol, and place them in a 50mL eggplant-shaped bottle. The mass ratio of phospholipids and cholesterol is 4:1, the drug-to-lipid ratio (mass ratio) is 2:75 (the lipid mass is the total mass of phospholipids and cholesterol), and the mass of fenofibrate is 6 mg. Add 3mL of chloroform and shake fully to dissolve the drug and lipid completely. The film was formed by rotary evaporation under reduced pressure at 37°C, and dried in vacuum at room temperature overnight, so that the film formed on the bottle wall was evenly dried. Add 10mL PBS buffer solution (the specific formula is NaCl137mmol/L, KCl2.7mmol/L, _{Na2HPO4 10mmol} /L, _KH2PO4 2mmol/L, PBS buffer solution concentration is 0.01mmol/L ₎ in the eggplant-shaped bottle, The hydration temperature is 37° C., and the hydration time is 30 minutes. In an ice-water bath, the probe is ultrasonicated to reduce the particle size, the ultrasonic frequency is 70Hz, and the ultrasonic power is 240W (30min/time, twice in total, with an interval of 10min), and filtered through a 0.45μm microporous membrane to homogenize the particle size, that is Obtain fenofibrate nano liposomes.

Embodiment 3, the quality evaluation of fenofibrate nano liposome

1. Determination of particle size and Zeta potential: Take the fenofibrate nanoliposome of Example 1 of the present invention, measure it with a Malvern laser particle size analyzer, and record the average particle size, polydispersity index (PDI) and Zeta potential. The average particle size is 122.1±1.40nm, the PDI is 0.293; the Zeta potential is -2.92mV, slightly electronegativity (as shown in Table 1).

Table 1. Characterization of Fenofibrate Nanoliposomes (n=3)

2. Determination of encapsulation efficiency and drug loading:

The method for measuring the encapsulation efficiency of the present invention is an ultracentrifugation method.

Accurately measure the fenofibrate nanoliposome 1mL of embodiment 1, with PBS buffer (formulation is _NaCl137mmol /L, _KCl2.7mmol /L, _{Na2HPO410mmol} /L, _KH2PO42mmol /L, The final concentration is 0.01mmol/L) diluted to 100mL, pipette 1mL of the diluted solution into an ultracentrifuge tube, centrifuge at 23000g at 4°C for 2h, and separate the supernatant immediately after centrifugation. After the supernatant was aspirated precisely, it was diluted to an appropriate concentration with methanol, and the free drug content (M _Free ) was detected by HPLC.

In addition, 1 mL of the fenofibrate nanoliposome of Example 1 of the present invention was accurately measured and diluted to 100 mL with PBS. Dilute 1 mL of diluent to 5 mL with PBS. Use 3 times methanol to break the emulsion and sonicate for 10min. The peak area was determined by HPLC, substituted into the regression equation to obtain the concentration of fenofibrate, and the total mass of fenofibrate in liposomes (M _Total ) was calculated.

According to the formula:

The encapsulation efficiency (%) of fenofibrate nanoliposomes was calculated. The average encapsulation efficiency was 96.6±1.60% (as shown in Table 1).

According to the formula:

Calculate the drug loading (%) of fenofibrate nanoliposomes. Wherein, M _Lipid is the total mass of lipid in the liposome prescription (the sum of the mass of phospholipid and cholesterol). The average drug loading was 7.44±4.39% (as shown in Table 1).

Embodiment 4, the in vitro release of fenofibrate nanoliposome

The PBS buffer containing 2% CremophorEL (the formula is 137mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na ₂ HPO ₄ , 2mmol/L KH ₂ PO ₄ , the final concentration is 0.01mmol/L) is used as the release medium. Add 0.5 mL of the fenofibrate nanoliposome or fenofibrate raw material drug suspension of Example 1 of the present invention into a dialysis bag, put it into 100 mL of release medium, the conditions are 37 ° C, 200 rpm, at different time points Take 1mL of the release medium outside the dialysis bag, and at the same time add an equal amount of fresh release medium at the same temperature. After the sample was centrifuged at 10,000 rpm for 10 min, the content of fenofibrate in the supernatant was detected by HPLC.

According to the formula:

Calculate the percent cumulative release. Wherein, C _n is the FNB concentration in the sample of each sampling; L is the theoretical dosage; V ₁ is the fixed volume of each sampling; V ₂ is the total volume of the dissolution medium, wherein the number of times n=3 of parallel experiments, the result As shown in Figure 1.

As can be seen from Figure 1, the release of the drug in the fenofibrate nanoliposomes of the present invention is relatively slow and relatively uniform, and the release reaches a plateau at 24h, and the cumulative release percentage is 82.8 ± 3.80%. Compared with fenofibrate liposome, the release rate of fenofibrate bulk drug suspension is very slow, and the release amount is very small, only 17.0±3.18% of the release amount at the 33rd hour time point.

Embodiment 5, the pharmacokinetics of fenofibrate nano liposome

Twelve male C57BL/6 wild-type mice (average body weight 23 g) were randomly divided into 2 groups according to body weight. After fasting for 12h, give the fenofibrate bulk drug or the fenofibrate nanoliposome of embodiment 1 respectively for intragastric administration, and the dose is calculated by fenofibrate 25mg/kg, after administration 5, 10 , 15, 20, 30 minutes and 1, 2, 10, 22 hours orbital blood, heparin anticoagulant, blood samples processed and HPLC detection, blood fenofibric acid concentration c, and draw the drug-time curve.

Compared with the fenofibrate bulk drug, the area under the drug-time curve of the fenofibrate nanoliposome group was significantly increased, indicating that the nanoliposome can significantly increase the absorption of fenofibrate, and it remained stable during 12 to 24 hours. Can make fenofibric acid maintain a higher concentration level in the blood (as shown in Figure 2).

Calculate T _max , t _1/2 , C _max and AUC _0→t with the software Kinetica5.1, and conduct statistical analysis, wherein C _max and AUC _0→t use one-way analysis of variance, and T _max and t _1/2 use Non-parametric test.

After one administration, the fenofibric acid plasma peak concentration _Cmax of the fenofibrate nanoliposome group is 34.9 times that of the fenofibrate raw material drug group, indicating that the nanoliposome can significantly improve the concentration of fenofibric acid in the The peak concentration in the blood; the AUC _{0 → t} of the fenofibrate nanoliposome group is 18.9 times that of the fenofibrate bulk drug group, indicating that the absorption of fenofibrate in the body is significantly improved (as in table 2 shown).

Table 2 Main pharmacokinetic parameters (n=6) of fenofibrate and fenofibrate nanoliposome once administered

Note: In Table 2, **P<0.01, ***P<0.001. n=6 means that the number of parallel experiments is 6 times.

Embodiment 6, the pharmacodynamics of fenofibrate liposome on mouse NAFLD model

The present invention uses a mouse NAFLD model induced by a methionine-choline deficient diet (MCD diet).

1. Preventive effect of fenofibrate liposomes on MCD diet-induced NAFLD

20 male C57BL/6 wild-type mice (average body weight 23g) were randomly divided into 4 groups according to body weight, namely control group, MCD model group, fenofibrate raw material group (20mg/kg/d) and fenofibrate Nanoliposome group (fenofibrate nanoliposome of Example 1 of the present invention, 20 mg/kg/d). During the whole experimental period, the control group was fed with common feed, the MCD model group and the administration group were fed with MCDdiet alone, and the mice in the administration group were given fenofibrate raw material or fenofibrate nano-lipid every day during the modeling. The plastids were intragastrically administered, and the control group and the MCD model group were given the same volume of normal saline by intragastric administration. After 7 days, the mice were harvested.

The oil red O staining of the liver section and the results of liver lipid extraction showed (as shown in Figure 3), that the liver of mice in the control group had no lipid accumulation, and the liver lipid accumulation of mice in the MCD model group was obvious. The fenofibrate nanoparticles of the present invention Compared with the MCD model group and the fenofibrate raw material drug group in the liposome group, liver TG decreased by 62.4% and 54.7% respectively, indicating that the fenofibrate nanoliposomes of the present invention have a significant inhibitory effect on liver lipid accumulation Superior to fenofibrate API.

As can be seen from Figure 4, compared with the fenofibrate bulk drug group, the mouse steady-state blood drug concentration of the fenofibrate nanoliposome group of the present invention is 11.8 times that of the fenofibrate bulk drug group, illustrating that the present invention is non-toxic Norofibrate nanoliposomes can significantly improve the oral absorption of fenofibrate, thereby increasing its effect of reducing liver fat accumulation.

2. The therapeutic effect of fenofibrate liposomes on MCD diet-induced NAFLD

30 male C57BL/6 wild-type mice (average body weight 23g) were randomly divided into 6 groups according to body weight, which were control group, MCD model group, low-dose fenofibrate raw material group (20mg/kg/d), high-dose Fenofibrate crude drug group (40mg/kg/d), low-dose fenofibrate nanoliposome group (fenofibrate nanoliposome of the embodiment of the present invention 1, 20mg/kg/d) and high Dosage of fenofibrate nanoliposome group (fenofibrate nanoliposome of Example 1 of the present invention, 40 mg/kg/d). During the whole experimental period, the control group was fed with common feed, and the MCD model group and the treatment group were fed with simple MCDdiet. After 7 days of simple MCDdiet feeding, continue the simple MCDdiet feeding and administration for 7 days at the same time, the mice in the treatment group were given fenofibrate raw material or fenofibrate nanoliposomes by intragastric administration every day, the control group and the MCD model group The same volume of normal saline was given by intragastric administration. After 14 days, the mice were harvested.

The results of Oil Red O staining of liver sections and liver lipid extraction (as shown in Figure 5) showed that there was no lipid accumulation in the liver of mice in the control group, and serious lipid accumulation in the liver of mice in the MCD model group. Fenofibrate API and low-dose fenofibrate nanoliposomes have a certain effect on reducing liver lipid accumulation, but high-dose fenofibrate nanoliposomes have the most obvious inhibitory effect on hepatic lipid accumulation. Compared with the MCD model group, high-dose fenofibrate API and low-dose fenofibrate nano-liposomes, the TG accumulated in the liver by high-dose fenofibrate nano-liposomes was 62.3%, 35.5% and 26.6%, respectively. %, and there was no significant difference with the control group.

Compared with the low-dose and high-dose fenofibrate raw material drug groups, the steady-state blood drug concentration of mice in the low-dose and high-dose fenofibrate nanoliposome groups was significantly improved, increasing by 10.6 times and 57.3 times respectively. times; while there was no significant difference in the steady-state plasma concentration of high-dose and low-dose fenofibrate raw material drug groups; The concentration increased by 6.7 times (as shown in Figure 6).

Claims (10)

1. a fenofibrate nano-liposome, is characterized in that: it comprises fenofibrate and nano-liposome; The nanoliposome is composed of phospholipids and cholesterol; The mass ratio of the cholesterol to the phospholipid is 1:3-15; The mass ratio of the fenofibrate to the phospholipid is 1:4-40. 2. fenofibrate nanoliposome according to claim 1, is characterized in that: the mass ratio of described cholesterol and described phospholipid is 1:4～12; The mass ratio of the fenofibrate to the phospholipid is 1:5-35. 3. the preparation method of fenofibrate nano liposome described in claim 1 or 2, comprises the steps: (1) phospholipid, cholesterol, fenofibrate are dissolved in solvent, mix, then remove described solvent , to get a mixture; (2) hydrating the mixture obtained in step (1) with a buffer solution to obtain a nanoliposome system; (3) The nanoliposome system obtained in step (2) is subjected to probe ultrasonic treatment, and then filtered to obtain fenofibrate nanoliposome. 4. preparation method according to claim 3, is characterized in that: described solvent is at least one in chloroform, methyl alcohol, dehydrated alcohol and methylene chloride; The volume ratio of the mass of the phospholipid to the chloroform is 1g:4-27mL. 5. according to the described preparation method of claim 3 or 4, it is characterized in that: remove described solvent and adopt the method for underpressure distillation, the temperature of described underpressure distillation is 30～50 ℃; Step (1) also includes the step of drying the mixture. 6. according to the preparation method described in any one in claim 3-5, it is characterized in that: described buffer solution is citric acid-disodium hydrogen phosphate buffer, bicarbonate buffer, phosphate buffer, phosphoric acid At least one of disodium hydrogen-citrate buffer, citrate buffer and acetate buffer; The volume ratio of the mass of the phospholipid to the buffer solution is 1g:13-75mL; The concentration of the buffer solution is 0.002˜0.02 mmol/L. 7. The preparation method according to any one of claims 3-6, characterized in that: the hydration temperature is 35-40°C; The hydration time is 20-50 minutes. 8. The preparation method according to any one of claims 3-7, characterized in that: ultrasonic treatment of the probe is carried out in an ice-water bath; The filtration is performed using a 0.45 μm microporous membrane. 9. according to the preparation method described in any one in claim 3-8, it is characterized in that: the condition that every 0.5g described nano liposome carries out described probe ultrasonic is: The frequency is 50~100Hz; The power is 120~300W; The ultrasound of the probe is performed 1-6 times; each time may be 10-40 minutes; each time interval is 5-20 minutes. 10. the application of the fenofibrate nano liposome described in claim 1 or 2 in the preparation treatment nonalcoholic fatty liver medicine.