KR100611559B1 - Intranasal pharmaceutical compositions containing calcitonin - Google Patents

Abstract

The present invention contains a calcitonin selected from salmon calcitonin, human calcitonin, eel calcitonin, carbo calcitonin, chicken calcitonin, porcine calcitonin as an active ingredient, aminoethylsulfonic acid as a stabilizer, hydroxypropylmethylcellulose as a penetration enhancer and It provides a pharmaceutical composition for nasal spray containing a pharmaceutically acceptable carrier, the pharmaceutical composition for nasal spray according to the present invention improves the stability of calcitonin in the liquid state, increases the absorption rate when applied to the nasal mucosa, non-mucosal irritant Is a novel pharmaceutical composition without.

Description

Intranasal pharmaceutical compositions containing calcitonin

The present invention relates to a novel pharmaceutical composition that improves the stability of calcitonin in the liquid state, increases the absorption rate when applied to the nasal mucosa, and has no nasal irritation.

Calcitonin is a hormone found in the thyroid gland and has a molecular weight of about 3400 polypeptides consisting of 32 amino acids, which lowers serum calcium and phosphoric acid. It promotes bone formation and increases urinary phosphate excretion in the kidney, which is used for deformative osteoarthritis, essential hypercalcemia in children, hyperparathyroidism in children, bone metastasis in cancer, and osteoporosis.

However, long-term intravenous, intramuscular, and subcutaneous administration of calcitonin in the treatment of chronic diseases such as osteoporosis entails a risk and discomfort, requiring another route of administration. During oral administration, since it is easily degraded by gastric juice and proteases such as pepsin and trypsin, it can be considered that nasal mucosa administration has a large amount of microvilli and a large absorption surface area, resulting in rapid action expression time of the drug. Factors influencing nasal mucosa absorption include physicochemical properties of drug such as intrinsic permeability, ionic strength, water distribution coefficient, molecular weight, presence of carrier transport, degradation by protease in nasal cavity and pathophysiological status of nasal mucosa. to be.

However, high molecular weight peptides do not pass well through the nasal mucosa, so the bioavailability is extremely low. As such, many studies have been conducted to increase the absorption of peptides using absorption accelerators.

Surfactants (EP115627, GB2127689), acylcarnitine (EP215697), acycorbates and salicylates (EP37943), cholineesters (EP214898), alpha-cyclodextrins (EP94157), chelating agents (US4476116) and the like are known as absorption enhancers. It is. These absorption enhancers are often not practical because they reduce the stability of calcitonin or irritate the nasal mucosa when applied to the formulation.

Therefore, it is important to develop an absorption accelerator that maintains the stability of the formulation, has no toxicity and irritation, expresses the effect of promoting rapid absorption, and ensures safety that does not damage mucosal tissues when used for a long time.

In the present invention, aminoethylsulfonic acid (taurine) enhances stability against calcitonin, and the composition is prepared with aminoethylsulfonic acid and hydroxymethylcellulose to increase systemic absorption of calcitonin when applied to the nasal cavity, which is a pharmacological action of calcitonin. Plasma calcium concentrations were found to be more pronounced and could be completed. In addition, the increase in in vitro permeability using the rabbit nasal mucosa was confirmed, and since there was no nasal irritation, the pharmaceutical composition containing calcitonin according to the present invention can be said to be a new nasal spray formulation of calcitonin with increased stability and absorption in the body. have.

Accordingly, it is an object of the present invention to improve the stability of calcitonin in the liquid state, to increase the absorption rate when applied to the nasal mucosa, and to provide a nasal spray pharmaceutical composition without nasal mucosal irritation.

More specifically, the present invention contains a calcitonin selected from salmon calcitonin, human calcitonin, eel calcitonin, carbo calcitonin, chicken calcitonin, and pork calcitonin as active ingredients, and aminoethylsulfonic acid as a stabilizing agent, and hydroxy as a penetration enhancer. It provides a pharmaceutical composition for nasal spraying containing propylmethylcellulose and a pharmaceutically acceptable carrier, the pharmaceutical composition for nasal spraying according to the present invention improves the stability of calcitonin in the liquid state, the absorption rate when applied to the nasal mucosa To provide new pharmaceutical compositions that are non-mucosal irritant.

The present invention relates to a new nasal spray composition which improves stability as a non-administrative solution containing calcitonin as an active ingredient and increases systemic absorption of calcitonin when applied nasal.

Recently, many studies have been conducted to investigate the decomposition mechanism of calcitonin and to reveal the structure of the degradation product. Among them, the rate and pathway of the decomposition of salmon calcitonin were found to be highly dependent on pH, and the mechanism of decomposition of salmon calcitonin in solution was not yet fully understood, but hydrolysis and deamination and trisulfide derivative formation and dimerization (J. Pharm. Science, 86 ( 3), 359-364, 1997) or by oxidation (Rapid Commun. In Mass Spectrometry, 10, 151-156, 1996). In Lee et al. (Pharm. Res., 9 (11), 1521-1523, 1992), which examined the chemical stability of calcitonin with respect to pH and temperature, calcitonin was most stable at pH 3.3 and harsh conditions of 50-80 ° C. Esau also fits well with Arrhenius.

In the present invention, an aminoethylsulfonic acid (taurine) was selected by examining an additive capable of stabilizing calcitonin at 60 ° C harsh conditions. Although amino acids are known to stabilize by reducing surface adsorption of peptides and preventing aggregation reactions, it is a newly discovered fact that aminoethylsulfonic acid is involved in the stabilization of salmon calcitonin. Aminoethylsulfonic acid is 0.01% or more, 0.1-1% of which is suitable, the optimum stabilization was confirmed that 0.3%.

In addition, hydroxypropyl methylcellulose was selected as a polymer to increase the systemic absorption of the drug by extending the contact time between the solution and the nasal mucosa. Hydroxypropylmethylcellulose did not affect the stability of calcitonin.

The stability of the liquid formulation containing aminoethylsulfonic acid and hydroxypropylmethylcellulose was compared to the control agent A and other commercially available formulations of other compositions except aminoethylsulfonic acid and hydroxypropylmethylcellulose under severe conditions of 25 ° C and 50 ° C. Formulation B and stability were compared. The results up to 12 weeks are shown in Example 8, and it was confirmed through experiments that the composition of the present invention further stabilized salmon calcitonin.

In addition, when the composition of the present invention was applied to the nasal cavity, systemic absorption and pharmacological action were compared with the control agent in rats, and the permeability through in vitro rabbit nasal mucosa was also compared with the control agent. The results are shown in detail in Examples 9-11, and it can be clearly seen that the body absorption of the composition of the present invention was increased compared to the control agent.

In the case of drugs applied to the nasal cavity, a topical point of view is as important as promotion of absorption. There have been reports of increased calcitonin uptake with the addition of surfactants or bile acids (Delivery system for peptide drugs, Plenum Press, 232-234, 1986), but these have not yet been used practically because of local irritation.

Comparison of nasal mucosal irritation obtained by repeated nasal administration to rabbits for 2 weeks showed no specific irritation in the general symptoms, lesions, and pathological examinations.

Therefore, the present invention improves the stability of salmon calcitonin in the liquid state, enhances the absorption rate when applied to the nasal mucosa, and provides a new nasal spray composition without nasal mucosal irritation.

The present invention can be applied to human calcitonin, eel calcitonin, carbocalcitonin, chicken calcitonin, porcine calcitonin in addition to salmon calcitonin.

The solutions according to the invention can be administered as nebulizers, inhalers, drops, or else by any suitable delivery system known in the art.

In the following examples, a composition containing salmon calcitonin as calcitonin is prepared and compared to explain the present invention in more detail, but this does not limit the scope of the present invention.

Examples 1-7

A nasal spray solution of calcitonin was prepared with the following composition.

Composition of each example

Example number

                                1 2 3 4 5 6 7

Salmon Calcitonin (5000 IU / mg, mg) 22 22 22 22 22 22 22

Benzalkonium Glolide (mg) 10 10 10 10 10 10 10

Sodium chloride (mg) 750 750 750 750 750 750 750

Aminoethylsulfonic acid (taurine, mg) 300 100 1000 3000 300--

Hydroxymethylcellulose (mg)-250 250 250 250 250-

Suitable volume making with 100 ml of distilled water

Suitable quantity to make with 0.1N HCl pH 3.7

Experimental Example 1

The stability of the formulation was confirmed by HPLC for 2 weeks at 60 ° C for the formulation of Example 1-7. The results are shown in Table 1 below.

TABLE 1

The stability of salmon calcitonin is greatly affected by temperature and pH, and the effects of additives can be compared when these conditions are constant. Aminoethylsulfonic acid showed a tendency to stabilize salmon calcitonin, especially at 0.3%. Hydroxypropylmethylcellulose did not significantly affect salmon calcitonin stabilization.

Experimental Example 2

Example 5 and Control A, excluding aminoethylsulfonic acid and hydroxymethylcellulose, and Control B, a commercially available preparation, B (salmon calcitonin (5000 IU / mg) 22 mg, 360 mg of citric acid, 451 mg of sodium citrate, 200 mg of ammonium stannate) , 80 mg of methylparaben, 20 mg of propyl paraben, and distilled water were added thereto to make 100 ml).

TABLE 2

       Stability of formulation when stored at 25 ° C (% retention)

TABLE 3

       Stability of formulation when stored at 50 ° C (% retention)

Experimental Example 3

Sprague-Dawley rats weighing 270 ± 20 g were anesthetized with 50 mg / kg of pentobarbital sodium intraperitoneally, followed by a polyethylene tube in the femoral artery, bronchus and esophagus. 6, respectively, in 1100 IU / ml of Example 5 and control A and B formulations and placebo inoculated with 20 μl of saline solution per placebo (0, 30 minutes, 1, 2, 3, 4) Blood was collected at 5, 6, and 6 hours and plasma calcium concentration was measured using an atomic absorption spectrometer.

The decrease rate (ΔD (%)) of plasma calcium concentration was calculated by the following equation.

ΔD (%) = (AUC _ca-plac -AUC _ca-sCT ) / AUC _ca-plac × 100

ΔD (%): The rate of decrease in the calcium concentration in plasma between 0 and 6 hours after administration

AUC _ca-plac : Area under the plasma calcium-time curve up to 6 hours upon placebo administration

AUC _ca-sCT : Area under the plasma calcium-time curve up to 6 h upon administration

Figure 1 shows the residual percentage of the plasma calcium concentration compared to the initial value, Table 4 shows the calculated AUC and ΔD (%).

TABLE 4

      Calculated AUC and ΔD (%) for Plasma Calcium Residual Percentage

Since the pharmacological action of calcitonin is the hypocalcium activity, the low AUC calculated for the plasma calcium residual percentage indicates a high pharmacological effect of the preparation. In addition, the decrease rate (ΔD (%)) of plasma calcium concentration compared to placebo clearly showed that the calcitonin pharmacological action of Example 5, the present invention composition, was significantly increased compared to other agents.

Experimental Example 4

Sprague-Dawley rats weighing 270 ± 20 g were anesthetized with 50 mg / kg of pentobarbital sodium intraperitoneally, followed by a polyethylene tube in the femoral artery, bronchus and esophagus. Blood was injected at 0, 5, 15, 30 minutes, 60, 120, 240 and 360 minutes after intranasal administration of 1100 IU / ml of Example 5 and 20 μl of Control A and B formulations, 2 shows the results of measuring the concentration of calcitonin in plasma by RIA kit (DSL-3600).

It can be seen that the systemic absorption after administration of Example 5, the composition of the present invention to the rat nasal cavity, was significantly increased compared to the control agent.

Experimental Example 5

Rabbit (Newzealand White, , 2.5-3.1 kg) of the nasal mucosa was removed and mounted in a Ussing chamber, and calcitonin preparation was added to the mucosal side, and 1 ml of buffered bicarbonate ringer solution was added to the serous side. 95% O ₂ -5% CO ₂ gas was injected into both solutions to ensure that the solution was well mixed and maintained at 37 ° C. 50 μl each was taken from the serous side at 0, 20, 40, 60, 90, 120, 180 and 240 minutes, and calcitonin was quantified using an ELISA kit. Calcitonin formulations used herein Example 5 and the control agents A and B of the composition of the present invention.

The transmission coefficient (P _eff , cm / sec) was calculated by the following equation.

V _R is the volume of the receiver (1 cm 3), A is the membrane surface area (0.636 cm 2), C ₀ is the initial drug concentration, 60 is 60 sec / min, dc / dt (pg / ml Cumulative dose graph shows the slope of the initial linear region.

Table 5 shows the results. In Example 5, the transmission coefficient was comparable to that of the control agent A, and the transmission coefficient was significantly higher than that of the control agent B (p <0.05). Therefore, it is considered that the composition of the present invention has superior nasal mucosal permeability than the control agent.

TABLE 5

In vitro Permeability Coefficient Using Extracted Rabbit Nasal Mucosa (n = 7)

Experimental Example 6

To determine the clinical symptoms and lesions of the upper respiratory system following nasal administration, three New Zealand male rabbits weighing 2.5-3 kg per group were negative control group (saline), group 5 of the present invention. A total of 4 groups of control A and B groups were administered, and 20 µl / kg (22 IU / kg) was repeatedly administered once a day for 2 weeks to observe general symptoms (respiratory state, nasal bleeding, presence of nasal mucus), and lesion observation. And histopathologic examination was performed. As a result, no clinical general symptoms were judged to be due to the test substance. In the lesion observation, one case of nasal patella was found in the control group B. No unusual gross abnormalities were observed in the other test groups. The histopathological examination results are shown in Table 6. In one negative control group, no lesion was found to be due to the test substance in all other test groups, except for the infiltration of inflammatory cells.

TABLE 6

       Histopathological examination after repeated administration of saline and calcitonin preparations for 2 weeks

() = Number of animals examined

Salmon calcitonin, human calcitonin, eel calcitonin, carbo calcitonin, chicken calcitonin, porcine calcitonin according to the present invention contain aminocysulfonic acid and hydroxypropylmethylcellulose, and contain a pharmaceutically acceptable carrier The pharmaceutical composition for the nasal spraying of calcitonin improves the stability of calcitonin in the liquid state, increases the absorption rate when applied to the nasal mucosa, and provides a new pharmaceutical formulation without nasal mucosal irritation.

FIG. 1 shows the residual percentage of plasma calcium concentration compared to the initial value of placebo, Example 5 composition of the present invention, Control A and Control B in rat intranasal administration, where each symbol is a placebo (▼). , Example 5 composition (●), control agent A (■) and control agent B (▲) are shown.

Figure 2 shows the concentration of systemically absorbed plasma calcitonin upon administration of Example 5 composition, Control A and Control B according to the invention in rat intranasal (n = 6), where each symbol is Example 5 (●). , Control A (■) and Control B (▲).

Claims (5)

A pharmaceutical composition for nasal spraying, comprising a stable, increased absorption from the nasal cavity, comprising salmon calcitonin, aminoethylsulfonic acid (taurine), hydroxypropylmethylcellulose, and a pharmaceutically acceptable carrier. The composition of claim 1 wherein the aminoethylsulfonic acid is 0.1 to 1% (w / w /) based on the total weight of the composition. The composition of claim 1 wherein the hydroxypropylmethylcellulose is 0.1 to 1% (w / w) based on the total weight of the composition. The composition of claim 1 in the form of a spray, inhalation or drop. The composition of claim 1 wherein the pH is from 3.0 to 5.0.