KR20090056543A - Pharmaceutical formulation comprising hepatitis b virus neutralizing human antibody - Google Patents

Abstract

A pharmaceutical agent containing a HBV(hepatitis B virus) neutralization human antibody, buffer, isotonic adjusting agent, and surfactant is provided to stabilize the antibody, and maintain the activity of antibody in a long time. A pharmaceutical agent comprises 0.1-50 mg/ml of human HBV(hepatitis B virus) neutralization antibody, 10-100 mmol/l of buffer, 20-250 mmol/l isotonic adjusting agent, and 0.001-1.0 weight% of surfactant. Antibody is a HBIG-Gene to HBV surface antigen which is produced from HBAb-49(KCLRF-BP-00054) cell line. The buffer is citrate salt, acetate salt, phosphate salt, or their free acid, base or salt or their mixture. The isotonic adjusting agent is sodium chloride, or potassium chloride. The surfactant is polyoxyethylene sorbitan fatty acid ester.

Description

PHARMACEUTICAL FORMULATION COMPRISING HEPATITIS B VIRUS NEUTRALIZING HUMAN ANTIBODY}

The present invention relates to a pharmaceutical preparation capable of stably storing hepatitis B virus (HBV) neutralizing human antibodies in a liquid phase.

Most protein therapeutics require stabilizers to prevent denaturation, precipitation and other forms of change in long term storage. Instability of protein therapeutics is evidenced by the formation of soluble / insoluble particles, which often increase with long term storage.

Techniques for stably preserving proteins in a state of biological activity are required to reflect the physical properties of the proteins. In particular, the technique of stably preserving the protein in the liquid phase is to protect many different functional groups of the protein and to maintain a three-dimensional structure related to activity.

Lyophilization (WO98 / 22136) has generally been used for the preservation of antibodies, a type of protein therapeutic. However, this method can cause significant errors in pre-use manufacturing since the user must restore the lyophilisate before use. In contrast, liquid formulations are simpler to administer, and thus, there is a greater need in the medical field.

As an example of such a liquid formulation, EP Patent 7371 discloses a formulation for intravenous administration with a pH of 3.5 to 5.0, containing immunoglobulins. However, such low pH values can lead to undesirable misfit reactions at the injection point.

In addition, US Pat. No. 6,171,586 discloses liquid antibody preparations having a pH of 4.48 to 5.5, including acetate buffers, surfactants, and polyols. However, the patent does not include isotonicity modifiers in the formulations, which can also cause inadequate reactions at the injection point.

In addition, Korean Patent Laid-Open Publication No. 2006-110305 discloses an aqueous pharmaceutical preparation containing an antibody against the epidermal growth factor receptor (EGF receptor), which contains an amino acid as an essential component for long-term preservation of the antibody. Doing.

In addition, Korean Patent Laid-Open Publication No. 2006-7016023 discloses a highly concentrated liquid formulation containing an anti-EGFR antibody, but the concentration step through ultrafiltration of the formulation is essentially included in the preparation of the formulation in a timely and economical manner. There is a burden.

An object of the present invention is to provide a pharmaceutical preparation capable of stably storing hepatitis B virus (HBV) neutralizing human antibodies in a liquid state for a long time.

In accordance with the above object, the present invention provides a pharmaceutical preparation for the prophylaxis or treatment of hepatitis B virus comprising hepatitis B virus (HBV) neutralizing human antibodies, buffers, isotonic regulators and surfactants.

The pharmaceutical preparations of the present invention can stably store hepatitis B virus (HBV) neutralizing human antibodies in a liquid state for at least one year and can be effectively used for the prevention or treatment of hepatitis B.

Formulations of the present invention include hepatitis B virus (HBV) neutralizing human antibodies, buffers, isotonicity modulators and surfactants.

At least some of the solvents present in the liquid formulations of the present invention consist of water.

The hepatitis B virus (HBV) neutralizing human antibody may be a recombinant antibody, preferably an antibody against the surface antigen of HBV produced from cell line HBAb-49 (KCLRF-BP-00054) (HBIG-Gene). .

In addition, the antibody may be a human antibody preferably comprising a heavy chain variable region (V _H ) having an amino acid sequence of SEQ ID NO: 1 and a light chain variable region (V _L ) having an amino acid sequence of SEQ ID NO: 2.

The antibody may be included in the formulation according to the invention at a concentration of 0.1 to 50 mg / ml, preferably 2 to 10 mg / ml, most preferably 5 mg / ml.

The buffer may be any substance that has physiological tolerability and has pH control, for example a citrate salt, acetate salt, phosphate salt, or free acid, base or salt thereof, or mixtures thereof. Here, the term mixture includes both mixtures of salts of different acids, such as mixtures of citrate salts and acetate salts, and mixtures of different salts of the same acid, such as mixtures of different citrate salts. The buffer may preferably be citrate salt, acetate salt or free acid thereof, and citrate salt, citric acid monohydrate, trisodium citrate dihydrate, tripotassium citrate monohydrate, and the like may be used. As the acetate salt free acid, acetic acid, sodium acetate, sodium acetate trihydrate, and the like can be used. The buffer in the formulation of the present invention may be included at a concentration of 10 to 100 mmol / L, preferably 2 to 50 mmol / L, most preferably 20 mmol / L.

As the isotonicity adjusting agent, salts having physiologically tolerant resistances such as sodium chloride or potassium chloride may be used, and sodium chloride may be preferably used. Isotonicity modifiers in the formulations of the present invention may be included at a concentration of 20 to 250 mmol / L, preferably 100 to 200 mmol / L.

As the surfactant, all surfactants commonly used in pharmaceutical preparations may be used, and polyoxyethylene sorbitan fatty acid ester (Tween ™ 80) may be preferably used. Examples of the polyoxyethylene sorbitan fatty acid esters include polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate or polyoxyethylene sorbitan monooleate. It is possible to use, preferably polyoxyethylene sorbitan monolaurate or polyoxyethylene sorbitan monooleate, and most preferably polyoxyethylene sorbitan monooleate.

In the formulation of the present invention the surfactant may be present in the formulation at a concentration of 0.001 to 1.0% by weight. When polyoxyethylene sorbitan fatty acid ester is used as the surfactant in the formulation of the present invention, it may be included in an amount of 0.005 to 0.1% by weight, preferably in an amount of about 0.05% by weight.

In addition, the pharmaceutical formulations of the present invention have an isotonic range, that is, an osmolality of 250 to 350 mOsmol / kg, in order to increase tolerability to parenteral administration. In this way, the preparation can be administered directly without substantial pain in intravenous or arterial, subcutaneous, muscle or the like. The pH of the formulation may be 4.0 to 7.0, preferably 5.0 to 6.0, most preferably 5.5.

In a particularly preferred embodiment of the invention, the pharmaceutical preparation comprises 10,000 units (5 mg / ml) of HBIG-Gene, 20 mmol / L acetate buffer (pH 5.5), 150 mmol / L sodium chloride and 0.05% polyoxy Ethylene sorbitan monooleate.

The pharmaceutical preparations according to the invention can be prepared by adding the remaining components of the preparation as an adjuvant to an acetate buffer containing said antibody (pH 5.5) or a solution comprising sodium chloride therein.

An amount of the stock solution containing the concentration of the auxiliaries may be added to the solution containing the concentration of the antibody, and if necessary, the mixture may be diluted to a pre-calculated concentration with water or a buffer. It is also possible to add these adjuvants as a solid to the starting solution containing the antibody. In addition, when the antibody is in solid form, for example lyophilisate, the formulation according to the invention first dissolves the antibody in water or in a liquid solution containing at least one adjuvant, and then in the adjuvant, solid form It can be prepared by adding a stock solution containing adjuvants and / or water in the required amount. The antibody can also be dissolved directly in a solution containing all adjuvants.

One or more adjuvants included in the pharmaceutical formulations of the present invention may optionally be added during or at the end of the preparation of the recombinant antibody. In one embodiment of the invention, HBIG-Gene may be dissolved directly in a liquid solution containing one or more or all adjuvants in the final stage of the purification carried out after its preparation. Formulations of the present invention may also contain adjuvants having physiological tolerability, for example antioxidants such as ascorbic acid, glutathione; Preservatives such as phenol, m-cresol, methyl- or propylparaben, glorobutanol, thiomersal, benzalkonium chloride; Polyethylene glycol (PEG) such as PEG 400, PEG 3000, PEG 3500, PEG 4000, PEG 6000; Disaccharides such as trihalose, saccharose; Or additional adjuvants, if desired, in small amounts such as hydroxypropyl-β-cyclodextrin, sulfobutylethyl-β-cyclodextrin, α-cyclodextrin, cyclodextrin such as γ-cyclodextrin. It is particularly preferred that these additional auxiliaries are dissolved directly in the liquid solution containing all such adjuvants in the final stage of purification carried out after the preparation of the antibody. In addition, when the solution comprising the antibody and the adjuvant does not have the desired pH, it can be adjusted by adding an acid or base, preferably an acid or base already present in the buffer system. In addition, bactericidal filtration may be performed on the pH-adjusted solution.

The pharmaceutical preparations of the present invention are physiologically well tolerated, simple to manufacture, high in dosage accuracy and high in safety to minimize the formation of degradation products and aggregates over storage periods. The formulations of the present invention can maintain the stability of the antibody even when the antibody is stored at a temperature of 2 to 8 ° C. for at least 1 year, and also stably store the antibody for 3 months or more at a temperature of 25 ° C. and a relative atmospheric humidity of 60%. have.

The pharmaceutical preparations according to the invention can be used for the prevention or treatment of hepatitis B, preferably for liver transplant surgery or the treatment of chronic hepatitis B through neutralization of HBV.

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.

Comparative Example 1A: Preparation of HBIG-Gene Formulation

10,000 units (5 mg / ml) HBIG-Gene (Green Cross, Korea); And hydrochloric acid was added to the solution prepared by dissolving maltose of 10% by weight in distilled water to adjust the pH to 4.0, and then sterile filtered and stored at 25 ℃, 60% relative atmospheric humidity.

Comparative Example 1B: Preparation of HBIG-Gene Formulation

A solution was prepared and stored in the same manner as in Comparative Example 1A, except that the solution was stored at 5 ° C.

Comparative Example 2A: Preparation of HBIG-Gene Formulation

10,000 units (5 mg / ml) HBIG-Gene; 10 mmol / L sodium phosphate buffer (1.2 g / L sodium dihydrogen phosphate), pH 7.2; And a solution containing 150 mmol / L sodium chloride were prepared and then sterile filtered and stored at 25 ° C. and 60% relative atmospheric humidity.

Comparative Example 2B: Preparation of HBIG-Gene Formulation

A solution was prepared and stored in the same manner as in Comparative Example 2A, except that the solution was stored at 5 ° C.

Comparative Example 3A: Preparation of HBIG-Gene Formulations

10,000 units (5 mg / ml) HBIG-Gene; 25 mmol / l citrate buffer with 7.3525 g / l citric acid tribasic dihydrate (pH 6.5); And a liquid solution containing 150 mmol / L sodium chloride were prepared and then filtered and stored at 25 ° C. and 60% relative atmospheric humidity.

Comparative Example 3B: Preparation of HBIG-Gene Formulation

The solution was prepared and stored in the same manner as in Comparative Example 3A, except that the solution was stored at 5 ° C.

Comparative Example 4A: Preparation of HBIG-Gene Formulation

10,000 units (5 mg / ml) HBIG-Gene; 20 mmol / L acetate buffer (containing 7.3525 g / L of 2.7216 g / L sodium acetate trihydrate) at pH 5.5; And a liquid solution containing 150 mmol / L sodium chloride were prepared and then filtered and stored at 25 ° C. and 60% relative atmospheric humidity.

Comparative Example 4B: Preparation of HBIG-Gene Formulation

A solution was prepared and stored in the same manner as in Comparative Example 4A, except that the solution was stored at 5 ° C.

Example 1A: Preparation of HBIG-Gene Formulations

10,000 units (5 mg / ml) HBIG-Gene; 10% by weight maltose; And 0.05% by weight of a polyoxyethylene (80) sorbitan monooleate solution was prepared to adjust the pH to 4.0 and then sterile filtered and stored at 25 ℃, 60% relative atmospheric humidity.

Example 1B: Preparation of HBIG-Gene Formulations

The solution was prepared and stored in the same manner as in Example 1A, except that the solution was stored at 5 ° C.

Example 2A: Preparation of HBIG-Gene Formulations

10,000 units (5 mg / ml) HBIG-Gene; 10 mmol / L sodium phosphate buffer (1.2 g / L sodium dihydrogenphosphate), pH 7.2; 150 mmol / L sodium chloride; And 0.05% by weight of a polyoxyethylene (80) sorbitan monooleate was prepared, followed by sterilization filtration and storage at 25 ° C. and 60% relative atmospheric humidity.

Example 2B: Preparation of HBIG-Gene Formulations

The solution was prepared and stored in the same manner as in Example 2A, except that the solution was stored at 5 ° C.

Example 3A: Preparation of HBIG-Gene Formulations

10,000 units (5 mg / ml) HBIG-Gene; 25 mmol / l citrate buffer (7.3525 g / l citric acid tribasic dihydrate), pH 6.5; 150 mmol / L sodium chloride; And 0.05% by weight of a polyoxyethylene (80) sorbitan monooleate was prepared, followed by sterilization filtration and storage at 25 ° C. and 60% relative atmospheric humidity.

Example 3B: Preparation of HBIG-Gene Formulations

The solution was prepared and stored in the same manner as in Example 3A, except that the solution was stored at 5 ° C.

Example 4A: Preparation of HBIG-Gene Formulations

10,000 units (5 mg / ml) HBIG-Gene; 20 mmol / L acetate buffer (2.7216 g / L sodium acetate trihydrate), pH 5.5; 150 mmol / L sodium chloride; And 0.05% by weight of a polyoxyethylene (80) sorbitan monooleate was prepared, followed by sterilization filtration and storage at 25 ° C. and 60% relative atmospheric humidity.

Example 4B: Preparation of HBIG-Gene Formulations

The solution was prepared and stored in the same manner as in Example 4A, except that the solution was stored at 5 ° C.

Test Example 1 Formulation Stability Experiment

For the solutions of Examples 1A to 4B and Comparative Examples 1A to 4B, before the storage and after a certain storage time, visual evaluation for property confirmation, protein content measurement by absorbance measurement (280 nm), and titer confirmation by ELISA measurement, respectively Changes in aggregates and degradation products by HPLC gel filtration size exclusion chromatography (SEC), reduction and non-reduction electrophoresis (SDS-PAGE), Western blot, isoelectric focusing (IEF) It measured and the result is shown in following Table 1 and Table 2. The specific measuring method is as follows.

(1) Visual evaluation for property identification

The color, appearance and transparency of the solution were evaluated by visually inspecting the vials containing each formulation at room temperature and white fluorescence.

(2) protein content measurement

For each solution, absorbance on a spectrophotometer was diluted 5 to 10 times, 15 to 30 times, or more with a solution of the same component (280 nm), 1.40 (mg / mL) ^-1 cm ^-1 The protein concentration was determined using the extinction coefficient of.

(3) potency check

Titers were measured according to the method disclosed in Shin et al. (Shin YW et al, Antiviral Research, 75, p113-120, 2007).

(4) Determination of contents of aggregates and degradation products

Size exclusion chromatography (SEC) was performed using a column (TSK G3000 SWXL ™, 7.8 × 300 mm), Gilson 321 pump, and UV / VIS-155 system. Dilute the solution to 1 mg / mL using mobile phase (50 mmole / L MES, 150 mmole / L sodium chloride, 1.5 mole / L L-arginine monohydrochloride; pH 6.0) and equilibrate at 0.5 mL / min for 30 minutes. Eluted with isocratic (injection volume: 50 μl). The eluate was monitored at absorbance 280 nm and integration was performed using Gilson Trillution ™ LC 1.4 software.

(5) Electrophoresis Analysis (SDS-PAGE)

After heating the solution under reduced or non-reduced buffer for 5 minutes at 70 ° C., 5 μg of the resulting protein was applied at 100 V on a protein electrophoresis gel (Invitrogen, USA) to within 5 mm of the lower end of the gel. Coomassie staining and discoloration observed the bands.

(6) western blot

After heating the solution under reduced or non-reducing buffer for 5 minutes at 70 ° C., 1 μg of the resulting protein was applied to a protein electrophoresis gel (Invitrogen, USA) at 100 V to within 5 mm of the lower end of the gel. It was. The protein developed on the gel is electrically transferred to nitrocellulose (NC) membrane, reacted with 5% skim milk powder / PBS for 1 hour, and then reacted with goat anti-human IgG peroxidase followed by PBST. Diluted 3 times for 5 minutes. The bands were identified by color development with TMB membrane substrate (KPL, USA).

(6) isoelectric focusing (IEF)

Isoelectric point analysis was performed on a Multiphor ™ II system (GE healthcare, Sweden) using an Ampoline PAGplate ™ pH 3.5-9.5 gel.

(7) pH measurement

The pH measurement of the solution was carried out according to the KEPCO General Test Method "pH measurement method".

In the case of the pharmaceutical formulations of Examples 1A, 2A, 3A and 4A, and Comparative Examples 1A, 2A, 3A and 4A stored at 25 ° C. and 60% relative humidity, the results were confirmed.

In addition, Comparative Examples 1B, 2B, 3B, and 4B containing no surfactant in the formulations stored at 5 ° C. were stored for up to 6 months, and the results were confirmed. Examples 1B, 2B, 3B, and 4B containing all the compositions were included. For 12 months, the results were confirmed.

As can be seen in Tables 1 and 2, the pharmaceutical formulations of Examples 1A, 2A, 3A and 4A, especially the pharmaceutical formulations of Example 4A, stored at 25 ° C. and 60% relative humidity, showed excellent results in terms of stability. Indicated.

In addition, when stored at 5 ℃ it was observed that the particles and precipitates produced in Comparative Examples 1B, 2B, 3B and 4B containing no surfactant, Examples 1B, 2B, 3B and 4B containing all compositions Stability remained until 12 months.

From these results, it can be seen that the formulation of the present invention can maintain the stability of the antibody even when the antibody is stored in the liquid state.

Test Example 2: Formulation Safety Experiment

To determine the safety of human formulations of the formulations according to the invention, the formulations of the invention were reacted with the blood of monkeys and humans to determine the hemolytic potential.

Specifically, the hemolytic potential of the antibody formulation was determined by the formulation of Example 4A (1, 5 and 10 mg / ml), the liquid composition excluding the antibody in the formulation (20 mM sodium acetate, 150 mM sodium chloride and 0.05% Tween ™ 80; pH 5.5), 1% saponin (positive control), monkey plasma (negative control); Alternatively, human plasma (negative control) was mixed with monkeys or human blood of the same volume, and then reacted with each other, followed by centrifugation, and the concentration of hemoglobin, a marker of hemolyzed red blood cells, was confirmed by spectrophotometric analysis. The results are shown in Table 3.

As can be seen in Table 3, the antibody of the present invention showed no hemolysis phenomenon when reacted with the same volume of monkey or human blood.

In addition, in order to confirm the compatibility with the plasma of the formulation of the present invention, the formulation of Example 4A (1, 5 and 10 mg / ml) or the liquid composition excluding the antibody in the formulation and the same volume of monkey or human plasma, respectively After reacting by mixing, the presence of agglomerates and precipitates was evaluated. The results are shown in Table 4.

As can be seen from the results in Table 4, the formulation of the present invention showed no abnormality in compatibility with monkey or human plasma. From these results, it was confirmed that the preparation according to the present invention is a safe preparation that does not hemolyze the blood of monkeys and humans.

Test Example 3: Formulation Safety Test

To confirm the safety of human administration of the formulation of the present invention, the formulation of the present invention was injected into the vein, perivenous and artery of 16-week-old male New Zealand white rabbits (Hra: (NZW) SPF rabbits) to evaluate local site tolerance. It was.

Specifically, as summarized in Table 5 below, the rabbits were divided into groups 1 and 2, and in the rabbits of group 1, the formulation of Example 4A (5 mg / mL (10,000 units)) was applied to the left ear vein (test site C). 1.0 ml, injected intravenously (0.21 ml) into the vein (test site D), and as a control liquid composition (20 mM sodium acetate, 150) excluding the antibody in the formulation in the right ear vein (test site A) and perivenous (test site B). mM sodium chloride and 0.05% Tween 80; pH 5.5) were injected 1.0 ml and 0.21 ml, respectively. Rabbits of Group 2 were treated with the formulation of Example 4A (5 mg / mL (10,000 units)) and the liquid composition (20 mM sodium acetate, 150 mM sodium chloride and 0.05% Tween 80; pH 5.5) excluding the antibody in the formulation. 0.5 ml each was injected into the artery (test site F). The incidence of edema and erythema was observed in 3 rabbits immediately before, 4 hours after injection, 2 days after injection, and 3 days after injection, respectively. Indicated.

As can be seen from the results shown in Tables 6 to 9, it was confirmed that the formulation of the present invention did not show serious side effects in intravenous, perivenous and arterial injection.

<110> KOREA GREEN CROSS CORPORATION <120> Pharmaceutical formulation comprising hepatitis B virus          neutralizing human antibody <130> FPD / 200710-0095 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Variable region of human antibody H chain <400> 1 Gln Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Ser Leu Thr Lys Tyr              20 25 30 Lys Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ser Ile Ser Ser Thr Ser Arg Asp Ile Asp Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe  65 70 75 80 Leu Gln Met Ser Ser Leu Arg Val Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Thr Arg Asp Gly Trp Leu Trp Gly Trp Asp Val Arg Ser Asn Tyr Tyr             100 105 110 Tyr Asn Ala Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser         115 120 125 Ser     <210> 2 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Variable region of human antibody L chain <400> 2 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Asn Ser              20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu          35 40 45 Tyr Ser Thr Ser Thr Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Thr Asn Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Val Thr Pro Glu                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105  

Claims (15)

Pharmaceutical preparations containing human hepatitis B virus (HBV) neutralizing antibodies, buffers, isotonicity modulators and surfactants. The method of claim 1, Pharmaceutical agent, characterized in that the antibody against the surface antigen of HBV produced from the cell line HBAb-49 (KCLRF-BP-00054) (HBIG-Gene). The method of claim 1, A pharmaceutical formulation, characterized in that the antibody is contained at a concentration of 0.1 to 50 mg / ml. The method of claim 1, Pharmaceutical formulation, characterized in that the buffer is a citrate salt, acetate salt, phosphate salt, or free acids, bases or salts thereof, or mixtures thereof. The method of claim 1, Pharmaceutical formulation, characterized in that the buffer is an acetate salt or free acid thereof. The method of claim 1, Pharmaceutical formulation, characterized in that the buffer is included at a concentration of 10 to 100 mmol / l. The method of claim 1, Pharmaceutical formulation, characterized in that the isotonicity regulator is sodium chloride or potassium chloride. The method of claim 1, Pharmaceutical formulation, characterized in that the isotonicity regulator is included in a concentration of 20 to 250 mmol / l. The method of claim 1, Pharmaceutical formulation, characterized in that the surfactant is polyoxyethylene sorbitan fatty acid ester. The method of claim 9, Pharmaceutical formulation, characterized in that the surfactant is polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate or polyoxyethylene sorbitan monooleate. The method of claim 1, Pharmaceutical formulation, characterized in that the surfactant is included in a concentration of 0.001 to 1.0% by weight. The method of claim 1, Pharmaceutical formulation, characterized in that it further comprises a physiologically tolerant adjuvant. The method of claim 13, Pharmaceutical formulation, characterized in that the adjuvant is an antioxidant, preservative, polyethylene glycol (PEG), disaccharide, or cyclodextrin. The method of claim 1, A pharmaceutical formulation having a molar penetration pressure concentration of 250 to 350 mOsmol / kg and a pH of 4.0 to 7.0. The method of claim 15, Pharmaceutical formulation, characterized in that the pH is 5.5.