Liquid formulation containing botulinum toxin and stabilizing agent, and preparation method therefor
11224640 · 2022-01-18
Assignee
Inventors
Cpc classification
A61K47/18
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61K31/167
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K47/26
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
A61K47/08
HUMAN NECESSITIES
A61P1/18
HUMAN NECESSITIES
C12Y304/24069
CHEMISTRY; METALLURGY
International classification
A61K47/18
HUMAN NECESSITIES
A61K31/167
HUMAN NECESSITIES
A61K47/08
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention relates to a liquid formulation containing botulinum toxin and stabilizing agent, and preparation method therefor. A liquid formulation containing botulinum toxin and stabilizing agent according to the present invention can be easily stored and distributed. It was proved a significant effect on the stabilization of botulinum toxin under suitable conditions according to the temperature and pH of the human body. Thus, it is expected that the pharmaceutical composition of the present invention will greatly contribute to the safe and convenient medical use of botulinum toxin.
Claims
1. A pharmaceutical formulation, comprising a neurotoxin, and gluconolactone, wherein the neurotoxin is botulinum toxin type A, wherein the botulinum toxin type A is either in a non-complex form or in a complex form with hemagglutinin proteins (HA) or non-toxic non-hemagglutinin proteins (NTNH).
2. The pharmaceutical formulation of claim 1, wherein the gluconolactone is provided in the form of a stabilizing buffer.
3. The pharmaceutical formulation of claim 1, wherein the concentration of gluconolactone per 100 units of the botulinum toxin type A is 0.01-1,000 mM.
4. The pharmaceutical formulation of claim 1, further containing polysorbate.
5. The pharmaceutical formulation of claim 1, which is liquid formulation.
6. A method for preparing a pharmaceutical formulation, comprising the steps of: (a) purifying a neurotoxin; and (b) adding gluconolactone to the neurotoxin, wherein the neurotoxin is botulinum toxin type A, wherein the botulinum toxin type A is either in a non-complex form or in a complex form with hemagglutinin proteins (HA) or non-toxic non-hemagglutinin proteins (NTNH).
7. The method of claim 6, wherein the gluconolactone is provided in the form of a stabilizing buffer.
8. The method of claim 6, which is liquid formulation.
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
(13) The results of the experiment performed in the present invention showed that the stabilizing effects of arginine and methionine on botulinum toxin were pH-dependent. Based on these results, the pH of liquid BoNT/A formulations was set to 6.0, and the concentration of arginine in the liquid BoNT/A formulations was changed to various concentrations. BoNT/A was added to the formulations to an initial potency of 80 units/ml, and then the formulations were incubated at 37° C. for 8 weeks, and the potency of the BoNT/A was measured by a DESCR assay. As a result, it was shown that the residual potency of the BoNT/A in the control group containing no stabilizer was 10% after 2 weeks, and the experimental group containing 50 mM methionine showed residual potencies of 67%, 47% and 27% after 2 weeks, 4 weeks and 8 weeks, respectively. This demonstrated that methionine has a significant stabilizing effect. Meanwhile, arginine showed a stabilizing effect greater than methionine at a concentration of 50 to 100 mM, and the residual potency of the BoNT/A in the formulation containing methionine was 31 to 65% even after 8 weeks.
MODE FOR INVENTION
(14) Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
(15) The sources of all reagents used in the examples of the present invention are listed in the following (*).
(16) *Gluconolactone (Sigma G2164); L(+)-tartaric acid (Merck 100804); lidocaine hydrochloride monohydrate (Sigma L5647); octanoic acid (Sigma C2875); L-methionine (Merck K45023607 414); L-arginine (Merck K45895542 534); glycine (Bioshop GLN001-1); L-Glutamic acid (Merck 100291); aspartic acid (Merck K45895542 534); maleic acid (Merck S6858580 534); butylated hydroxyanisole (Sigma SLBM1210V); propyl gallate (Sigma P3130); sodium bisulfite (Sigma MKBR6468V); thioglycolic acid (Sigma T3758); L-cystein hydrochloride (K46446495 513); succinic acid (Merck K46618782 533); sodium phosphate monobasic (Sigma S5011); sodium phosphate dibasic (Sigma S7907); sodium chloride (Merck K47013904 548); polysorbate (Sigma P7949); and DL-dithiothreitol (Sigma D0632).
(17) The abbreviations and the composition of the buffer used in the examples of the present invention are listed in the following (**).
(18) **L-methionine (Met, or “M”), L-arginine (Arg, or “R”), L-Glutamic acid (Glu, or “E”), aspartic acid (Asp, or “D”), Buffer P (10 mM NaPO.sub.4, pH 6.0, 45 mM NaCl, and 0.05% polysorbate), Buffer G (10 mM gluconolactone, pH 6.0, 45 mM NaCl, and 0.05% polysorbate), Buffer R (buffer G supplemented with 50 mM arginine).
Example 1: Development of Botulinum Toxin Stabilizer Using BoTest
Example 1-1: Preparation of Experiments
(19) A botulinum toxin used in the present invention was one produced by Hugel Pharma Co., Ltd. (Korea) and adjusted to a concentration of 0.1 mg/ml and potency of 1,529 units/ (based on the BoTest). In all experiments for identification of additives having a stabilizing effect, the botulinum toxin was used after it was diluted to 50 units/
with a “protein dilution buffer” having a composition (50 mM NaPO.sub.4, pH 7.0, 1 mM DTT, 0.05 wt % polysorbate, and 20 wt % glycerol). For identification of additive candidates having a stabilizing effect, each experimental group (100
) was prepared by diluting 200 units of the botulinum toxin and a stabilizer additive candidate in a “stabilizing liquid composition (10 mM NaPO.sub.4 (pH 5.5-7.0), 0.01 wt % polysorbate, 130 mM NaCl)”. Then, each experimental group was incubated at a temperature of 37° C. for 1-11 weeks, and a portion (25%) thereof was used for measurement of the residual potency thereof. The potency of the botulinum toxin was measured using a BoTest® Botulinum Neurotoxin Detection Kit (BioSentinel, USA). For this, 5 mM HEPES-NaOH (pH 7.1), 0.1 wt % polysorbate, 10 uM ZnCl.sub.2 (Sigma 229997), 0.2 uM BoTest A/E reporter, and 25
stabilizing experimental group were mixed with one another to prepare a final reaction solution (100
), and the reaction solution was incubated at 37° C. for 21 hours. The CFP/FRET ratio of the incubated reaction solution was measured using a Synergy Neo2 Multi-Mode Reader (BioTek, USA) system and applied to a standard curve, thereby determining the residual potency of the botulinum toxin. In addition, all reagents used in the study on stabilization of the botulinum toxin according to the present invention were dissolved in triple-distilled water and adjusted to a pH of 5.5-7.0 by the addition of hydrochloric acid and sodium hydroxide.
Example 1-2: Comparison of Stabilizing Effects of Arginine or Methionine at Varying pHs
(20) The effects of arginine and methionine as stabilizers on the stabilization of botulinum toxin at a pH ranging from 5.5 to 7.0 were comparatively examined.
(21) For this, botulinum toxin was added to the stabilizing liquid composition described in Example 1, and 50 mM of arginine or methionine was further added to the liquid composition. The resulting composition was incubated at 37° C. for 28-56 days, and then the residual potency of the botulinum toxin was measured. The results of the measurement are shown in Table 1 below and
(22) TABLE-US-00001 TABLE 1 0 day 28 days 56 days pH 5.5 50 mM arginine 100 34.2 27.5 50 mM methionine 100 1.22 9.01 Negative control 100 0.97 −0.34 pH 6.0 50 mM arginine 100 28.2 32.6 50 mM methionine 100 7.5 −0.66 Negative control 100 8.38 −0.63 pH 6.5 50 mM arginine 100 23.6 16.2 50 mM methionine 100 13.6 8.78 Negative control 100 −1.06 −0.8 pH 7.0 50 mM arginine 100 19.6 1.72 50 mM methionine 100 1.63 −0.94 Negative control 100 −0.83 0.11
(23) As can be seen from the experimental results, in the negative control group containing no stabilizing candidate, the botulinum toxin was unstable at all the pHs, and thus the residual potency of the botulinum toxin was not substantially detected after 28 days. The experimental group containing methionine as a stabilizer a residual potency of about 10% at pH 5.5 to 7.0, whereas the experimental group containing arginine showed a residual potency of up to 30%. In addition, it was measured that the effect of arginine on the stabilization of the botulinum toxin was higher at a pH ranging from 5.5 to 6.0 than at a pH ranging from 6.5 to 7.0.
Example 1-3: Comparative Examination of Stabilizing Effects of Arginine or Methionine
(24) In order to verify the effect of arginine or methionine on the stabilization of botulinum toxin, a botulinum toxin composition containing arginine (50 mM) or methionine (50 mM) as a stabilizing additive was incubated for 56 days, and the residual potency of the botulinum toxin was measured in three independent experiments. The results of the measurement were statistically processed, thereby comparatively verifying the effects of arginine and methionine. As a control group, a sample containing no additive was used. To determine the significance between the three experimental groups, the one-way ANOVA method was used. When the significance probability (p-value) was 0.05 or less, it was determined that there was a significant difference between the three experimental group, and post-hoc analysis was performed by the LSD (Least Significant Difference) method. The results of the measurement are shown in Table 2 below and
(25) TABLE-US-00002 TABLE 2 Stabilization at 37° C. pH 0 day 14 days 28 days 56 days 50 mM arginine 6.0 100 90.1 ± 5.5 71.6 ± 7.5** .sup. 69.1 ± 14** 6.5 100 71.9 ± 2.1* 70.6 ± 9.6** 38.4 ± 8.2** 7.0 100 70.1 ± 9** 55.8 ± 8.2*** 16.9 ± 6.8* 50 mM methionine 6.0 100 57.6 ± 12 27.2 ± 8.3 22.6 ± 8.5 6.5 100 .sup. 55 ± 4.4 36.7 ± 3.3 11.1 ± 3.8 7.0 100 40.2 ± 2.7 17.1 ± 4.3 0.35 ± 1.4 Negative control 6.0 100 47 ± 14 21.7 ± 5.6 −1.22 ± 0.2 6.5 100 29.1 ± 7.5 17.3 ± 6.9 −1.27 ± 0.3 7.0 100 5.16 ± 4.8 3.37 ± 2.7 1.37 ± 0.6
(26) The results obtained in this Example indicate that arginine and methionine all show the high stabilizing effect at a pH of 6.0, and these experimental results are consistent with the above-described experimental results. However, under the same pH condition, arginine showed a higher stabilizing effect compared to methionine in all the experimental groups. For example, in the control group containing no stabilizer, the potency of the botulinum toxin was not measured under all conditions after 56 days of incubation, but in the experimental group containing methionine, a residual potency of 22.6% was measured at a pH of 6.0, and in the experimental group containing arginine, a potency of 69.1% was measured under the same condition.
(27) The significance of the relative stabilizing effect of arginine and methionine was examined using the one-way ANOVA method, and as a result, it was shown that, at a pH of 6.0, the stabilizing effect values were significant in all the experimental groups except for the experimental group incubated for 14 days. For such results, post-hoc analysis was performed by the LSD (Least Significant Difference) method, and as a result, it was shown that the stabilizing effect of arginine was significantly better than that of methionine at a level of p<0.05 to p<0.001. For example, the comparison of values measured after 28 days of incubation at a pH of 7.0 indicated that the experimental group containing methionine showed a residual potency of 17.1% and the experimental group containing arginine showed a residual potency of 55.8%, and thus there was a significant difference of p<0.001 in the stabilizing effect between the two groups. In addition, the comparison of values measured after 56 days of incubation at a pH of 6.0 indicated that the experimental group containing methionine showed a residual potency of 22.6% and the experimental group containing arginine showed a residual potency of 69.1%, and thus there was a significant difference of p<0.01 in the stabilizing effect between the two groups.
(28) And the present inventors examined the effect of the surfactant property of polysorbate on the stabilizing effect of botulinum toxin when methionine or arginine is added. For this, the botulinum toxin and 50 mM of arginine or methionine were added to a stabilizing liquid composition containing 0-0.05 wt % of polysorbate, and the resulting composition was incubated at 37° C. for 28-56 days, after which the residual potency of the botulinum toxin was measured. From the experimental results, in the case of the stabilizing composition containing methionine, at a pH ranging from 5.5 to 6.0, the stabilizing effect showed a tendency to increase in proportion to the concentration of poly sorbate, and at a pH ranging from 6.5 to 7.0, the effect of polysorbate generally did not appear. In the case of the experimental group containing arginine, at a pH ranging from 5.5 to 6.5, the addition of polysorbate showed a tendency to contribute to the stabilizing effect, but the effect of polysorbate was not so significant. Such results suggest that the addition of polysorbate to a liquid botulinum toxin formulation containing arginine as a stabilizer is not essential.
Example 1-4: Identification of New Stabilizers for Liquid Formulation of Botulinum Toxin
(29) The results of Examples 1-2 to 1-3 indicate that arginine has a better effect on the stabilization of a liquid formulation of botulinum toxin compared to methionine. However, detection of new additives showing effects similar to that of arginine enables the development of various products. For this, the present inventors comparatively examined the botulinum toxin-stabilizing effects of various stabilization candidates shown in Table 3 below. The results of the examination are shown in
(30) TABLE-US-00003 TABLE 3 Candidate for stabilization of botulinum toxin Stabilizing effect Arginine +++ Gluconolactone ++ Glycine − Tartaric acid ++ Sodium bisulfite − Cysteine − Propyl gallate − Sodium hydrosulfite + Thioglycolate −
(31)
Example 2. Development of Botulinum Toxin Stabilizer Using DESCR
Example 2-1. Preparation of Experiments
(32) In Example 1, the liquid formulation sample containing the botulinum toxin having a relatively high potency (200 units/0.1 ml) was prepared in the polypropylene tube, and the residual potency of the botulinum toxin was measured using the BoTest assay. However, this botulinum toxin concentration significantly differs from the concentration of botulinum toxins which are commercially available for clinical use. Hence, in the present invention, studies on formulations for stabilization of botulinum toxin were performed using formulations having compositions similar to those of currently commercially available botulinum products, that is, formulations in which the initial potency of botulinum toxin is 40 to 80 units/ml. Since the BoTest assay requires a potency of at least 50 units, it cannot measure the residual potency of botulinum toxin in the liquid formulations prepared under the above-described conditions. Therefore, the present inventors have developed a DESCR (Direct ELISA coupled with in vitro SNAP25 cleavage reaction) method capable of quantitatively measuring the potency of BoNT/A present in trace amounts. The DESCR method will be described in detail in Example 2-2 below.
(33) Briefly, although 200 units of BoNT/A was added to 100 μl of each liquid formulation sample and the residual potency thereof in all the formulation samples was measured by the BoTest assay in Example 1, 40 to 80 units/ml of BoNT/A was added to 0.1 to 1 ml of each liquid formulation sample and the residual potency thereof in all the formulation samples was measured by the DESCR method (newly developed by the present inventors) in Example 2.
(34) For identification of additive candidates having a stabilizing effect, a sample of each experimental group, which contained 10 mM NaPO.sub.4 (pH 6.0), 10 mM tartaric acid (pH 6.0) or 10 mM gluconolactone (pH 6.0) as a buffer, was used as a negative control, and all the formulation samples commonly contained 0.05% polysorbate, 45 to 130 mM NaCl, botulinum toxin and a stabilizer additive candidate. These formulation samples were incubated at 37° C. for 2 to 8 weeks, and 10 μl (0.4 units) of each formulation sample was used for the measurement of the residual potency of the botulinum toxin. Experimental methods whose detailed description was omitted were as described in Example 1 above.
Example 2-2. Development of DESCR Measurement Method
(35) DESCR (Direct ELISA coupled with in vitro SNAP25 cleavage reaction) consists of the following two steps:
(36) (1) performing an in vitro enzymatic reaction between BoNT/A and a highly pure recombinant protein (GST-SNAP25) as a substrate (in vitro SNAP25 cleavage reaction); and
(37) (2) quantitatively measuring the degree of the enzymatic reaction by enzyme-linked immunosorbent assay (ELISA).
(38) The degree of the reaction is detected by a color reaction. Specifically, it can be detected by a color reaction using a primary antibody, which reacts specifically with a form (SNAP25197) cleaved by BoNT/A, and a HRP (horseradish peroxidase)-conjugated secondary antibody. Each of the steps is performed as follows.
(39) (1) In Vitro SNAP25 Cleavage Reaction
(40) The botulinum toxin used in the experiment was diluted to various concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.2 and 1.6 units) and subjected to an enzymatic reaction in 20 μl of a buffer solution (20 mM HEPES-NaOH (pH 7.1), 0.1% Tween 20, 10 μM ZnCl.sub.2, and 1 μg GST-SNAP25) at 37° C. for 21 hours.
(41) (2) ELISA
(42) 80 μl of RSB (Reaction Stop Buffer; 125 mM carbonate (pH 9.6, Sigma S6014), and 6.25 mM EDTA) was added to the reaction solution in order to stop the BoNT/A reaction, and the reaction solution was transferred onto Maxisorp Immuno-plate (NUNC, Cat No. 170-6531), followed by coating at 37° C. for 2 hours. Each well was washed three times with WB (Washing Buffer; 1×PBS containing 0.05% Tween-20, and 0.2 M NaCl), and then blocked with BS (Blocking Solution; 5% skim milk in 1×PBS) at 37° C. for 15 minutes. Next, each well was washed once with WB, and 100 μl of SNAP25197-specific antibody (1:250 dilution, R&D) diluted in BS was dispensed into each well and allowed to react at 37° C. for 1 hour. After washing three times with WB, 100 μl of HRP-conjugated secondary antibody (1:1000 dilution, AbFrontier, LF-SA8001) diluted in BS was dispensed into each well and allowed to react at 37° C. for 1 hour. After washing three times with WB, 100 μl of TMB substrate (Thermo-fisher, Cat No. 34028) was dispensed into each well to induce a color reaction. The reaction was stopped by adding the same amount of 2M sulfuric acid (Sigma, Cat No. 258105), and the absorbance at 450 nm was measured using an absorption analyzer (Multi-Mode Reader Synergy Neo2, BioTek) system, and the AU value was calculated. These steps are schematically shown in
Example 2-3. Comparison of the Effects of Arginine and Methionine on Stabilization of Liquid Botulinum Formulation
(43) The results of Example 1 showed that the stabilizing effects of arginine and methionine on botulinum toxin were pH-dependent. Specifically, in an experimental group containing methionine as a stabilizer, the botulinum toxin showed a residual potency of up to about 10% in the pH range of 5.5 to 7.0, but in an experimental group containing arginine, a residual potency of up to about 30% was measured. Furthermore, it was shown that the effect of arginine on the stabilization of BoNT/A was higher at a pH of 5.5 to 6.0 than at a pH of 6.5 to 7.0. In a negative control group containing stabilizer additive candidate, BoNT/A tended to be unstable under all the pH conditions, and thus the residual potency of BoNT/A was not detected after 28 days.
(44) Based on these results, the pH of liquid BoNT/A formulations was set to 6.0, and the concentration of arginine in the liquid BoNT/A formulations was changed to various concentrations. BoNT/A was added to the formulations to an initial potency of 80 units/ml, and then the formulations were incubated at 37° C. for 8 weeks, and the potency of the BoNT/A was measured by a DESCR assay. The results of the measurement are shown in
(45) As a result, it was shown that the residual potency of the BoNT/A in the control group containing no stabilizer was 10% after 2 weeks, and the experimental group containing 50 mM methionine showed residual potencies of 67%, 47% and 27% after 2 weeks, 4 weeks and 8 weeks, respectively. This demonstrated that the methionine has a significant stabilizing effect. Meanwhile, arginine showed stabilizing efficacy higher than methionine at a concentration of 50 to 100 mM, and the residual potency of the BoNT/A in the formulation containing methionine was measured to be 31 to 65% even after 8 weeks. The difference in the relative stabilizing effects of methionine and arginine was also observed in formulations containing 0.3% lidocaine. In other words, after 8 weeks, an experimental group containing methionine showed a residual potency of 27%, and an experimental group containing arginine showed a residual potency of 44 to 62%.
(46) The above results show that: (1) the stabilizing effect of arginine is verified regardless of various measurement methods, including the BoTest assay and the DESCR assay; (2) arginine exhibits a stabilizing effect even under the conditions of formulations containing a botulinum toxin having a potency similar to those of products which are actually prepared/distributed for clinical use; and (3) the stabilizing effect of arginine is maintained even in formulations containing lidocaine.
Example 2-4. Evaluation of the Effect of Liquid Formulation Container Material on Stabilization of BoNT/A Efficacy
(47) Example 2-3 above shows the results obtained by preparing the botulinum toxin-containing formulations in polypropylene tubes. However, since botulinum toxin liquid formulations that are actually distributed for clinical use are prepared in glass containers, the stabilizing effects of methionine and arginine on the residual potency of botulinum toxin were evaluated again using glass containers. Specifically, BoNT/A was prepared into liquid formulations having an initial potency of 40 units/ml and containing 20 mM methionine or 100 mM arginine, and the residual potency of the BoNT/A in the formulations was measured by the DESCR assay while the formulations were incubated at 37° C. for 8 weeks. The results of the measurement are shown in
(48) As a result, the residual potency of the BoNT/A in the formulation containing methionine was measured to be 41%, 15% and 8% after 2 weeks, 4 weeks and 8 weeks, respectively. Meanwhile, the residual potency of the BoNT/A in the formulation containing arginine was measured to be 84%, 64% and 43% after the same periods, and showed a great difference from the residual potency of the BoNT/A in the formulation containing methionine.
(49) Even when the liquid formulations contained lidocaine, methionine and arginine all showed a stabilizing effect. Specifically, the residual potency of the BoNT/A in the formulation containing methionine was measured to be 47%, 21% and 16% after 2 weeks, 4 weeks and 8 weeks, respectively, and the residual potency of the BoNT/A in the formulation containing arginine was measured to be 79%, 71% and 55%, indicating that the stabilizing effect of arginine significantly differs from that of methionine.
(50) The above results show that: (1) the potency of BoNT/A in a formulation sample prepared in a polypropylene tube is more stably maintained than the potency of BoNT/A in a formulation sample prepared in a glass container, even if the formulations have the same composition; and (2) the stabilizing effect of arginine on a BoNT/A liquid formulation prepared in a glass container is better than that of methionine.
Example 2-5. Evaluation of the Effect of Buffer and Glutamic Acid on the Stabilizing Effect of Arginine
(51) Example 1-4 above indicated that tartaric acid and gluconolactone as buffers had a stabilizing effect and all exhibited the optimum effect at around pH 6.0. Accordingly, the stabilizing effect of arginine on BoNT/A formulations containing tartaric acid or gluconolactone as a buffer was examined using a glass container. A buffer used as a negative control was sodium phosphate (NaPO.sub.4, pH 6.0) which is most generally used. The results of the comparison are shown in
(52) As a result, it was shown that the stabilizing effect of arginine showed similar patterns in all the liquid formulations, and the residual potency of BoNT/A in the formulations containing arginine was measured to be 57 to 70% even after 8 weeks. The stabilizing effect of arginine on the formulation containing gluconolactone among all the buffers used in the experiment was significantly high, and the residual potency of BoNT/A in this formulation was measured to be about 10% higher than those in other formulation samples. This tendency also appeared in formulations containing lidocaine.
(53) In addition, the effect of arginine as a stabilizer in formulations containing glutamic acid in addition to a tartaric acid or gluconolactone buffer was evaluated. The results of the evaluation are shown in
(54) Based on the experimental results, in order to select the optimum buffer for stabilization of BoNT/A liquid formulations containing both arginine and glutamic acid, the efficacy of BoNT/A in formulations containing sodium phosphate, tartaric acid or gluconolactone was compared using glass containers. As a result, it was shown that the residual potency of DESCR, measured by the DESCR assay after 2 weeks, 4 weeks and 8 weeks, was significantly high in the formulation containing gluconolactone as a buffer. Specifically, when the formulation contained no lidocaine, the residual potencies after 2 weeks, 4 weeks and 8 weeks reached 96%, 87% and 71%, respectively, and when the formulation contained lidocaine, the residual potencies were measured to be 96%, 86% and 68%.
(55) Finally, in order to evaluate the optimal concentration of glutamic acid that contributes to the stabilizing effect of arginine, 50 mM arginine and 10 to 50 mM glutamic acid were added to formulations containing gluconolactone as a buffer, and the efficacy of BoNT/A in the formulations was measured in polypropylene tubes. The results of the measurement are shown in
(56) The above results suggest that the following important fact. Glutamic acid has the effect of stabilizing BoNT/A in liquid formulations, but the effect of glutamic acid alone is insignificant compared to the effect of arginine alone. A formulation containing both glutamic acid and arginine shows no synergistic effect even after a relatively long storage period of 8 weeks, and the residual potency of BoNT/A in this formulation is maintained at a constant level (60 to 80%).
Example 2-6. Evaluation of the Effect of Aspartic Acid on the Stabilizing Effect of Arginine as Stabilizer
(57) The effect of aspartic acid, which is an acidic amino acid such as glutamic acid, on the BoNT/A stabilizing effect of arginine, was examined, and the results are shown in
(58) TABLE-US-00004 TABLE 4 Stabilize at 37° C. 0 weeks 2 weeks 4 weeks 8 weeks Buffer G Without 100 62.84 ± 10.78 29.66 ± 2.01 15.19 ± 2.37 aspartic acid (73.07 ± 7.70) (48.66 ± 4.67) (36.59 ± 10.22) +50 mM 100 87.91 ± 4.25 72.66 ± 5.69 51.92 ± 5.47 aspartic acid (85.29 ± 4.16) (75.08 ± 3.24) (51.72 ± 6.04) Buffer R Without 100 82.01 ± 6.89 75.96 ± 4.65 61.19 ± 4.20 aspartic acid (91.40 ± 6.30) (80.36 ± 6.53) (61.54 ± 3.88) +10 mM 100 92.19 ± 3.98 85.28 ± 5.79 73.05 ± 79.67 aspartic acid (86.83 ± 3.10) (85.49 ± 2.33) (79.01 ± 6.61) +25 mM 100 93.37 ± 4.53 86.92 ± 7.08 58.05 ± 3.37 aspartic acid (94.76 ± 3.94) (84.87 ± 4.48) (65.49 ± 11.48) +50 mM 100 .sup. 100 ± 0.24 94.90 ± 3.72 61.07 ± 1.63 aspartic acid (94.79 ± 4.49) (89.70 ± 4.21) (73.85 ± 4.43)
(59) TABLE-US-00005 TABLE 5 Stabilize at 37° C. 0 weeks 2 weeks 4 weeks 8 weeks Buffer G Without 100 62.84 ± 10.78 29.66 ± 2.01 15.19 ± 2.37 glutamic acid (73.07 ± 7.70) (48.66 ± 4.67) (36.59 ± 10.22) +50 mM 100 87.41 ± 5.42 78.30 ± 6.22 40.87 ± 6.04 glutamic acid (90.29 ± 4.95) (71.09 ± 3.35) (37.42 ± 4.78) Buffer R Without 100 82.01 ± 6.89 75.96 ± 4.65 61.19 ± 4.20 glutamic acid (91.46 ± 6.30) (80.36 ± 6.53) (61.54 ± 3.88) +10 mM 100 93.34 ± 4.55 88.21 ± 5.69 65.76 ± 4.53 glutamic acid .sup. (100 ± 2.63) (92.39 ± 7.38) (72.11 ± 3.92) +25 mM 100 87.69 ± 3.21 76.38 ± 3.73 64.85 ± 5.93 glutamic acid (92.53 ± 12.79) (87.68 ± 6.37) (67.83 ± 15.29) +50 mM 100 83.68 ± 5.85 81.34 ± 6.50 55.98 ± 3.93 glutamic acid (89.18 ± 5.39) (86.83 ± 6.60) (50.36 ± 3.40)
Example 2-7. Confirmation of Stability of BoNT/A Product Prepared from Novel Liquid Formulation
(60) A novel liquid composition for BoNT/A, established based on the results of systematically and comparatively analyzing a variety of liquid injectable additives whose safety was confirmed by the present inventors, comprises 10 mM gluconolactone (pH 6.0), 45 to 130 mM sodium chloride, 50 mM arginine, 50 mM aspartic acid, and 0.05% polysorbate. In order to verify the safety of a liquid formulation by mouse LD50 assay, a BoNT/A formulation having the above-described composition was prepared using a glass container so as to have an initial potential of 40 units/ml. A liquid formulation product containing methionine instead of arginine was used as a control, and the stabilities of the products were comparatively examined. The results of the examination are shown in
INDUSTRIAL APPLICABILITY
(61) Botulinum toxin inhibits the exocytosis of acetylcholine at the cholinergic presynapse of a neuromuscular junction in animals having neurological function to thereby cause asthenia. Thus, efforts have recently been made to use the neurotoxicity of botulinum toxin for cosmetic or therapeutic purposes. However, botulinum toxin, a protein agent, has a problem in that it is not easy to formulate into pharmaceutical compositions and is also not easy to store, distribute and manage. This is attributable to the instability of the protein, and the problem is serious in the case of protein agents such as botulinum toxin, which are formulated into pharmaceutical compositions at a very low concentration.
(62) A liquid formulation containing botulinum toxin and stabilizing agent according to the present invention can be easily stored and distributed. It was proved a significant effect on the stabilization of botulinum toxin under suitable conditions according to the temperature and pH of the human body. Thus, it is expected that the pharmaceutical composition of the present invention will greatly contribute to the safe and convenient medical use of botulinum toxin.