Patent Application
20160229891
The present invention belongs to the field of pharmaceutical technology and relates to salt of polypeptide vaccine, preparation method therefor, and pharmaceutical preparation comprising said salt, in particular to KIFGSLAFL acetate, the preparation method and a pharmaceutical preparation containing such acetate.
Breast cancer (BCa) is the most common cancer diagnosed in female and the second leading factor of death related to cancer for female (RiesLAG et al., SEER Cancer Statistics Review, 1975-2003, National Cancer Institute, Bethesda, Md.). During the past 20 years, the major progress for the treatment of breast cancer improves the disease-free survival (DFS) rate significantly. For example, the antibody therapy reactive to tumor-associated antigen has been used for blocking specific cellular process to slow the progression of disease or to prevent the disease recurrence. Although the treatment of breast cancer progressed in recent years, a fair amount of patients ultimately died from recurrent diseases. Therefore, the treatment for preventing, slowing or inhibiting recurrent disease is necessary.
Vaccine is the noticeable model for such treatment and prevention because of the convenience of administration and the high success rate observed in infectious diseases. The fundamental principle for constructing cancer vaccine is direct theoretically. However, the success rate is limited in the development of effective cancer vaccine for solid tumor practically.
KIFGSLAFL (nonapeptide with the amino acid sequence of Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu, also known as HER2/neu 369-377), the peptide sequence of HER2/neu proto-oncogene family, responds to cytotoxicity T lymphocyte (CTL), is intended for vaccine aiming at preventing and/or treating cancer and is relatively used in clinical study at present (Zaks T et al., Immunization with a peptide epitope (369-377) from HER-2/neu leads to peptide specific cytotoxic T lymphocytes that fail to recognize HER-2/neu+tumors, Cancer Research, 1998, 58(21):4902-8; Knutson K L et al., Immunization of cancer patients with HER-2/neu, HLA-A2 peptide, p 369-377, results in short-lived peptide-specific immunity, Clin Cancer Res 2002, 8(5): 1014-1018).
However, KIFGSLAFL samples used in available clinical trial of disclosured technologies are in the state of DMSO solution, with poor stability at room temperature and should be kept at low temperature of −20° C. and thawed before using. So far, no compound or mixture of such polypeptide stable at room temperature has been reported.
One aim of the invention is to provide KIFGSLAFL acetate to improve the purity, water solubility and stability of raw materials significantly.
Wherein, the said KIFGSLAFL means nonapeptide with the following amino acid sequence:
Which is also known as HER2/neu 369-377, and the molecular formula of the said KIFGSLAFL acetate is C50H78N10O11.C2H4O2.
The above KIFGSLAFL acetate preferably prepared and obtained by the method of the present invention has very high medicinal purity. The purity is ≧95%, preferably ≧98% and more preferably ≧99%. In ensuring the efficacy and safety of medicine, it is important with high commercial development value.
Because large numbers of impurities generate from the synthesis of KIFGSLAFL, there is obvious technical difficulty for preparing high-purity KIFGSLAFL salt and it is difficult to obtain pure single salt with high purity.
The invention preferably provides the method for preparing KIFGSLAFL acetate through a lot of experiments, comprising the following steps:
Synthesize KIFGSLAFL peptide resin according to the amino acid sequence of (9→1) with the method of Fmoc solid-phase synthesis, crack it with trifluoroacetic acid to obtain KIFGSLAFL crude, which specifically includes the following steps:
(1.1) Use the acid sensitive resin and connect with Fmoc-Leu-OH (such as Wang resin or 2-chloro-trityl-resin) as raw material to react with piperidine solution, take off Fmoc protecting group and obtain Leu-resin after washing with solvent.
(1.2) In accordance with the solid-phase peptide synthesis method, Leu-resin reacts with Fmoc-protected amino acids successively to obtain the nonapeptide KIFGSLAFL-resin. Conduct transpeptidase reaction with condensing agent successively during the period, wash with solvent, take off Fmoc protecting group, then wash with solvent, carry out the condensation of next Fmoc-protected amino acid.
The Fmoc-protected amino acids for further condensation reaction with Leu-resin are: Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Ile-OH and Fmoc-Lys(Boc)-OH in order.
The condensing agents HATU, HBTU, BOP, PyBOP, DIC/HOBt, DIC/HOAt, etc. are preferable. (1.3) Treat KIFGSLAFL-resin with trifluoroacetic acid lysis solution for the trifluoroacetic acid solution of KIFGSLAFL, add diethyl ether for precipitation, centrifuge and wash for KIFGSLAFL crude.
The trifluoroacetic acid lysis solution means the carbocation trapping reagent contains trifluoroacetic acid 50-95% (v/v) and the rest dichloromethane or the further added 1-10% (v/v) of water, phenol, p-cresol, anisole, thioanisole, 1,2-dithioglycol, triisopropylchlorosilane (TIS) and other carbocation capture reagents.
Dissolve the KIFGSLAFL crude prepared and obtained from step (1) in the mixed solution of acetonitrile and water, filter, purify the filtrate with reverse-phase high-performance liquid chromatography to obtain pure KIFGSLAFL acetonitrile/water solution.
For the purification of reverse-phase high-performance liquid chromatography, preferably use C4, C8 or C18 alkyl-bonded silica as stationary phase, more preferably C18 alkyl-bonded silica gel; the column temperature is preferably 20-35° C., more preferably 25-30° C.; the mobile phase is the acetonitrile/water solution containing trifluoroacetic acid, wherein, the concentration of trifluoroacetic acid is 0.02%-0.5% (v/v), preferably 0.05%-0.2% (v/v). The preferable volume percentage of acetonitrile in the mobile phase is between 10% and 80% for the gradient elution from low concentration to high concentration, and more preferable between 20% and 50%.
(3) Salt-Transformation (Transform into Acetate)
Perform reverse-phase high-performance liquid chromatography for the pure KIFGSLAFL prepared and obtained from step (2) with acetic acid in acetonitrile/water solution as the mobile phase to obtain KIFGSLAFL acetate solution.
For the reverse-phase high-performance liquid chromatography used for salt-transformation, preferably use C4 or C8 or C18 alkyl-bonded silica gel as stationary phase, more preferably C18 alkyl-bonded silica gel; the column temperature is preferably 20-35° C., more preferably 25-30° C.; the mobile phase is the acetonitrile/water solution containing acetic acid, wherein, the concentration of acetic acid is 0.02%-0.5% (v/v), preferably 0.05%-0.2% (v/v). The preferable volume percentage of acetonitrile in the mobile phase is between 2% and 80% for the gradient elution from low concentration to high concentration, and more preferable to process at least one column volume firstly by isocratic or gradient elution between 2% and 4%, followed by isocratic or gradient elution from low gradient to high gradient between 20% and 50%.
One method is to concentrate under reduced pressure and dry the KIFGSLAFL acetate solution prepared and obtained from the above step (3) to obtain the solid KIFGSLAFL acetate.
Another method is to concentrate (under reduced pressure) the KIFGSLAFL acetate solution prepared and obtained from the above step (3), precipitate out KIFGSLAFL acetate, centrifugate for precipitation, vacuum dry to obtain the solid KIFGSLAFL acetate.
Another aim of the invention is that the provided KIFGSLAFL acetate is amorphous solid, and its X-ray diffraction pattern has no crystal characteristic absorption peak. The KIFGSLAFL acetate of amorphous solid can obviously improve water solubility and stability, thus is easier for medicine preparation, in particular for the use of preparation injection.
The invention provides the method for preparing amorphous solid KIFGSLAFL acetate through a lot of experiments, comprising the following steps:
Adjust the pH value of the KIFGSLAFL acetate solution prepared and obtained from the above step (3) to pH 4 with acetic acid, concentrate under reduced pressure until the KIFGSLAFL acetate precipitates out, lower the temperature to below 5° C., centrifugate for precipitation, vacuum dry under 30° C. to obtain the uniform KIFGSLAFL acetate amorphous solid.
Wherein, the X-ray diffraction pattern is obtained by the following detection method. Instrument model: Empyrean X-ray diffractometer; test condition: Cu target Kα1 ray, voltage of 40 kv, current of 40 mA, 2θ range of 3°-50°, divergence slit of 1/32°, anti-scattering slit of 1/16°, anti-scattering slit of 7.5 mm, step length of 0.02°, and dwell time per step of 40S.
The said amorphous solid KIFGSLAFL acetate is detected under the IR spectrum detection condition by KBr pellet pressing method, and its infrared absorption reaches peaks at 3288 cm−1, 3065 cm−1, 2958 cm−1, 1632 cm−1, 1527 cm−1, 1404 cm−1 and 698 cm−1.
The said amorphous solid KIFGSLAFL acetate is detected by ESI mass spectrometry, with the molecular ion peak of 995.9, which is basically the same as the calculated molecular weight for KIFGSLAFL.
The KIFGSLAFL acetate amorphous solid is dissolved in the mixed solution of acetonitrile and water (volume ratio=5:95) and is analyzed under the following detection condition of high-performance liquid chromatography: using C18-bonded silica gel (250×4.6 mm, 5 μm) as the stationary phase, mobile phase A as acetonitrile, mobile phase B as sodium dihydrogen phosphate solution 0.1 mol/L (adjusting the pH value of phosphoric acid to 3.0), conducting gradient elution (towards mobile phase A from 5% to 45% with the elution time of 20 min; then changing into 45% isocratic elution of mobile phase A), with the flow velocity of 1 mL/min. The detection wavelengths are 195 nm (0-8 min) and 230 nm (8-50 min). The elution time for acetic acid and target peptide KIFGSLAFL are approximately 4.6 min and 21 min, respectively.
Another aim of the invention is to provide a pharmaceutical preparation of peptide vaccine containing the above KIFGSLAFL acetate which is applicable for reformulated into an injectable medical product for patients with pharmaceutically acceptable solvent. The pharmaceutically acceptable solvent comprises water for injection, sterile water, etc.
The efficacy and safety of such pharmaceutical preparation is improved significantly as the KIFGSLAFL acetate of the invention, in particular its amorphous solid has good purity and stability and superior water solubility.
The said pharmaceutical preparation contains one or above medicinal excipients or not. The pharmaceutical preparation of the invention can be in any powder form of parenteral administration, for example, freeze-dried powder, suspension or diary product injection, etc.
The said pharmaceutical preparation can be preferably used for the treatment of breast cancer and breast cancer recurrence.
Unless otherwise specified, the KIFGSLAFL acetate of the invention shall be that with the molecular formula of C50H78N10O11.C2H4O2.
Abbreviations of various reagents in the invention are defined as follows:
Compared to the known technologies, the invention has the following prominent advantages and beneficial effects:
1. Compared to KIFGSLAFL and KIFGSLAFL sulfate, the KIFGSLAFL acetate provided in the invention improves the drug purity, stability and water solubility greatly and ensures the medical safety and efficacy sufficiently.
2. The amorphous solid KIFGSLAFL acetate provided in the invention has excellent water solubility and stability, accelerates the redissolving of drug in use by means of injection administration and has better stability for dissolved KIFGSLAFL acetate solution than injection solvent (such as dimethyl sulfoxide). According to the dissolving and stability properties, the amorphous solid KIFGSLAFL acetate provided in the invention belongs to the form more advantageous for preparing pharmaceutical preparation, can sufficiently ensure the convenient use of pharmaceutical preparation, and will be safe and effective for use.
3. The invention provides a method for preparing high-purity KIFGSLAFL acetate and overcomes the technical difficulties in available technologies for preparing salt and improving purity. The method can be carried out in a mild condition, the method condition is easy to be reproduced and can reduce industrialized cost greatly.
The following embodiments and attached figures illustrate the invention, but in no way limit the invention.
1. Weighed 40 g of Fmoc-Leu-Wang resin (with the degree of substitution: 0.8 mmol/g), added 300 mL of DMF for swelling the resin for 30 min. Removed DMF by suction filtration, and added 300 mL of 20% piperidine/DMF solution for reaction for 30 min. Performed suction filtration, and washed the resin with 6×150 mL of DMF.
2. Added 24.8 g of Fmoc-Phe-OH, 8.6 g of HOBt and 150 mL of DMF, stirred well, added 9.5 mL of DIC, and stirred in water bath under 28-30° C. for 4-5 h, Detected the resin to be colorless by Kaiser method, performed suction filtration, and washed the resin with 2×200 mL of DMF, added 300 mL of 20% piperidine/DMF solution for reaction for 30 min, performed suction filtration, and washed the resin with 6×200 mL of DMF.
3. Referred to step 2, coupled next amino acid in order. The sequence and dosages of subsequent amino acids were: Fmoc-Ala-OH 19.9 g, Fmoc-Leu-OH 22.6 g, Fmoc-Ser(tBu)-OH 24.5 g, Fmoc-Gly-OH 19.0 g, Fmoc-Phe-OH 24.8 g, Fmoc-Ile-OH 22.6 g and Fmoc-Lys(Boc)-OH 30.0 g.
4. Washed the resin with dichloromethane and methyl alcohol in turn, vacuum dried to get KIFGSLAFL peptide resin.
5. Weighed 10 g of peptide resin, added 100 mL of lysis solution (TFA/TIS/H2O=95/2.5/2.5, v/v/v), and stirred for 2 h at room temperature, filtered the resin, and washed the resin with appropriate quantity of lysis solution, combined the washing liquid with filter liquor, added 1 L of diethyl ether under stirring for precipitation, centrifuged and washed with diethyl ether for several time, vacuum dried to get KIFGSLAFL crude.
Dissolved 5 g of KIFGSLAFL crude obtained from embodiment 1 in 100 mL of water containing 10% of acetonitrile in volume ratio, and filtered, purified the filtrate by C18 column with the filling height of 250 mm and the diameter of 15 mm, conducted gradient elution (B: 20-50%, elution time: 60 min) with the mobile phase A of water containing 0.1% of trifluoroacetic acid and the mobile phase B of acetonitrile containing 0.1% of trifluoroacetic acid with the flow velocity of 600 mL/min and the detection wavelength of 230 nm, collected the main peak ingredients, detected the purification of collected liquid by analytical high-performance liquid chromatography, combined the portions with the purity higher than 98% to get the pure KIFGSLAFL solution, detected the purification of collected liquid by analytical high-performance liquid chromatography, and got the purity of 98.6% and the yield of 45%.
The condition for detecting the contents of trifluoroacetic acid and target peptide by the analytical high-performance liquid chromatography was as follows: high-performance liquid chromatograph: Chuangxin TongHeng LC300, stationary phase: C18-bonded silica gel (250×4.6 mm, 5 μm), mobile phase A: acetonitrile, and mobile phase B: sodium dihydrogen phosphate solution 0.1 mol/L (adjusted the pH value of phosphoric acid to 3.0). Performed gradient elution (transferred mobile phase A from 5% to 45%, with the elution time of 20 min; then changed to 45% isocratic elution of mobile phase A) with the flow velocity of 1 mL/min and the detection wavelengths of 195 nm (0-8 min) and 230 nm (8-50 min). The elution time for trifluoroacetic acid and target peptide were approximately 4.9 min and 21 min, respectively.
The condition for detecting the purity of target peptide by the analytical high-performance liquid chromatography was as follows: high-performance liquid chromatograph: Chuangxin TongHeng LC300, stationary phase: C18-bonded silica gel (250×4.6 mm, 5 μm), mobile phase: the mixed liquor of sodium dihydrogen phosphate solution 0.1 mol/L (adjusted the pH value of phosphoric acid to 3.0) and acetonitrile (with the volume ratio of 7:3), with the flow velocity of lmL/min and the detection wavelength of 230 nm. The elution time for target peptide was 10 min, approximately.
Cooled the KIFGSLAFL solution obtained from embodiment 2 to 0-5 CC and dropwise added 5% of NaOH solution until pH value reached to 10 under stirring, concentrated under reduced pressure until precipitated out a mass of gelatinous precipitates, centrifuged, washed with a small amount of ice water, precipitated and vacuum dried to obtain white solid with the purity of 98.1%.
The KIFGSLAFL solution obtained from embodiment 2 was purified by C18 column after proper concentration, with the filling height of 250 mm and the diameter of 150 mm. Eluted with the water solution containing 0.1% of acetic acid in volume ratio and 3% of acetonitrile for 15 min, with the flow velocity of 800 mL/min. Then carried out gradient elution (B: 20%-40%, elution time: 50 min) with the mobile phase A of water containing 0.1% of acetic acid and the mobile phase B of acetonitrile containing 0.1% acetic acid, with the detection wavelength of 230 nm, collected the main peak ingredients for KIFGSLAFL acetate solution with the purity of 99.6% and the yield of 90%.
Adjusted pH value of the KIFGSLAFL acetate solution obtained from embodiment 4 to pH 4 by acetic acid, concentrated under reduced pressure until a mass of gelatinous precipitates precipitated out, cooled to 0-5° C., centrifuged for precipitates, vacuum dried under 30° C. to obtain white solid.
According to the detection of analytical high-performance liquid chromatography of embodiment 2, the elution time of acetic acid was 4.6 min approximately. As being measured, the weight percent of acetic acid in the sample was 5.7%. The purity of KIFGSLAFL was 99.5%.
The condition for X-ray diffraction detection was as follows: instrument model: Empyrean X-ray diffractometer; test condition: Cu target Kα1 ray, voltage: 40 kv, current: 40 mA, 20 range: 3°-50°, divergence slit: 1/32°, anti-scattering slit: 1/16°, anti-scattering slit: 7.5 mm, step length: 0.02°, and the dwell time per step: 40S. X-ray diffraction spectrum was shown in
The IR spectrum obtained by KBr pellet pressing method was shown in
The result of ESI mass spectrometric detection was shown in
Cooled the KIFGSLAFL solution obtained from embodiment 2 to 0-5 and dropwise added NaOH solution in the weight ratio of 5% until pH value reached to 10 under stirring, concentrated under reduced pressure until precipitated out a mass of gelatinous precipitates, centrifuged and washed with a small amount of ice water, dissolved the solid in the acetonitrile solution with the volume ratio of 50%, added H2SO4 in the proportion of 0.5 mol H2SO4 per mol E75, stirred well (pH=6), and carried out rotary evaporation and concentration under reduced pressure in water bath of 30° C. for 2˜3 h, freeze dried to obtain the solid product.
Studied the stability of the following samples under the sterile condition at 25° C. and RH 50%: Sample 1 was the amorphous solid product of embodiment 5. Sample 2 was the solution of embodiment 4, which was diluted to approximately 1 mg/mL with dimethyl sulfoxide and filtered through the filter membrane with the aperture of 0.2 microns. Sample 3 was the solid product of embodiment 3. Sample 4 was the solid product of embodiment 3, which was dissolved in dimethyl sulfoxide, diluted to approximately 1 mg/mL and filtered through the filter membrane with the aperture of 0.2 microns. Sample 5 was the solid product of embodiment 6. The purity of each sample was detected by high-performance liquid chromatography, and the results were as follows:
It could be seen from the experimental results in the table above that: the stabilities of KIFGSLAFL solid (sample 3) and DMSO solution (sample 4) in available technologies were obviously inferior to those of KIFGSLAFL acetate amorphous solid (sample 1) and KIFGSLAFL acetate DMSO solution (sample 2); and the stability of KIFGSLAFL acetate amorphous solid (sample 1) was obviously better than those of DMSO solution (sample 2) and KIFGSLAFL.H2SO4 (sample 5).
Added the solid products of embodiments 3, 5 and 6 into a certain amount of water for injection of 25° C. in small test tubes, respectively, placed in constant temperature shakers for vibration, observed the dissolving conditions. The dissolving conditions were as follows:
It could be seen from the experimental results in the table above that: the water solubility of KIFGSLAFL acetate amorphous solid (sample of embodiment 5) improved greatly, compared to the KIFGSLAFL solid of available technologies and KIFGSLAFL.H2SO4, and was more convenient for the use of preparation.
Prepared the KIFGSLAFL acetate by the operation of embodiment 5 under sterile condition, weighed accurately (1 mg/dose by KIFGSLAFL), subpackaged and sealed in glass bottles to get peptide vaccine product.
Appropriately concentrated the KIFGSLAFL solution obtained from embodiment 2, purified by C18 column, with the filling height of 250 mm and the diameter of 150 mm. Carried out gradient elution (B: 20%-40%, elution time: 50 min) with mobile phase A of water containing 0.1% of acetic acid in volume ratio and mobile phase B of acetonitrile, with the flow velocity of 800 mL/min and the detection wavelength of 230 nm, collected the main peak ingredients and obtained KIFGSLAFL solution, concentrated under reduced pressure and freeze dried to obtain white solid. According to the HPLC detection of embodiment 2, the weight percentage of acetic acid was 4.8%, and the weight percentage of trifluoroacetic acid was 1.9%.
Conducted chromatographic purification with the method of embodiment 2 for the KIFGSLAFL crude obtained from embodiment 1 to collect the main peak ingredients. Detected the purity of the collected liquid by analytical high-performance liquid chromatography, combined the portions with purity higher than 92% to get KIFGSLAFL solution, detected the purity of the collected liquid by analytical high-performance liquid chromatography to get the purity of 95.1%, performed salt-transformation by the chromatographic process in embodiment 4 and collected the main peak ingredients to get KIFGSLAFL acetate solution with the purity of 95.3%, concentrated the obtained KIFGSLAFL acetate solution under reduced pressure to dry, vacuum dried under 40° C. to obtain white solid. The total yield was 60%. The purity of KIFGSLAFL was detected to be 94.1% by the HPLC of embodiment 2.
Conducted chromatographic purification with the method of embodiment 2 for the KIFGSLAFL crude obtained from embodiment 1 to collect the main peak ingredients. Detected the purity of the collected liquid by analytical high-performance liquid chromatography, combined the portions with purity higher than 92% to get KIFGSLAFL solution, detected the purity of the collected liquid by analytical high-performance liquid chromatography to get the purity of 95.1%, performed salt-transformation by the chromatographic process in embodiment 4 and collected the main peak ingredients to get KIFGSLAFL acetate solution with the purity of 95.3% and the total yield of 60%, processed with the method in embodiment 5 to obtain white solid, which was amorphous KIFGSLAFL acetate with the purity of 95.3%.
Conducted chromatographic purification with the method of embodiment 2 for the KIFGSLAFL crude obtained from embodiment 1 to collect the main peak ingredients, Detected the purity of the collected liquid by analytical high-performance liquid chromatography, combined the portions with purity higher than 96% to get KIFGSLAFL solution, detected the purity of the collected liquid by analytical high-performance liquid chromatography to get the purity of 97.5%, performed salt-transformation by the chromatographic process in embodiment 4 and collected the main peak ingredients to get KIFGSLAFL acetate solution with the purity of 98.1% and the total yield of 45%, processed with the method in embodiment 5 to obtain white solid, which was amorphous KIFGSLAFL acetate with the purity of 98.0%.
As large numbers of impurities generated from the synthesis of KIFGSLAFL, the industrial preparation of high-purity KIFGSLAFL salt was obviously difficult in technology and it was hard to obtain the pure single salt with high purity. As mentioned in embodiment 10, the synthetic product might easily generate mixed salts, such as acetic acid, trifluoroacetic acid, etc., which were difficult to be separated and purified for the pure single salt.
The inventor could only obtain products with the purity less than 95% by the preferred schemes of conventional separation and purification methods in the field, such as the scheme in embodiment 11 which went against the medical application of product. However, the purity of product was improved to more than 95% or even 98% by preferred purification schemes obtained through a large number of experiments, such as embodiments 12 and 13, which overcame the difficulty of obtaining high-purity KIFGSLAFL salt and made the industrialization and medical application of such product possible.
The above embodiments are preferred execution modes of the invention, but will not be limited by these embodiments. Any other changes, modifications, substitutions, combinations and simplifications without deviation from the spirit and principle of the invention shall be equivalent displacement modes and will be included in the protection scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201310447114.6 | Sep 2013 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2014/087506 | 9/26/2014 | WO | 00 |
