Method for obtaining partricin a or b and partricin a and b obtained by method

Abstract

The invention refers to method for manufacturing pure antibiotics from the group of aromatic polyenemacrotides with potentially high therapeutic index — partricin A and partricin B from a commercial preparation - particin complex- substrate comprising partricin complex among other substances, compositions or from an extract from the fermentation broth of a strain producing partricin A and B - that comprises particin complex, through their isolation. The invention also covers products obtainable by this method.

Description

[0001] (16.06.2025)

[0002] 1

[0003] Method for obtaining partricin A or B and partrici n A and B obtained by method

[0004] The invention refers to method for manufacturing pure antibiotics from the group of aromatic polyene macrolides with potentially high therapeutic index — partricin A and partricin B from a commercial preparation - particin complex - substrate comprising partricin complex among other substances, compositions or from an extract from the fermentation broth of a strain producing partricin A and B - that comprises particin complex, through their isolation. The invention also covers products obtainable by this method.

[0005] Systemic fungal infections pose a serious challenge to modem chemotherapy. Their effective treatment is hindered by two major problems: the low selectivity of existing antifungal drugs and the molecular similarities between human and fungal cells. Amphotericin B, commonly known as the "last-resort drug," exhibits most features of an "ideal drug." However, natural analogs of Amphotericin B with higher biological activity exist — aromatic polyene macrolides (AHM). Representatives of this group include partricin A (syn. gedamycin) and partricin B (syn. vacidin), which display the highest antifungal activity among aromatic polyene macrolides. Unfortunately, fundamental and application-driven research aiming to improve the selective toxicity index of these antibiotics is hindered by their complex nature. Partricins A and B are produced by Streptomyces aureofaciens bacteria in the form of a complex called partricin (syn. aureofacin), in which both antibiotics occur in similar quantities. The preparation containing the partricin complex is obtained through known methods from the fermentation broth of Streptomyces aureofaciens via extraction of the mycelium or entire fermentation broth using lower aliphatic alcohols (most commonly n-butanol). The extract is then concentrated under reduced pressure, and the precipitate is filtered, dried, or lyophilized. Often, the extraction process is preceded by pH modification of the fermentation broth to enhance the efficiency of antibiotic complex extraction. The preparation can be obtained using the aforementioned and well-established method, (by fermentation of Streptomyces aureofaciens under controlled conditions) and also there are commercially available partricin preparations that require the isolation of a specific antibiotic. However, such preparations containing the partrici n complex or extracted solutions exhibit a low concentration of either the entire partricin complex (comprising partricin A and partricin B) or only one of these components. This is attributed to the presence of additional polyene macrolides, degradation byproducts, minor quantities of unidentified natural compounds, as well as certain constituents of the fermentation broth and trace amounts of metal ions.

[0006] The method for isolating partricin A is known from publication CN102453062 A. The publication discloses a separation process that includes the following steps: adjusting the pH of the fermentation broth containing partricin, sedimentation and filtration, suspending the precipitate in a polar organic solvent, followed by next filtration; adjusting the appropriate pH of the filtrate, concentrating at a temperature of 25-35°C to a water content of 30-35%, sedimentation and filtration to obtain a crude partricin complex preparation; dissolving the crude preparation in a methanol solution containing 25-30% water and loading it onto a column filled with styrene macroporous adsorption reverse phase resin. The preparation containing partricin is then isolated on the column using gradient elution in a methanol-water solvent system, where impurities are initially removed using methanol containing 18-23% water (v / v), followed by elution of partricin A using methanol containing 13-17% water. The method employs a styrene macroporous resin adsorption reverse phase column. In the next step, the collected fractions of partrici n A are concentrated to the solution containing 25-30% water in methanol and then subjected to another separation on a column filled with styrene macroporous resin, where methanol containing 17-19% water is used to remove impurities, followed by reducing the water content in methanol to 10-14% and eluting partricin A fractions, which are then precipitated. The procedure is time-consuming (involving two isolation steps on a column and a concentration step between them) and requires significant amounts of chemical solvents. Additionally, the adsorption resin undergoes gradual degradation, necessitating replacement or replenishment over time.

[0007] The method for isolating partricin B is known from publication CN 102453063. In this method, the crude preparation containing the partricin complex is dissolved in a methanol solution containing 25-30% water, and the resulting partricin solution is separated in styrene macroporous resin adsorption reverse phase column using methanol-water solvent system. Initially, impurities are removed using methanol containing 18-23% water (v / v), followed by elution of partricin B using methanol containing 13-17% water (v / v). The next step involves concentrating the partricin B fractions to a solution containing 40-50% water in methanol, followed by sedimentation and filtration. The publication discloses a separation process that includes the following steps: adjusting the pH of the fermentation broth containing partricin, sedimentation and filtration, suspending the precipitate in a polar organic solvent, followed by another filtration; collecting the filtrate and adjusting the appropriate pH, concentrating at a temperature of 25-35°C to a water content of 30-35%, sedimentation and filtration to obtain a crude partricin complex preparation. Like the method for isolating partricin A, this procedure requires significant amounts of chemical solvents, and the adsorption resin undergoes gradual degradation, necessitating replacement or replenishment over time.

[0008] Other isolation methods rely on classical column chromatography, but they focus on isolating the entire not pure partricin complex rather than separating partrici n A or partricin B individually.

[0009] Known methods for obtaining pure antibiotics - partricin A and partricin B, aside from the previously mentioned drawbacks such as labor intensity and high solvent consumption, which impact on high isolation costs, additionally have a negative environmental impact.

[0010] The partricin complex obtained through known methods or purchased commercially comprises a lot of additional compounds, including other polyene macrolides, their degradation products, small amounts of other natural compounds whose structures have not been identified, as well as certain fermentation broth components and trace amounts of metal ions, all of which must be removed thoroughly. It is necessary therefore to perform isolation and additionally purification of the desired compounds - in this invention it is partricin A and partricin B. Alternatively, a more efficient and effective fermentation process followed by better - more effective isolation process from the fermentation broth must be provided. On the other hand, in the case of the separation of the partricin complex itself, the slight structural differences between partricin A and B set a problem and challenge - goal for effective isolation of partricin A and partricin B separately, not in the same process / time, from preparations comprises the partricin complex - substrate for particins. Hence, there is a need to provide an effective method for obtaining both partricins in pure form. Currently, no such effective methods are available, and as a result, partricin A and partricin B are not easy commercially available what significantly limiting their research potential and hindering structural, biological, application studies and in line, the antibiotics in desire form are not easy available.

[0011] In the field of centrifugal partition chromatography (CPC), there is a known method that involves introducing the stationary phase first, followed by the sample solution, and finally the mobile phase without establishing - obtaining equilibrium / balance state in the column before sample introduction. This is referred to as the "sandwich method."

[0012] The goal of the invention was to provide a procedure for obtaining pure partricin A and pure partricin B, enabling their separate isolation or combined with high efficiency - effective methods for its obtaining - while eliminating the disadvantages of known isolation, obtaining methods. Research experimental studies for achieving the goal focused on provision of a method for obtaining both compounds from the multi-component partricin complex - substrate for particins. The goal of the invention was to provide an effective and cost-efficient isolation method for partrici n A and partricin B, ultimately leading to the production of valuable antifungal antibiotics and substrates for the synthesis of Mepartricin, in which particin set active substrate for required pharmaceutical product used in the treatment of prostate gland hypertrophy. The inventors of the invention have performed research and studies on the partricin complex, purchased from Tarchomin Pharmaceutical Works POLFA S.A, which also comprises a lot of other compounds, composition, substances - among other there are polyene macrolides. The invention is also used to preparations - substrate (extracts) obtained from the fermentation broth of Streptomyces aureofaciens comprising partricin A and B. In line with this the methods according to the invention can be used for substrate from fermentation broth and commercial available preparations partricin complex. During studies and research in order to provide on the invention, the inventors observed that difficulties in obtaining partricin A and B from substrate, partricin complex were due, among other factors, to the low solubility of the partricin complex in commonly used solvents in the field. Consequently, chromatographic systems exhibited low capacity, necessitating multiple separation cycles to obtain the desired quantities of compounds. Additionally, methods based on preparative high- performance liquid chromatography (prepHPLC) were found to be highly expensive due to the irreversible adsorption of macrolides on chromatographic columns, leading to permanent damage to costly preparative columns. Furthermore, the aforementioned adsorption resulted in losses of the separated partricins and are not effective therefore.

[0013] In the description, the term "partricin complex" — the substrate for the procedure according to the invention refers to a commercially purchased preparation comprising partrici n A and B or a preparation (extract) obtained from the fermentation broth of Streptomyces aureofaciens comprises not only partrici n A and B, but a lot of other previously mentioned compounds, ingredients, substances such as other polyene macrolides, their degradation products, small amounts of other natural compounds whose structures have not been identified, certain fermentation broth components, and trace amounts of metal ions.

[0014] The stationary phase — also referred to as the lower phase is formed according to the invention from a three-component mixture of selected solvents and predominantly - mainly comprises chloroform and methanol, while the mobile phase — also referred to as the upper phase is also formed from this mixture, primarily - mainly comprising of buffer and methanol.

[0015] The invention refers to the isolation stage - step of partricin A or partricin B from a dry preparation complex comprising the partricin complex, which is obtained through known methods (extract from the fermentation broth) or ordered from commercial sources - particin complex - preparation. The invention thus concerns the subsequent stage of obtaining partricin A or B, meaning that the method enables to obtain partricin A or partricin B with high purity from an available substrate: commercial preparation with the partricin complex, an extract from the fermentation broth. It is also possible to obtain both partricins but separately in a single method by adjusting different separation parameters according to established ranges for partrici n A and separately for partrici n B based on the research.

[0016] The invention refers to the isolation - also known as obtaining methods - of partricin A and partricin B with high purity from a commercial preparation comrising the partricin complex or an extract obtained from the fermentation broth of Streptomyces aureofaciens, collectively referred to in the description as the partricin complex, using centrifugal partition chromatography (CPC). The invention, i.e. method enables the production of pure antibiotics from the group of aromatic polyene macrolides, specifically partricin A or partricin B.

[0017] The method for obtaining partrici n A or B is characterized by the preparation of a biphasic three-component solvent system with a developed composition of chloroform: methanol: buffer at a pH range of 7.5 to 9.5, in percentage amounts relative to the total mixture forming a volumetric mixture of these solvents, namely: 15-52% (v / v) chloroform, 15-52% (v / v) methanol, and 15-45% (v / v) buffer at a pH range of 7.5-9.5. A buffer with the indicated pH, preferably borate buffer at pH 8.2, is used. The composition and quantities were selected based on research conducted on the invention. These solvents, in the specified proportions, are mixed, resulting in two phases — one stationary and one mobile — both forming a chromatographic column in the CPC apparatus, where partricin A or partricin B will be separated. The stationary phase is predominantly composed of chloroform and methanol, while the mobile phase is primarily composed of buffer and methanol.

[0018] This solution is introduced into the CPC column before introducing the partricin complex in such a way that after obtaining the biphasic three-component solvent mixture, the stationary phase (lower phase) is first introduced into the CPC column in an amount necessary to fill the entire column volume, followed by the introduction of the mobile phase (upper phase) until equilibrium is established between them, thereby balancing the chromatographic column.

[0019] Another possible method for conducting the chromatographic process in CPC technology involves introducing the stationary phase first, followed by the sample to be separated, and finally the mobile phase (the so-called "sandwich method"). During research on the invention, the inventors observed that using this method did not yield the expected results for isolating partricin A and partricin B and based on experimental studies, they developed a method in which the sample to be separated (partricin complex) is introduced into the CPC apparatus only after equilibrium has been established in the CPC system. According to the invention, the stationary phase (lower phase) is introduced first, followed by the mobile phase (upper phase), and then the sample dissolved in a mixture of selected solvent and one of the phases, as described in the scope of protection and examples. Other methods were also tested, including dissolving the sample in a selected solvent alone, which showed results, but not as significant as when adding the selected phase.

[0020] In the next step, after equilibrium is established in the column, the solution of the partricin complex is introduced, prepared in two selected variants, preferably one of them, among the four variants tested and described further in the document. Thus, the available substrate for separation, namely the preparation comprising the partricin complex, is introduced into the column.

[0021] In the first variant of the invention, the partricin complex is dissolved in a selected solvent — dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) — or in a mixture of these solvents. The inventors also studied, tested methods using methanol or ethanol, as well as other commonly used organic solvents in the fireld, but these were not as effective as the two selected solvents according to the invention. In CPC chromatography, the use of DMSO or DMF is not a standard approach for dissolving the preparation, as it is typically dissolved in the pure upper phase or a mixture of the upper and lower phases. However, based on experimental studies and research, the inventors observed that dissolving the complex in the selected solvent or its mixture yielded - lead to effective results. In line with this this observation was unexpected.

[0022] The solution in the selected variant is mixed with the mobile phase (described above) in percentage amounts relative to the total mixture forming the volumetric mixture of solvents, namely: 1-99% (v / v) DMSO or DMF (or their mixture) : 1-99% (v / v) mobile phase — upper phase, preferably in volumetric proportions of 10:90 (v / v) (10% DMSO or DMF or their mixture : 90% mobile phase — upper phase). It was also investigated whether, in an alternative variant of possibilities, the partricin complex is dissolved in a solvent in the form of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) or in a mixture of these solvents. It was determined that this option - variant of the invention also yields - lead to effective results.

[0023] In the third variant of possibilites, the process was tested wherein the partricin complex dissolves in the mobile phase, which is primarily composed of methanol and buffer.

[0024] In the fourth variant of possibilities, the dissolution of the partricin complex in the stationary phase was examined.

[0025] The inventors also investigated the dissolution of the complex in an organic solvent — methanol and / or ethanol or in the mobile phase or the stationary phase. However, it was found during the studies and research that these possibilities did not produce significant results.

[0026] The amount of added complex and phases depends on the volume of the CPC column and the quantity of partricin A or partricin B to be obtained. This is calculated using known methods — technical knowledge of a person skilled in the art. Specific quantities of the "input," i.e., the partricin complex, are described in the example, namely 80 mg. It was also verified that the method is effective for larger quantities.

[0027] The prepared solution of partricins is introduced into the CPC column. The separation process is performed using centrifugal partition chromatography (CPC) in ascending mode (ASC), with column rotation set between 500-2400 rpm and a volumetric flow rate of the mobile phase between 1-15 mL / min. For the isolation of partrici n A, the separation is performed using elution through 0.6-2 column volumes, preferably within the range of 1.1-1 .4 column volumes, preferably followed by an extrusion step of the stationary phase, preferably covering one column volume. For the isolation of partricin B, the separation is performed using elution through 0.3- 1.1 column volumes, preferably within the range of 0.5-0.8 column volumes. Depending on the size of the apparatus (purchased column), this column volume will vary. For a 250 mL column, it will be 250 mL, whereas for a 1 L column, it will be 1 liter, etc. This is known to a person skilled in the art. The separation process is monitored at a wavelength range of 220-450 nm, preferably 378 nm, which corresponds to the maximum absorption of the chromophore of both partricins. It was determined that partricin A elutes from the column within 0.6-2 column volumes, preferably 1.1-1 .4 column volumes, while partricin B elutes within 0.3-1.1 column volumes, preferably 0.5-0.8 column volumes. Then, fractions containing only partricin A or only partricin B are processed separately according to known method - the collected fractions are concentrated under reduced pressure, followed by extraction using n-butanol from the aqueous layer. The organic phase is then desalted with ultrapure water, further concentrated, and precipitated using diethyl ether, petroleum ether, hexane, or acetone. The precipitate is centrifuged, the supernatant is discarded, and the residue is dried under vacuum, protected from light.

[0028] The invention enables the isolation of pure partricin A and pure partricin B from substrate - preparation comprising the partricin complex or from an extract from the fermentation broth of a strain producing partricin A and B in a fast and efficient manner, with relatively low financial investment. The method is also easily scalable for preparative or industrial applications. The purity of the obtained antibiotics exceeds 82%, as confirmed by HPLC analysis.

[0029] The invention is described in detail in Examples 1 and 2. Fig. 1 presents the CPC-DAD chromatogram obtained for the separation of partricin A, confirming the selectivity of the chromatographic system and the effective separation of partrici n A from other components of the complex. The purity of obtained preparation of partricin A has been proven on Fig. 2, which shows the HPLC-DAD chromatogram of the obtained partricin A, where the highest peak, with a retention time of 9.5 min, corresponds to partricin A. Fig. 3 presents the CPC-DAD chromatogram obtained for the separation of partricin B, confirming the selectivity of the chromatographic system and the effective separation of partricin B from other components of the complex.

[0030] List of figures:

[0031] Fig. 1. CPC-DAD chromatogram obtained as a result of separating the antibiotic mixture used to isolate partricin A. Fig. 2. HPLC-DAD chromatogram of the standard solution of partricin A preparation obtained by using the invention.

[0032] Fig. 3. CPC-DAD chromatogram obtained as a result of separating the antibiotic mixture used to isolate partricin B.

[0033] Fig. 4. HPLC-DAD chromatogram of the standard solution of partricin B preparation obtained by using the invention.

[0034] Example 1

[0035] The isolation of partricin A from a commercial partricin preparation produced by Tarchomin Pharmaceutical Works POLFA S.A, in the amount of 80 mg and with a purity of approximately 45% (mass percentage of the partrici n A and B complex in the preparation), was performed using a Gilson CPC-250 centrifugal partition chromatograph, equipped with a 250 mL column, an Ecom flash 14 DAD 600 detector, and a Gilson LS-5600 fraction collector (entire apparatus from Gilson Inc., Middleton, Wl, USA).

[0036] A biphasic solvent system — chloroform: methanol: borate buffer (pH=8.2) (v / v / v) — was prepared by mixing appropriate volumes of solvents at room temperature (500 mL: 500 mL: 375 mL). The borate buffer was obtained using a known method by dissolving 3.09 g of boric acid and 3.73 g of potassium chloride in 940 mL of water, followed by the addition of 60 mL of a 0.1 M potassium hydroxide solution, yielding 1 liter of buffer at pH=8.2.

[0037] The separation was performed in ascending mode (ASC), using upper phase as the mobile phase. First, the lower phase, comprising mainly of chloroform and methanol, was introduced into the column in the volume of 360 mL, at a flow rate of 30 mL / min and a rotation speed of 500 rpm for 12 minutes. Next, the upper phase, primarily composed of buffer and methanol, was pumped through the stationary phase for an additional 13 minutes, at a flow rate of 5 mL / min and a rotation speed of 800 rpm, until equilibrium was achieved at a pressure of approximately 50 bar. Subsequently, 80 mg of the commercial preparation comprising the partricin complex (a mixture of antibiotics), dissolved in 1 mL of dimethyl sulfoxide (DMSO) and diluted with the mobile upper phase to a final volume of 10 mL, was introduced into the column for separation. The upper phase was pumped for 72 minutes, at a flow rate of 5 mL / min and a rotation speed of 800 rpm, collecting 12 mL per fraction (total 360 mL). Following this, the lower phase was introduced into the column in the amount of 300 mL, at a flow rate of 30 mL / min and a rotation speed of 500 rpm for 10 minutes (extrusion). Partricin A eluted from the column within 57 to 70 minutes of separation, corresponding to 1.1-1 .4 column volumes, as confirmed by the chromatogram. The separation was performed at room temperature, with monitored wavelength of A=378 nm. Data was collected and processed using Gilson Glider CPC V5.1 d.01 software. The purity of the fractions was confirmed using HPLC-DAD analysis (Luna C18(2) column, 150x4 mm, 5pm, 100A; mobile phase: 38% ACN, 62% CH3COONH4 (5.5 mmol, pH=4.5), v / v; flow rate co=1.O mL / min; concentration C=1 mg / mL; injection volume v=20μL; monitored wavelength A=378 nm; room temperature). In the final step, the fractions comprising partricin A were combined and concentrated under reduced pressure (5-7 mbar) at a temperature not exceeding 35°C, removing chloroform and methanol. The antibiotic was then extracted from the remaining aqueous layer (comprising borate buffer) using 200 mL of n-butanol. The obtained organic phase comprising the antibiotic was desalted by washing with 100 mL of ultrapure water (2x50 mL) and further concentrated to a volume of approximately 20 mL. Next, the antibiotic was precipitated from the butanol solution using 30 mL of diethyl ether and centrifuged in a preparative centrifuge for 5 minutes at 2400 rpm. The supernatant was then discarded, and the antibiotic-containing precipitate was washed with 30 mL of diethyl ether and centrifuged again under the previously described conditions. The supernatant was once again discarded, 30 mL of diethyl ether was added, and the centrifugation process was repeated under the same conditions, after which the supernatant was discarded once more. Finally, the precipitate containing partricin A was dried in a vacuum desiccator for 24 hours, protected from light. The final yield was 8.3 mg of partricin A, with a purity of 85% (HPLC-DAD analysis).

[0038] From Figure 1 , it is evident that introducing the partricin complex solution into the CPC column under the described conditions resulted in the successful separation of partricin A from the remaining compounds in the complex. High-purity partricin A (85%) was obtained between 57 and 70 minutes of separation. To obtain the graph (the CPC-DAD chromatogram) in Figure 1 , a CPC 250 m L rotor and a biphasic solvent system (chloroform: methanol: borate buffer, pH=8.2, 4:4:3, v / v / v) were used. 80 mg of the partricin complex, dissolved in 10 mL of a DMSO- upper phase mixture (1 :9, v / v), was introduced into the column. Conditions: a rotation speed of 800 rpm, ASC mode, mobile phase flow rate co=5 mL / min, room temperature, monitored wavelength A=378 nm.

[0039] From Figure 2, which presents the HPLC-DAD chromatogram, it is confirmed that the partricin A preparation obtained using the described invention, i.e. method exhibits high purity — 85%. The peak at a retention time of 9.5 minutes corresponds to partricin A. To obtain the HPLC-DAD chromatogram in Figure 2, the following conditions were used: Luna C18(2) column, 150x4 mm, 5pm, 100A; mobile phase: 38% ACN, 62% CH3COONH4 (5.5 mmol, pH=4.5), v / v; flow rate w=1.0 mL / min; concentration C=1 mg / mL; injection volume v=20μL; room temperature; monitored wavelength A=378 nm.

[0040] Example 2

[0041] The isolation of partricin B from a commercial partricin preparation produced by Tarchomin Pharmaceutical Works POLFA S.A., in the amount of 80 mg and with a purity of approximately 45% (mass percentage of the partricin A and B complex in the preparation), was performed using a Gilson CPC-250 centrifugal partition chromatograph, equipped with a 250 mL column, an Ecom flash 14 DAD 600 detector, and a Gilson LS-5600 fraction collector (entire apparatus from Gilson Inc., Middleton, Wl, USA). A biphasic solvent system — chloroform: methanol: borate buffer (pH=8.2) (v / v / v) — was prepared by mixing appropriate volumes of solvents at room temperature (500 mL: 500 mL: 375 mL). The borate buffer was obtained using a known method by dissolving 3.09 g of boric acid and 3.73 g of potassium chloride in 940 mL of water, followed by the addition of 60 mL of a 0.1 M potassium hydroxide solution, yielding 1 liter of buffer at pH=8.2. The separation was performed in ascending mode (ASC), using the upper phase as the mobile phase. First, the lower phase, comprising mainly of chloroform and methanol, was introduced into the column in the amount of 360 mL, at a flow rate of 30 mL / min and a rotation speed of 500 rpm for 12 minutes. Next, the upper phase, primarily composed of buffer and methanol, was pumped through the stationary lower phase for an additional 13 minutes, at a flow rate of 5 mL / min and a rotation speed of 800 rpm, until equilibrium was achieved at a pressure of approximately 50 bar. Subsequently, 80 mg of the commercial preparation comprising the partricin complex (a mixture of antibiotics), dissolved in 1 mL of dimethyl sulfoxide (DMSO) and diluted with the mobile upper phase to a final volume of 10 mL, was introduced into the column for separation. The upper phase was pumped for 45 minutes, at a flow rate of 5 mL / min and a rotation speed of 800 rpm, collecting 12 mL per fraction (total 225 mL). Partricin B eluted from the column within 25 to 40 minutes of separation, corresponding to 0.5- 0.8 column volumes, as confirmed by the chromatogram. The separation was performed at room temperature, with monitored wavelength of A=378 nm. Data was collected and processed using Gilson Glider CPC V5.1d.01 software. The purity of the fractions was confirmed using HPLC-DAD analysis (Luna C18(2) column, 150x4 mm, 5pm, 100A; mobile phase: 38% ACN, 62% CH3COONH4 (5.5 mmol, pH=4.5), v / v; flow rate co=1.O mL / min; concentration C=1 mg / mL; injection volume v=20μL; monitored wavelength A=378 nm; room temperature). In the final step, the fractions comprising partricin B were combined and concentrated under reduced pressure (5-7 mbar) at a temperature not exceeding 35°C, removing chloroform and methanol. The antibiotic was then extracted from the remaining aqueous layer (comprosing borate buffer) using 200 mL of n-butanol. The obtained organic phase comprising the antibiotic was desalted by washing with 100 mL of ultrapure water (2x50 mL) and further concentrated to a volume of approximately 20 mL. Next, the antibiotic was precipitated from the butanol solution using 30 mL of diethyl ether and centrifuged in a preparative centrifuge for 5 minutes at 2400 rpm. The supernatant was then discarded, and the antibiotic-containing precipitate was washed with 30 mL of diethyl ether and centrifuged again under the previously described conditions. The supernatant was once again discarded, 30 mL of diethyl ether was added, and the centrifugation process was repeated under the same conditions, after which the supernatant was discarded once more. Finally, the residue containing partricin B was dried in a vacuum desiccator for 24 hours, protected from light. The final yield was 19.4 mg of partricin B, with a purity of 83% (HPLC-DAD analysis). From Figure 3, it follows that the introduction of the partricin antibiotic complex solution into the CPC column under the conditions described above resulted in the separation of partricin B from the remaining compounds in the complex. High-purity partricin B (83%) was obtained between 25 and 40 minutes of separation.

[0042] To obtain the graph (the CPC-DAD chromatogram) in Figure 3, a CPC 250 mL rotor and a biphasic solvent system consisting of - chloroform: methanol: borate buffer (pH=8.2) (4:4:3, v / v / v) were used. A total of 80 mg of the partrici n complex dissolved in 10 mL of a DMSO-upper phase mixture (1 :9, v / v) was introduced into the column. The conditions were as follows: a rotation speed of 800 rpm, ASC mode, volumetric flow rate of the mobile phase co = 5 mL / min, room temperature, monitored wavelength A=378 nm.

[0043] From Figure 4, which presents the HPLC-DAD chromatogram, it is confirmed that the partricin B preparation obtained using the described invention, i.e. method exhibits high purity — 83%. The peak at a retention time of 5.7 minutes corresponds to partricin B. To obtain the HPLC-DAD chromatogram in Figure 4, the following conditions were used: Luna C18(2) column, 150x4 mm, 5pm, 100A; mobile phase: 38% ACN, 62% CH3COONH4 (5.5 mmol, pH=4.5), v / v; flow rate w=1.0 mL / min; concentration C=1 mg / mL; injection volume v=20μL; room temperature; monitored wavelength A=378 nm.

Claims

Claims1. Method for obtaining partricin A or partricin B comprising step of isolation of partricin A or partricin B from the partricin complex, which constitutes a commercial preparation comprising the partricin complex or an extract obtained from the fermentation broth of a strain producing partricins, characterized in that the following steps:- in the first step, a biphasic three-component solvent system is prepared, comprising: chloroform, methanol, and a buffer with a pH range of 7.5 to 9.5, in the following volumetric proportions relative to the total mixture: 15-52% (v / v) chloroform, 15-52% (v / v) methanol, and 15-45% (v / v) respectively, resulting in preparation of two phases that one sets the stationary phase forming the lower phase, predominantly composed of chloroform and methanol, while the another sets the mobile phase forming the upper phase, predominantly composed of buffer and methanol, while in the following step, the obtained solution is used to form a chromatographic column in a centrifugal partition chromatograph (CPC) operating in ascending mode (ASC) by introducing the stationary phase forming the lower phase firstly, followed by the mobile phase forming the upper phase, until equilibrium is established within the column;- in the second step:- the partricin complex is dissolved in an organic solvent in the form of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) or their mixture, then mixed with the prepared mobile phase in volumetric proportions relative to the total obtained solvent mixture of 1-99% (v / v) DMSO and / or DMF: 1-99% (v / v) mobile phase, preferably in a volumetric ratio of 10:90, or- in an organic solvent in the form of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) or their mixture:•• in the third step, the previously obtained solution comprising the partricin complex, preferably in the mobile phase, is introduced into the chromatographic column, and the separation of the partricin complex is performed using centrifugal partition chromatography (CPC) in ascending mode (ASC), applying columnrotation in the range of 500-2400 rpm, with a volumetric flow rate of the mobile phase of 1-15 mL / min, while in the case of obtaining partricin A, the separation is performed in elution mode through 0.6-2 column volumes, and preferably in the following step an extrusion step with the lower phase is performed, and the separation process is monitored at a wavelength range of A-220-450 nrn until partricin A is obtained, while in the case of obtaining partricin B, the separation is performed in elution mode through 0.3-1.1 column volumes, and the separation process is monitored at a wavelength range of A-220-450 nm until partricin B is obtained.

2. The method according to claim 1, wherein the separation is performed in elution mode through 1.1 to 1.4 column volumes for obtaining partricin A, white the separation is performed in elution mode through 0.5 to 0.8 column volumes for obtaining partricin B.

3. The method according to claims 1 -2, wherein borate buffer is used.

4. The method according to claims 1-3, wherein the partricin complex is dissolved in an organic solvent in the form of dimethyl sulfoxide (DMSO), then it is mixed with the prepared mobile phase in volumetric proportions relative to the total obtained solvent mixture of 1-99% (v / v) organic solvent: 1-99% (v / v) mobile phase, preferably in a votemetric ratio of 10%:90%,5. The method according to claims 1-4, wherein the obtained partricin A or the obtained partricin B is concentrated and extracted using a known method,6. The method according to claims 1-5., wherein the obtained organic layer is desalted by washing with water and further concentrated, after which the antibiotic is precipitated using a known method.

7. The method according to claims 1-6, wherein the preparation comprising the complex is dissolved in the upper phase obtained by mixing solvents in the composition of chloroform, methanol, and a buffer with a pH range of 7.5 to 9.5, in volumetric proportions relative to the total mixture of 15 -52% (v / v) chloroform, 15 - 52% (v / v) methanol, and 15-45% (v / v) of the appropriate buffer.8, The method according to claims 1-6, characterized in that the solution comprising the complex is dissolved in the tower phase obtained by mixing solvents in the composition of chloroform, methanol, and a buffer with a pH range of 7.5 to 9.5, in volumetric proportions relative to the total mixture of 15-52% (v / v) chloroform, 15-52% (v / v) methanol, and 15-45% (v / v) of the appropriate buffer.

9. Partrioin A or partrioin B obtained by the method described in the claims 1-8.

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