Patent Application
20110171691
The present invention relates to improved microbial strains and to their use in a biotransformation process for improving the yields of anthracycline antitumor antibiotics, particularly epirubicin and idarubicin.
Daunomycins are a group of antitumor antibiotics produced by several Streptomyces sp., such as S. peucetius, S. coerulorubidus, S. griseus, Streptomyces sp. C5, S. peucetius var. caesius, and S. bifurcus. The basic compound of the group is daunomycin (DiMarco et al., 1964).
Daunomycins may be described by the general formula I
Its most important derivatives are shown in Table 1.
Semisynthetic derivatives of daunorubicin, epirubicin and idarubicin could be classified as unnatural anthracycline antibiotics as the natural occurring strains do not produce these anthracyclines. Epirubicin is clinically used for many types of cancer. The market is growing as it competes with doxorubicin. New formulations, conjugates and new combinations with other cancer drugs also expand the usage of epirubicin. Epirubicin is manufactured by a process which comprises producing daunorubicin by fermentation and synthetically modifying the aglycone and sugar moiety, disclosed e.g. in U.S. Pat. No. 5,874,550.
Idarubicin (4-demethoxy-daunorubicin) is used to treat certain types of cancer, including leukemia, lymphoma, and other diseases of the bone marrow. It is claimed to cause less side effects than doxorubicin. The global annual market for idarubicin is, however, no more than 20 kg, presumably because of its exceptionally high price, which is due to a very complicated manufacturing process. Idarubicinone is manufactured started from daunomycinone, obtained from fermentation of daunorubicin with subsequent acidic hydrolysis. Daunorubicinone is further synthetically modified to idarubicinone. A sugar residue, daunosamine, is attached by a complicated synthetic reaction series, as described in e.g. U.S. Pat. No. 4,325,946.
In fermentation production of important anthracyclines such as daunorubicin and epidaunorubicin intermediates accumulates in fermentation broth. A typical intermediate in both cases is ε-rhodomycinone (Strohl et al., 1989). However, as epidaunorubicin may be produced by fermentation by using a genetically modified strain, 13-dihydro-epidaunorubicin (referred to as 13-DHED) is concomitantly accumulated to the fermentation broth. Typically these metabolites are considered as waste. U.S. Pat. No. 5,652,125 discloses the use of ε-rhodomycinone to produce daunorubicin, and Dickens et al. (1997) have described the oxidization of 13-dihydro-anthracyclines to their 13-keto forms. No publications describing biotransformation in processes for un-natural anthracyclines have been located.
We have discovered a new process for obtaining higher yields of commercially important unnatural anthracyclines, epirubicin and idarubicin at lower cost by utilization of side products obtained in fermentation production of daunomycin and epidaunorubicin.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
In the drawings:
The present invention relates to a microbial strain, which converts anthracycline metabolites, such as epidaunorubicin, 13-dihydroepidauno-rubicin, 4′-epi-feudomycin and ε-rhodomycinone, into non-natural anthracycline antibiotics, such as epirubicin and idarubicin. Preferably such strains are selected from the genera Streptomyces, more preferably from the species Streptomyces peucetius, and most preferably from the subspecies Streptomyces peucetius var. caesius. In a preferred embodiment of the present invention, said strain comprises a heterologous resistance gene snorO, isolated from the species Streptomyces nogalater.
The present invention also relates to a process for converting anthracycline metabolites such as 13-dihydroepidaunorubicin, and ε-rhodomycinone, into non-natural anthracycline antibiotics, such as epirubicin and idarubicin using a microbial strain according to the present invention. In a preferred embodiment according to the present invention, in said process a resin, preferably selected from the group consisting of ionic and non-ionic adsorbents, more preferably from polystyrenes, and most preferably from the group consisting of XAD-7 and Diaion HP-20, is used at any time to adsorb the anthracycline metabolites or anthracycline antibiotics.
The present invention relates to improved Streptomyces strains, which are able to convert modified anthracycline intermediates into important antitumor anthracycline drugs, idarubicin and epirubicin. Said strains are useful in a process for converting anthracycline metabolites into the final products.
It is thus an object of the present invention to provide an improved Streptomyces strain, modified to be able to stably conversion of 4-deoxy-ε-rhodomycinone into idarubicin at a high rate. Such a strain, blocked to produce daunorubicin, was derived from strain G001 (DSM 12245) as described in Example 1 (Strain G001 was derived by mutagenization of wild type S. peucetius var. caesius ATCC 27952). This improved mutant strain derived from strain G001 is able to convert 4-deoxy-ε-rhodomycinone into idarubicin whereas repeatability was poor. In order to improve conversion, resistance genes were introduced into this strain.
The gene snorO was isolated from S. nogalater (ATCC 27952) and is suggested to be responsible for the resistance to nogalamycin (Torkkell 2001). Based on sequence analysis, the gene product, SnorO is a multifunction gene product for resistance, with domains for the excision repair protein UvrA, and ABC transporter ATP-binding protein. Heterologous expression of snorO in S. lividans TK24 showed that the gene protected the strain from all tested different anthracyclines, nogalamycin, aclarubicin and daunorubicin. Introduction of the gene snorO into the improved strain derived from G001 provided a new biotransformation strain, which exhibits increased resistance to idarubicin. Surprisingly, snorO, when introduced into improved strain derived from G001 in the E. coli vector, improved the conversion rate and stabilized the strain to maintain the conversion within ±10%, even in six sequences of cultivations.
Said new biotransformation strain may be fed by natural anthracycline intermediates such as aklavinone and ε-rhodomycinone and natural anthracyclines, daunomycins are formed.
Surprisingly, the strain is able to carry out conversion of unnatural metabolites and feeding with un-natural 4-deoxy-ε-rhodomycinone resulted in formation of idarubicin. Additionally, the strain is able to convert 13-DHED into epidaunorubicin. Synthetic conversion of ε-rhodomycinone into 4-deoxy-ε-rhodomycinone is presented. In accordance with the present invention 4-deoxy-ε-rhodomycinone is subsequently biotransformed into idarubicin by the above mentioned new biotransformation strain, which does not produce daunomycin metabolites. 13-DHED was further converted into epidaunorubicin by biotransformation using the same mutant strain.
Important aspects of a biotransformation strain suitable for use in the present invention are:
i) natural and unnatural substrates are accepted for conversion;
ii) the strain has increased resistance against natural and unnatural anthracyclines;
iii) the strain accomplish essential functions needed for conversion, and
iv) the strain does not accumulate detectable quantities of natural daunomycins.
Any suitable strain can be used for biotransformation of the 4-deoxy-semisynthetic intermediate into idarubicin. Nevertheless, it is critical that the strain has either endogenous or transferred expressible genes for the following reactions: modifications of glucose to form daunosamine, 10-esterase activity to remove a methyl group with the connected 10-decarboxylase activity, 13-oxygenase and the suitable glycosyl transferase activity. Furthermore, the downstream process after biotransformation is cost-effective only if no or minor amounts of natural anthracycline metabolites are accumulated by the strain used as a host in biotransformation.
It is advantageous to use a S. peucetius var. caesius mutant blocked in the early stage of daunomycin biosynthesis; preferably a mutant blocked in minimal PKS (minimal PolyKetideSynthase catalyzes the first reactions, polyketide assembly in anthracycline and other Type II polyketide pathway).
The new biotransformation strain derived from wild type Streptomyces peucetius var. caesius ATCC 27952 as described above is a highly preferred strain, but any strain sharing the following characteristics is suitable for the conversion process according to the present invention.
1. Blocked in early pathway for daunomycins. Disruption of dpsG gene of minimal PKS (data not shown) based on complementation experiment of the corresponding gene.
2. Does not produce detectable quantities of daunomycins, whereas accumulates an acidic yellow substance on solid medium. The structure of the compound is not known, but it is not a member of anthracyclines.
3. On ISP4+tsr- and ISP2+tsr-plates biotransformation strain is normally non-sporulating and forms colourless aerial hyphae.
4. Express resistance to several different anthracyclines as a consequence of snorO function.
5. Converts 4-deoxy-ε-rhodomycinone into two main products: idarubicin and 13-dihydroidarubicin. Some other idarubicin metabolites are found in small quantities.
The present invention further relates to a process for production of un-natural anthracycline antibiotics, epirubicin and idarubicin by exploiting the shunt products, ε-rhodomycinone formed in the fermentation production of daunorubicin and epidaunorubicin, and 13-DHED formed in the fermentation production of epidaunorubicin.
It is thus a further object of the present invention to provide a process for preparing commercially useful anthracycline antibiotics by means of biotransformation using an improved bacterial host strain according to the present invention. Preferably, such a host strain is the new biotransformation strain described above, derived from genus Streptomyces, preferably derived from S. peucetius var. caesius.
In the art, it is known that idarubicinone can be converted into idarubicin by a complicated chemical synthesis series, as described in e.g. U.S. Pat. No. 7,053,191. In the process according to the present invention an endogenous biosynthetic reaction series of a bacterial strain to convert 4-deoxy-ε-rhodomycinone into idarubicin is preferably used. It is known that biosynthesis proceeds in the sequence shown in
However, the gene products for these modifications need a substrate in which the substituent at C-10 is a COOCH3— group.
ε-rhodomycinone, obtained as a shunt product by a fermentation process of daunomycin metabolites, is processed into 4-deoxy-ε-rhodomycinone by synthetic chemistry. Various synthetic paths are possible to remove a hydroxyl group from position 4 of ε-rhodo-mycinone. However, we prefer to carry out the four reaction series starting by protection of C7 and C9-hydroxyl groups by ketalization. After that, trifylation of the OH— group at C4 takes place following by reduction to remove the substituent at C4. After each step the product is isolated or purified by precipitation/crystallization and the overall yield of >20%, preferably >30%, most preferably >40% is obtained. The purity of 4-deoxy-ε-rhodomycinone obtained in this process is >60%, preferably >80%, most preferably >90% being a suitable substrate for biotransformation.
ε-rhodomycinone is typically accumulated in the fermentation broth in large quantities and purification by conventional methods is successful. Synthetic conversion of ε-rhodo-mycinone provides >20%, preferably >30%, most preferably >40% of pure 4-deoxy-ε-rhodomycinone, which is fed to the non-producing mutant strain of S. peucetius var. caesius. The efficiency of biotransformation of 4-deoxy-ε-rhodomycinone into idarubicin was >20%, preferably >30%, most preferably >40%. More than 100 mg of the semisynthetic intermediate could be fed to a litre of the culture of the biotransformation strain. The timing to feed the intermediate is not critical. Any fermentation conditions allowing growth of streptomycetes and secondary metabolism could be used whereas it is advantageous to use E1-medium and temperature range of 25-35° C. in pH between 6 to 8.
Recovery of idarubicin from the culture can be carried out with any suitable method, such as centrifuging, filtration or by a suitable ionic or non-ionic adsorbent. For insertion of idarubicin, any water-soluble organic solvent could be used, whilst acidic alcohols are preferred. Aglycones are removed with acidic extraction after which glycosides are extracted back from water phase to chloroform phase in high pH. Depending on the profile of the compounds after evaporation of the last extract, idarubicin is finally purified by crystallization or, preferably, by chromatography and crystallization. It is advantageous to use a silica column for purification.
Conversion of 13-DHED to EPIDAUNORUBICIN
It is well known that 13-dihydro-anthracyclines accumulates to fermentation broth together with 13-keto forms. However, 13-DHED is not commercially useful and based on its cytotoxic nature, it shall be handled as a toxic waste. To our surprise, this metabolite even though not a natural one was converted to epidaunorubicin by the biotransformation strain in a useful rate, e.g. >20%, preferably >30%, most preferably >40%.
13-DHED, a side product in epidaunorubicin fermentation is easily separated from glycosidic fraction by chromatography followed by crystallization alongside with epidaunorubicin separation. 13-DHED could be adsorbing to a resin added to the culture broth of Streptomyces strain with capabilities for daunomycin synthesis and especially the late steps. Even though any strain is suitable, it is advantageous to use the strain which is blocked in early biosynthetic pathway and unable to accumulate daunomycin metabolites.
In a preferred embodiment of the present invention, the new biotransformation strain, derived from genus Streptomyces, preferably derived from S. peucetius var. caesius is used for biotransformation to convert 13-DHED to epidaunorubicin.
Conversion rates in the conditions described for production of epidaunomycins according to the present invention are >20%, preferably >30%, most preferably >40%. Epidau-norubicin obtained in this way may be purified from other metabolites bound to the resin by any conventional methods used for anthracyclines recovery whereas chromatography and especially reverse-phase chromatography is preferably used to provide adequately purified epidaunorubicin for synthesis.
Conversion of Epidaunorubicin to Epirubicin
Crude, epidaunorubicin (purity >60%, preferably >80%, most preferably >90%) is used as a starting material for synthetic chemistry to obtain epirubicin, which is a frequently used cancer drug. Any synthetic or biocatalytic reaction series may be used for the 14-hydroxylation.
There are several possibilities to convert epidaunorubicin into its 14-hydroxylated form, epirubicin. The endogeneous gene product alone, 14-hydroxylase, is not sufficiently active in the cultural conditions to convert all epidaunorubicin formed into epirubicin even though minor amounts of epirubicin is found in the culture broth. According to our experiments (data not shown), even high copies of the gene for 14-hydroxylase, failed to complete the process for epirubicin production. Nevertheless, two US patents, U.S. Pat. No. 5,955,319 and U.S. Pat. No. 6,210,930 disclose the conversion of daunorubicin into doxorubicin in a low level by the gene product of doxA. Apparently, the bioconversion is highly dependent on the conditions, and we have not succeeded in repeating the process.
There are various possibilities to add a hydroxyl group at the C-14 of the intact epidaunorubicin, analogously to synthesis of doxorubicin from daunorubicin.
Purification of epirubicin is carried out by chromatography and/or by crystallization after extraction of epirubicin from the synthesis mixture. However, to achieve the quality requested for active pharmaceutical ingredient, chromatography separation to give epirubicin in >97% purity is essential.
A more detailed description of the present invention is given in the examples below.
It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described below but may vary within the scope of the claims.
a discloses a biosynthetic pathway for daunorubicin. Baumycins are formed from daunorubicin. The biosynthetic pathway is branched to (i) doxorubicin and to (ii) baumycin after daunorubicin. These steps are not shown in the figure.
b discloses a biosynthetic pathway for dTDP-daunosamine, which is used for the C-7-glycosylation in
For mutagenization strain G001, derived from wild type S. peucetius var. caesius ATCC 27952 by mutagenization was cultured in 50 ml TSB-medium in 250 ml Erlenmeyer flask. All the flasks for mutagenesis contained a string in the bottom of the flask to disperse mycelia during cultivation. Cultivation was carried out for two days at 30° C. and 330 rpm in a shaker. One ml of the culture was further inoculated to the next bottle containing 50 ml TSB and cultivation was continued for one day (30° C., 330 rpm). This younger culture was adapted to alkaline pH with NaOH and NTG was added to act on the cells for 20 minutes at >30° C. The mutagenized culture of said strain was divided into two tubes and pelleted by centrifugation (300 rpm, 10 minutes). Combined pellets were used to inoculate 50 ml of TSB medium. After one day (30° C., 330 rpm), the titre of the cell suspension was determined by plating suitable dilutions, said 1:10-1:100000 on ISP4-plates. Colonies of mutagenized cultures were compared to the wild type and those exhibiting distinct features from the wild type were selected for further characterization.
The mutants were selected based on pale colour from the dark red-pigmented wild type on the ISP4-agar plate.
The gene snorO (Torkkell, 2001) was introduced into the mutants with E. coli vector, pCNB3033. Integration was suggested to occur by attP-site. The obtained improved biotransformation strain gave repeated conversion rates for >20%, preferably >30%, most preferably >40% of fed 4-deoxy-ε-rhodomycinone into idarubicin metabolites as is detailed described in Example 5.
Suitable mutants selected as described in example 1 were cultivated in 50 ml of E1-medium supplemented with adsorbent resin (15 g/l). (E1: Per litre of tap water: glucose 20 g; soluble starch 20 g; Peptide 5 g; Yeast extract 2.5 g; K2HPO4.3H2O 1.3 g; MgSO4.7H2O 1 g; NaCl 3 g; CaCO3 3 g; pH 7-7.5).
To determine the anthracycline metabolites the compounds were extracted at the tenth day of incubation. For analysis, the adsorbent resin was decanted with water from one cultivation flask and washed resin was extracted with 40 ml of acidic alcohol shaking for at least 30 min. The HPLC was used for analysing the samples. The use of adsorbent resin in E1-medium was known to increase production of anthracycline metabolites in said conditions.
The changes in colony morphology were not found at the end of cultivation in the presence of the adsorbent.
Production of anthracycline metabolites were not detected in culture broth of the mutant strains.
However, mutant strains have the functional biosynthetic genes for the glycosylation and modification of aglycones according to biosynthesis pathway of
The improved biotransformation strain was able to convert natural aglycones, 8-rhodomycinone and aklavinone as was expected. It also converted un-natural analogous biosynthetic intermediate, 4-deoxy-ε-rhodomycinone and 13-DHED to idarubicin and to epidaunorubicin, as is described in the example 4 below.
Purified ε-rhodomycinone of >60%, preferably >80%, most preferably >90% chromatographic purity was in use for synthesis of 4-deoxy-ε-rhodomycinone.
The synthesis of 4-deoxy-ε-rhodomycinone consists of four steps:
A) protection, B) trifylation, C) reduction and D) deprotection.
After each step the product was isolated by precipitation/crystallization from chloroform-methanol mixtures. Synthesis reactions were monitored with TLC.
A) Protection
ε-rhodomycinone was dissolved in chloroform at room temperature. 2 eqv. of 2-methoxypropene was added to a mixture followed by 1 eqv. of TMSCl. Reaction was followed by TLC. When the reaction was finished as was detected on TLC plate, the mixture was allowed to cool down and the precipitate was collected and dried for the next step.
B) Trifylation
A starting material from protection step was dissolved in chloroform. NMP (N-methyl pyrrolidone) and di-isopropylethylamine (DIPEA) were added and finally PhNTf2. Reaction was monitored with TLC. The compound was precipitated by adding water and citric acid to mixture. Precipitate was filtered and washed with KHSO4. Crystals were filtered and dried under vacuum.
C) Reduction
Material from trifylation was suspended to ACN under argon. (Ph3P)4Ph was added followed by addition of (Ethyl)3N and HCOOH. Reaction mixture was heated up and reaction was followed by TLC. After 1 h reaction was completed. Reaction mixture was allowed to cool down and the product was filtered off.
D) Deprotection
The product from reduction was dissolved in chloroform and 1 eqv. of TsOH×H2O was added at RT. Reaction proceeds within 0.5 h. Solution was washed with 1M NaHCO3, dried with MgSO4 and evaporated to dryness.
4-deoxy-ε-rhodomycinone Purification
A deprotection reaction mixture (CHCl3/TsOH×H2O) was diluted with chloroform, and washed with 1M NaHCO3. The chloroform fraction was dried with anhydrous MgSO4, filtered and evaporated to dryness. Crystallization was done from chloroform-methanol. Crystals were filtered and dried under vacuum.
Fermentation broth for production of epidaunorbicin contains three major metabolites in this order: Epidaunorubicin, 13-DHED and epi-feudomycin (Ref epi-patent application). The substituents adsorbed to a resin were decanted from the 20 litre culture broth obtained from fermentation. The resin was washed to remove cell debris by water. Pellet was extracted with alcohol for one to five times. The aglycones were extracted with chloroform by adding chloroform to the combined alcohol extracts. Solvent and water layers were separated. Glycosides in water phase were extracted to chloroform at lightly alkaline pH and pH was stabilized with saturated NaHCO3. Salts were removed by washing with water. Finally the chloroform-phase was filtrated through a cartridge filter.
Silica gel chromatography was carried out to separate the metabolites. The filtrated chloroform was pumped into a silica column and purified by chromatography using chloroform-methanol-solution as a mobile phase. Pure fractions of each three metabolites were collected, and fractions of each product were pooled for crystallization. The fractions of 13-DHED were crystallized by ethanol-water solutions. The crystals were filtrated and adsorbed to adsorbent resin.
Seed culture was made by cultivating a biotransformation strain in two flasks of 400 ml of the E1 medium for three days. The cultures were combined and the 800 ml of the culture broth were used to inoculate a 20 litre E1-medium supplemented with XAD-7. Fermentation was carried out in 20 litres volume for 8 days at the temperature of 30° C., 350 rpm with the aeration of 10 l/min. pH has to be kept slightly acid, which ensures more stable conversion of fed 4-deoxy-ε-rhodomycinone into idarubicin. 4-deoxy-ε-rhodomycinone is feeded continuously 4 days started 24 hours after inoculation with at least 5 mg/ml 4-deoxy-ε-rhodomycinone in EtOH. Feeded 4-deoxy-ε-rhodomycinone amount is at least 100 mg/l.
During biotransformation with mutant strain two main secondary metabolites were produced from the fed 4-deoxy-ε-rhodomycinone; namely idarubicin and 13-dihydro-idarubicin. Other minor metabolites were idarubicinone, 13-dihydroidarubicinone, baumycins and 13-deoxy-Idarubicin and its aglycone. Stable titre of Idarubicin is >20 mg/L, in addition >20 mg/L of previous intermediate 13-DHI is produced; thus altogether the amount is >40 mg/L. Baumycins can be hydrolysed to Idarubicin by heating in acidic conditions (+45° C., 1 hour).
The chromatograph of the products obtained from biotransformation strain is shown in
Pre-cultivation of biotransformation strain was done as detailed described in Example 5. 13-DHED adsorbed to resin corresponding to at least 50 mg/1 of the culture broth was added to the cultivation after one day and cultivation was continued for four days. Fermentations were carried out in flasks containing 50 ml E1-medium, at 34° C., 300 rpm.
According to the chromatographic analysis, 50% of 13-DHED was converted to epidaunorubicin. Minor amounts of epirubicin, in the range of <10%, were detected.
TLC
1. 0.5 MQ-water
2. 0.1 HCOOH
3. 25 MeOH
4. 75 CHCl3, mix with solutions 1, 2 and 3 carefully
HPLC
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
This application is a Section 371 of International Application No. PCT/EP2008/007460, filed Sep. 11, 2008, which was published in the English language on Mar. 18, 2010, under International Publication No. WO 2010/028667 A1 and the disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/007460 | 9/11/2008 | WO | 00 | 3/10/2011 |
