Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
  • C12P35/06—Cephalosporin C; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66

Definitions

• This invention relates to methods of preparing metabolites of compounds, and more particularly to the preparation of such metabolites by biotransformation.
• TDM therapeutic dose monitoring
• Metabolites are formed when enzymes, commonly liver enzymes, work to break down or modify a drug so that the drug can be more easily eliminated from the body.
• the parent compound is rapidly metabolized, it may be most convenient to measure the levels of a metabolite for the purposes of TDM. Frequently, immunoassays are used for such measurements.
• a TDM assay for measuring a metabolite is an immunoassay
• the drug, or an isolated, purified metabolite of the drug maybe used for generating and/or selecting for an antibody having the desired specificity for use in that assay.
• the purified metabolite may be used to define an antibody specific for the parent compound, i.e., an antibody that exhibits minimal cross-reactivity between the parent compound and metabolites of the parent compound. Therefore, efficient methods for producing isolated metabolites are needed in order to obtain a quantity of metabolite suitable for use in producing antibodies for TDM.
• Metabolites may also have uses that are independent of TDM. Metabolites may have pharmaceutically important activities. For example, metabolites may exhibit beneficial characteristics such as improved pharmacokinetics, increased pharmacological activity or improved bioavailability.
• a metabolite of a parent drug compound may itself be a useful therapeutic.
• A77 1726 is the active metabolite of leflunomide
• hydroxy-tert- butylamide is the active metabolite of the HIV drug, nelf ⁇ navir
• 4-OH-tamoxifen is the active metabolite of tamoxifen.
• efficient methods of producing large quantities of the metabolite may be desired.
• one or more of the metabolites may be toxic.
• knowledge about how a drug is metabolized, its resulting metabolites, and the activity of these metabolites is important for understanding the activity of a drug. This information may also be required prior to drug approval. In order to identify the metabolites and their properties, a sufficient quantity of the metabolites must be produced and isolated.
• One method for producing metabolites is to administer the drug to a mammal, such as a human, then collect blood, urine, bile or other body fluids, and extract, purify, and isolate metabolites from these fluids.
• a mammal such as a human
• biotransformation the conversion of a drug to metabolites of the drug, is achieved in human patients in the liver by the liver cytochrome P450 enzymes (CYP450 or P450).
• the P450 enyzme family includes an estimated 70 or so enzymes which act to render a compound more soluble for excretion in the bile or urine.
• a parent compound may be tagged so that the metabolites may be recognized.
• a drug having a similar structure may be analyzed in parallel when the results are monitored by high pressure liquid chromatography separation and mass spectral analysis.
• a second method for biotransformation of a parent compound is to use a whole organ, a tissue slice, or cultured cells such as hepatocytes as a biotransforming system.
• microsomes prepared from mammalian cells may be used. These approaches use animal isolates, thus risking introduction of unwanted contaminants into the metabolites. These methods are difficult to scale up when larger quantities of one or more of the metabolites is desired.
• biotransformations using microorganisms to convert the parent compound into metabolites may also be used.
• the present invention provides methods of producing metabolites of xenobiotic compounds by biotransformation using a microorganism.
• the xenobiotic compound may be delivered to the microorganism in a mixture with a surfactant.
• the method can be scaled up to produce large quantities of metabolites by, for example, biotransformation in a reactor.
• the metabolites produced by the present method can be used, for example, for antibody production, as standards in therapeutic dose monitoring, or in pharmaceutical applications.
• one aspect of the present invention provides a method for producing at least one metabolite of a xenobiotic compound in a microorganism, comprising the steps of:
• the mixture may comprise the xenobiotic compound, a solvent, and the surfactant.
• the solvent may an alcohol, such as ethanol.
• the solvents may comprise more than one substance.
• the solvent comprises both an alcohol and dimetheyl sulfoxide (DMSO).
• the microorganism may be any microorganism that is capable of metabolizing the xenobiotic compound, preferably one that possesses the same metabolizing pathway for the xenobiotic compound as the human does.
• the microorganism is selected from the group consisting of Actinoplanes sp., Streptomyces griseus, Streptomyces setonii, and Saccharopolyspora erthyraea.
• the microorganism may also be Cunningham ellaechinulata, Nerospora crassa, or Actinoplanes sp.
• the surfactant may be any suitable surfactant, which can be identified by a skilled artisan based on teachings of the present disclosure.
• the surfactant may be selected from the group consisting of polyethylene glycol (PEG) 400, castor oil, isopropyl myristate, glycerine, Cremophor® (polyoxyl castor oil), Labrasol® (caprylocaproyl macrogolglycerides), and TWEEN® 40.
• the xenobiotic compound is preferably a compound with a low solubility in aqueous solutions.
• the xenobiotic compound is selected from the group consisting of immunosuppressants and anti-bacterial compounds, preferably a cyclosporin compound, more preferably IS A247 or cyclosporin A.
• the metabolite is preferably selected from the group consisting of Ml-d-1, IMl-d-2, IMl-d-3, IMl-d-4, Ml-c-1, IMl-c-2, IMl-e-1, IMl-e-2, IMl- e-3, IM9, IM4, IM4n, IM6, M46, M69, and IM49.
• the method of the present invention may optionally further comprise the step of isolating the metabolite from the culture.
• Another aspect of the present invention provides a method for identifying a microorganism suitable for use in a biotransformation system comprising: a) comparing the structure of a compound to be metabolized with a known enzyme activity; b) identifying an enzyme that expresses the known enzyme activity; c) identifying a microorganism that expresses the identified enzyme; and d) using the microorganism that expresses the identified enzyme in a biotransformation system to make metabolites of the compound, hi certain embodiments, the microorganism may be identified by comparing the genomic sequence of various microorganisms to the sequence of the identified enzyme, thereby identifying at least one microorganism that expresses the enzyme.
• Figure 1 illustrates the structure of ISA247.
• the amino acid residues in ISA247 are indicated by numbers. Greek letters indicate the carbon positions of amino acid 1.
• Figures 2A and 2B provide the structures of the trans (E-) and cis (Z-) isomers of ISA247 molecule, respectively.
• Figure 3 is an HPLC scan showing the profile of the ISA247 metabolites isolated from human whole blood of a subject who had received a 50:50 mixture of cis ⁇ trans ISA247.
• Figure 4 is an HPLC scan illustrating the profile of the ISA247 metabolites isolated from the biotransformation method described in Example 4.
• Figure 5 is a graph showing the effects on IS A247 metabolite production of different surfactants in a biotransformation system.
• Figure 6 shows the LC-MS profile of the ISA247 metabolites isolated from human whole blood of a subject who had received an ISA247 formulation that contained predominantly the trans-isomev of ISA247.
• Figures 7 and 8 compare the effects of Media 3 and Media 16 on ISA247 metabolite production by Actinoplanes sp. (ATCC 53771) and Saccharopolyspora erythraea (ATCC 11635), respectively.
• Figure 9 demonstrates the effects of different solvents and surfactants on ISA247 metabolite production by Beauvaria bassiana .
• the present invention provides methods of producing metabolites of xenobiotic compounds by biotransformation using a microorganism. Specifically, the xenobiotic compound is delivered to the microorganism in a mixture with a surfactant.
• the method can be scaled up to produce large quantities of metabolites by, for example, biotransformation in a reactor.
• the metabolites produced by the present method can be used, for example, for antibody production, as standards in therapeutic dose monitoring, or in pharmaceutical applications.
• biotransformation refers to the process of metabolizing a compound by a living cell, particularly a cell of a microorganism.
• a "xenobiotic compound,” or “xenobiotic,” is a compound that is not native with respect to a microorganism.
• a xenobiotic compound may be pharmaceutically active.
• the xenobiotic compounds of this invention are preferably not readily soluble in water.
• the compound may have a water solubility, at 25 0 C, of 1 mg/ml or less, 0.75 mg/ml or less, 0.5 mg/ml or less, 0.25 mg/ml or less, 0.1 mg/ml or less, 0.08 mg/ml or less, 0.06 mg/ml or less, 0.04 mg/ml or less, or 0.02 mg/ml or less.
• a "cyclosporin compound” is a cyclosporin, or derivative thereof, that has immunosuppressive activities.
• the term encompasses the naturally occurring cyclosporins, cyclosporin A to Z, ISA247, synthetic and artificial dihydro- and iso-cyclosporins such as those disclosed in US Pat. Nos. 4,108,985; 4,210,581 and 4,220,641; derivatized cyclosporins such as shown in US Pat. Nos.
• IS A247 IS ATX247 or ISA
• ISA247 is a cyclic undecapeptide consisting almost entirely of hydrophobic amino acids. Many of these amino acids are not normally found in mammalian proteins.
• Figure 1 illustrates the structure of IS A247 and the 11 amino acid residues that comprise the cyclic peptide ring of this molecule. As shown, the amino acid residues are numbered in a clockwise direction. As shown in Figure 1, seven amino acids of the eleven amino acids of the 11-membered amino acid ring are N-methylated.
• CsA has a solubility of about 0.04 mg/ml at 25° C. Due to its low water-solubility, the bioavailability of cyclosporin A is known to be 30% or less when orally administered to humans.
• ISA247 exhibits a similarly low water solubility.
• ISA247 contains a sarcosine residue (whose three letter abbreviation is Sar; sarcosine is a methylated glycine residue and may also be abbreviated MeGIy), one each of a D- and an L- alanine (Ala) residue, an ⁇ -amino butyric acid residue (Abu), a valine (VaI) residue, an N-methyl valine (MeVaI) residue, four N-methyl leucine (MeLeu) residues, and an alkene-containing 9- carbon, ⁇ -hydroxylated amino acid unique to the cyclosporins called (4R)-4-[(E) ⁇ 2-butenyl]-4,N- dimethyl-L-threonine (MeBmt).
• sarcosine residue whose three letter abbreviation is Sar; sarcosine is a methylated glycine residue and may also be abbreviated MeGIy
• MeGIy a
• ISA247 is cyclo ⁇ (E)- and (Z)- (2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6,8-nonodienoyl ⁇ -L-2-aminobutyryl-N- methyl-glycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L- leucyl-N-methyl-L-leucyl-N-methyl-L-valyl ⁇ .
• Its empirical formula is C63H11 INl 1O12.I. It has a molecular weight of about 1214.85.
• ISA247 is known to exist in two isomeric forms, cz>s-ISA247 (or Z-ISA247) and trans- ISA247 (or E-ISA247).
• Figures 2A and 2B illustrates the trans and cis forms of ISA247.
• a mixture of cis and trans forms of the ISA247 compound has been found to be less toxic and more potent than CsA (See U.S. Pat. Nos. 6,605,593 and 6,613,739).
• ISA247 has been found to be less toxic and more potent than CsA as a mixture of cis and trans forms when the mixture contains a predominant proportion of the trans isomer.
• ISA247 is a mixture of the cis and trans isomers, and that the mixture may be enriched in the trans isomeric form of the compound.
• the isomeric compounds may be present in a mixture, ranging from 1:99 cis'.trans to 99: 1 cis:trans.
• R is selected from the group consisting of:
• R is selected from the group consisting of CH 3 and H; where R lis selected from the group consisting of CH2CH(CH3)2 and CH2C(CH3)2OH; and where R4 is selected from the group consisting of CH(CH3)2 and C(CH3)2OH.
• ISA247 metabolites which are modifications at amino acid-1 of the ISA247 compound are illustrated in Table 1.
• the boxes represent amino acids 2-11 which form the ring portion of the cyclosporin structure with the modified amino acid-1, see Figure 1.
• Table 1 is not an exhaustive list of ISA247 metabolites that are modified at amino acid-1.
• amino acid 1 metabolites may include 5, 6, 7 or 8 member rings.
• ISA247 metabolites include N-demethylated metabolites where the N-demethylation occurs at at least one methylated nitrogen of the amide linkage of an amino acid, for example, IM4n, (or ISA247 Metabolite, N-demethylation at amino acid-4). N-demethylation can occur at amino acid-3 (IM3n), amino acid-4 (IM4n), amino acid-6 (IM6n), amino acid-9 (IM9n), amino acid- 10 (IMlOn) or amino acid-11 (IMl In).
• IM3n amino acid-3
• IM4n amino acid-4
• IM6n amino acid-6
• IM9n amino acid-9
• IM9n amino acid- 10
• IMlOn amino acid-11
• IMl In amino acid-11
• ISA247 metabolites also include hydroxylated metabolites where the hydroxylation occurs at at least one methyl leucine amino acid, for example amino acids 4, 6, 9 or 10 (IM4, IM6, IM9 or IMlO), or at valine residue 5 (IM5) or at methyl valine residue 11 (IMl 1).
• IM46 are hydroxylated at both amino acids 4 and 6
• IM49 are hydroxylated at both amino acids 4 and 9, and so on.
• Combinations of N-demethylated and hydroxylated metabolites can occur, as well as combinations of the metabolites which are alterations at amino acid-1, as shown in Table 1, with N-demethylations or hydroxylations.
• ISA247 metabolites also include metabolites which are glucuronide, sulfonide, glycosylated and phosphorylated derivatives of hydroxylated metabolites of ISA247.
• U.S. Pat. Application No. (Attorney Docket Number 16593-009001, filed December 19, 2005) co-pending and commonly assigned to the assignee provides IS A247 metabolites and uses thereof.
• FIG. 3 is an HPLC scan illustrating the metabolite profile of metabolites isolated from the whole blood of this subject. Using organic extractions on human whole blood, metabolites were extracted, dried, reconstituted in methanol and identified using chromatographic techniques coupled with mass spectrometry. As shown in Figure 3, at least three diol , two hydroxylated and three N- demethylated metabolites were detectable in human whole blood.
• a dog liver microsome preparation was also used to produce ISA247 metabolites (Example T). While ISA247 metabolites can be produced in this manner, the yield was low and the cost was high. Therefore, it is not practical to obtain meaningful quantities of ISA247 metabolites using this approach.
• cytochrome P450 enzymes are known to fonn metabolites from CsA. It has been found that cytochrome P450 enzymes also act to form ISA247 metabolites. Specifically, the cytochrome P450 enzyme CYP3A4 has been identified as the enzyme responsible for cyclosporin and ISA247 metabolism. In order to produce a metabolite profile that is similar to that obtained from humans, the biotransformation system must utilize a microorganism which has the microbial equivalent of the human cytochrome P450 enzyme, grown in a medium and under culture conditions suitable for active growth and metabolism of the microorganism.
• ISA247 metabolites were detected: IM4n (ISA247 Metabolite that is N-demethylated at amino acid-4), IM9 (ISA247 Metabolite that is hydroxylated at amino acid-9), IM4 (ISA247 Metabolite that is hydroxylated at amino acid-4), IMl-c-l(See table 1), IMl-d-1 (Table 1), IMl- d-2 (Table 1) and IMl-d-3 (Table 1).
• IM4n ISA247 Metabolite that is N-demethylated at amino acid-4
• IM9 ISA247 Metabolite that is hydroxylated at amino acid-9
• IM4 ISA247 Metabolite that is hydroxylated at amino acid-4
• IMl-c-l(See table 1) IMl-d-1 (Table 1)
• IMl- d-2 Table 1
• IMl-d-3 Table IMl-d-3
• the use of different surfactants or solvents may result in increased amount of metabolites or an improved production profile.
• PEG 400 and glycerol led to the production of greater amounts of metabolites when Saccharopolyspora erytheraea was used (Example 4), while TWEEN® 40 significantly increased the number of different metabolites produced by Beauvaria hassiana (Example 6).
• one aspect of the present invention provides a method for producing at least one metabolite of a xenobiotic compound in a microorganism, comprising the steps of:
• the method may optionally further comprise the step of isolating the metabolite from the culture.
• Certain embodiments of the present invention provide an in vitro biotransformation system for producing significant quantities of metabolites of poorly soluble compounds such as those listed herein, especially immunosuppressive compounds such as cyclosporins (for example, ISA247 and CsA), macrolide lactones (for example, FK506), and triene macrolides (for example, rapamycin).
• immunosuppressive compounds such as cyclosporins (for example, ISA247 and CsA), macrolide lactones (for example, FK506), and triene macrolides (for example, rapamycin).
• Suitable xenobiotic compounds are discussed in greater detail below.
• the bioreaction is allowed to proceed for a time and under conditions which permit the parent compound to be metabolized.
• the metabolites are extracted from the bioreaction mixture, purified by separation, for example by chromatography such as high pressure liquid chromatography and mass spectral analysis (HPLC-MS). Nuclear magnetic resonance analysis may be used to verify that the individual metabolites have been isolated from one another and to verify the structure thereof. Individual metabolites that have been verified as separate chemical entities may be used as standards in subsequent assays.
• a purified metabolite may be used in a TDM assay.
• ISA247 may be administered to an organ transplant patient in a dose sufficient to achieve immunosuppression and prevent the rejection of a transplanted organ.
• a blood sample may be obtained from the patient at intervals. Blood levels of IS A247 may be measured, hi addition, blood levels of at least one metabolite may be monitored to ensure that the patient's body is metabolizing the drug in a predictable manner.
• Quantification may be achieved by, for example, immunoassay or by HPLC-MS. Similarly, antibodies specific for ISA247 or one of its metabolites may be developed.
• ISA247 in ethanol is mixed with glycerol and then added to a biotransformation system containing Saccharopolyspora erytheraea ⁇ e.g., ATCC 11635).
• PEG 400 is mixed with ISA247 in ethanol prior to the addition of ISA247 to the biotransformation system.
• castor oil is mixed with ISA247 in ethanol prior to the addition of ISA247 to the biotransformation system.
• isopropyl myristate is mixed with IS A247 in ethanol prior to the addition of ISA247 to the biotransformation system.
• Cremophor® is mixed with ISA247 in ethanol prior to the addition of ISA247 to the biotransformation system
• Labrasol® is mixed with IS A247 in ethanol prior to the addition of IS A247 to the biotransformation system
• TWEEN® 40 is mixed with IS A247 in ethanol prior to the addition of ISA247 to the biotransformation system.
• drugs which are poorly soluble in aqueous solutions include: analgesics/antipyretics (e.g., aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine, propoxyphene hydrochloride, propoxyphene napsylate, meperidine hydrochloride, hydromorphone hydrochloride, morphine, oxycodone, codeine, dihydrocodeine bitartrate, pentazocine, hydrocodone bitartrate, levorphanol, diflunisal, trolamine salicylate, nalbuphine hydrochloride, mefenamic acid, butorphanol, choline salicylate, butalbital, phenyltoloxamine citrate, diphenhydramine citrate, methotrimeprazine, cinnamedrine hydrochloride, and meprobamate); antiasthamatics ⁇ e.g., ketotifen and
• Suitable microorganisms for a successful biotransformation may be chosen based on the presence of microbial enzymes, such as cytochrome P450 enzymes, having the capacity to metabolize the parent compound.
• microbial enzymes such as cytochrome P450 enzymes
• Microorganisms that may be useful for biotransformation methods include bacteria, fungi and actinomycetes which possess cytochrome P450 activity.
• CYP3 A4 is a human P450 enzyme that can be characterized by its ability to hydroxylate testosterone, thereby producing 6 ⁇ - hydroxytestosterone.
• the enzyme is inhibited by such compounds as clotrimazole, and naringenin. It is induced by carbamazipine, phenobarbital, and rifampin.
• An organism growing in growth media, which expresses an enzyme which has cytochrome P450 activity, should produce 6 ⁇ -hydroxytestosterone when testosterone is introduced into the media, and this production should be affected by the known inhibitors and inducers.
• Other substrates 5 metabolized by CYP3 A4 include, for example, acetaminophen, diazepam, theophylline, warfarin, taxol, and nifedipine.
• CYP3 A4 include, for example, acetaminophen, diazepam, theophylline, warfarin, taxol, and nifedipine.
• Known and characterized enzymes have known and characterized activity. By i " o comparing the structure of the compound to be metabolized with the known activities of enzymes, enzymes can be identified that will be active in metabolizing the compound. Microorganisms can be screened for the presence of the identified enzyme.
• an aspect of the present invention provides a method of identifying a microorganism suitable for use in a biotransformation system where the method has the steps: a) comparing the structure of a 15 compound to be metabolized with a known enzyme activities; b) identifying an enzyme that expresses the desired enzyme activity; c) identifying a microorganism that expresses the identified enzyme; and d) using the microorganism that expresses the identified enzyme in a biotransformation process to make metabolites of the compound.
• genetic sequence data may be used to identify potentially useful organisms by comparing the genomic sequence of an organism to the sequence of a known mammalian gene which encodes a cytochrome enzyme, for example CYP3 A4. Microorganisms which have the appropriate genetic sequences, grown in the proper conditions, should express the target enzyme.
• cytochrome enzyme for example CYP3 A4.
• 25 particular human P450 enzyme can be tested in both systems.
• Drugs which are known to be metabolized by specific cytochrome enzymes include: (1) acetaminophen, aromatic amines, caffeine, estradiol, imipramine, phenacetin, theophylline and warfarin, broken down by CYP 1A2; (2) amitryptiline, bufurolol, captropril, clozepine, debrisopuine, flecainide, fluoxetine, haloperidol, metoprolol, mexiletine, sparteine, timolol, tomoxetine, propranolol and codeine, broken down by CYP2D6; (3) acetaminophen, diazepam, amiodarone, benzphetamine, carbemazepine, cyclosporine, digitoxin, diltiazem, erythromycin, etopiside, flutamide, imipramine, lidocaine, loratidine
• Microorganisms that express the CYP3A4 enzyme, and that may be useful for biotransformation methods include but are not limited to: Actinoplanes sp. ⁇ e.g., ATCC No. 53771), Streptomyces griseus (e.g.e.g., ATCC 13273), Saccharopolyspora erythraea ⁇ e.g., ATCC No. 11635), and Streptomyces setonii ⁇ e.g., ATCC No. 39116).
• Other useful microorganisms that may express cytochrome P450 enzymes include Amycolata autotrophica ⁇ e.g., ATCC No.
• Saccharomyces cerevisiae ⁇ e.g., ATCC No. 20137 or ATCC No. 64667) Aspergillus nidulans ⁇ e.g., ATCC No. 32353) Cimninghamella echinulata var. elegans ⁇ e.g., ATCC No.36112), Rhizopus stolonifer, ⁇ e.g., ATCC No. 6227b), Candida apicola ⁇ e.g., ATCC No. 96134), 'Coprinus cireneus, ⁇ e.g., ATCC No. MYA-727, MYA-726, MYA-728, MYA-729, MYA-730, MYA-731).
• Selection of appropriate culture time, culture conditions, extraction and purification methods is known to those of skill in the art. Growth of the chosen organism may be achieved by a skilled artisan, for example, by the use of an appropriate growth medium containing nutrients such as carbon and nitrogen, a buffering system, and trace elements and of conditions of pH, temperature, and aeration conducive to growth.
• exemplary carbon sources include glucose, maltose, dextrin, starch, lactose, sucrose, molasses, soybean oil, and the like.
• Suitable nitrogen sources include soybean meal, cotton seed meal, fish meal, 'yeast, yeast extract, peptone, rice bran, meat extract, ammonium nitrate, ammonium sulfate and the like.
• Inorganic salts may be added such as phosphates, sodium chloride, calcium carbonate and the like. Different growth media may be used depending upon the stage of growth of the organism.
• Exemplary conditions and media for growth of microorganisms suitable for use in the bioconversion of cyclosporins and cyclosporine derivatives thereof by Saccharopolyspora erythraea (for example, ATCC 11635), Saccharopolysora hirsute (for example, ATCC 20501), Amycolata autotrophic** (for example, ATCC 35204), are provided in Corconan, Methods in Enzymology 43: 487-498 (1975), US Pat. Nos. 5,124,258; 6,043,064; and 6,331,622. Conditions for growth of Actinoplanes sp. (for example, ATCC No. 53771) are exemplified in US Pat. No. 5,270,187.
• Exemplary growth conditions for microbial bioconversion of macrolides to their hydroxylated and/or demethylated metabolites include: 1) growth conditions as disclosed for the demethylation of L-679,934 (FK-506) to its metabolite, L-683,519, using Actinomycete sp.
• Bacillus subtillis ATCC 55060 and Nocardia asteroids ATCC 3318 may be used to produce hydroxylated (for example, 24-OH rapamycin) and/or demethylated metabolites (for example, 39-O-demethylrapamycin) of rapamycin (Kuhnt, M., et al, 1997, Enzyme and
• Suitable surfactants for use in an embodiment of the inventive method may be able to withstand autoclaving prior to being introduced into a microbial growth environment.
• Suitable surfactants are biocompatible surfactants and include but are not limited to nonionic surfactants such as polyethylene glycols for example PEG 300, PEG 400, PEG 600 (also known as Lutrol® E 300, Lutrol® E 400, Lutrol® E 600 Lutrol® F 127, and Lutrol® F 68 from BASF); caprylocaproyl macrogol-8 glycerides such as Labrasol® (Gatte Fosse, Cedex France); polyoxyethylene sorbitan fatty acid esters such as Tween® 20, Tween® 21, Tween® 40, Tween® 80, Tween® 80K, Tween® 81 and Tween® 85 (ICI Americas Inc., Bridgewater NJ, obtained from Aldrich Chemical Company Inc., Milwaukee Wis.); glycerine (BDH Fine Chemicals, Toronto On
• surfactants that may be used include those that can act as lubricants or emulsifiers such as tyloxapol [4-(l,l,3,3-tetramethylbutyl)phenol polymer with formaldehyde and oxirane]; polyethoxylated castor oils such as Cremophor® A25, Cremophor® A6, Cremophor® EL, Cremophor® ELP, Cremophor® RH from BASF and Alkamuls EL620 from Rhone Poulenc Co; polyethoxylated hydrogenated castor oils, such as HCO-40; and polyethylene 9 castor oil.
• tyloxapol [4-(l,l,3,3-tetramethylbutyl)phenol polymer with formaldehyde and oxirane]
• polyethoxylated castor oils such as Cremophor® A25, Cremophor® A6, Cremophor® EL, Cremophor® ELP, Cremophor® R
• surfactants that may be used include; polysorbate 20, polysorbate 60, and polysorbate 80; Cremophor® RH; poloxamers; Pluonics LlO, L31, L35, L42, L43, L44, L61, L62, L63, L72, L81, LlOl, L121, L122; PEG 20 almond glyceride; PEG 20 corn glyceride; and the like.
• Suitable surfactants also include alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyethylene alkyl ethers; polyoxyethylene alkylphenols; polyethylene glycol fatty acids esters; polyethylene glycol glycerol fatty acid esters; polyoxyethylene-polyoxypropylene block copolymers; polyglycerol fatty acid esters; polyoxyethylene glycerides; polyoxyethylene sterols; polyoxyethylene vegetable oils; polyoxyethylene hydrogenated vegetable oils; polyoxyethylene alkylethers; polyethylene glycol fatty acids esters; polyethylene glycol glycerol fatty acid esters; polyoxyethylene sorbitan fatty acid esters; polyoxyethylene-polyoxypropylene block copolymers; polyglycerol fatty acid esters; polyoxyethylene glycerides; polyoxyethylene vegetable oils; polyoxyethylene hydrogenated vegetable oils; reaction mixtures of polyols such as PEG
• oils such as almond oil; babassu oil; borage oil; blackcurrant seed oil; canola oil; coconut oil; corn oil; cottonseed oil; evening primrose oil; grapeseed oil; groundnut oil; mustard seed oil; olive oil; palm oil; palm kernel oil; peanut oil; rapeseed oil; safflower oil; sesame oil; shark liver oil; soybean oil; sunflower oil; hydrogenated castor oil; hydrogenated coconut oil; hydrogenated palm oil; hydrogenated soybean oil; hydrogenated vegetable oil; hydrogenated cottonseed and castor oil; partially hydrogenated soybean oil; soy oil; glyceryl tricaproate; glyceryl tricaprylate; glyceryl tricaprate; glyceryl triundecanoate; glyceryl trilaurate; glyceryl trioleate; glyceryl trilinoleate; glyceryl trilinolenate; glyceryl tricaprylate/caprate; glyceryl tricaprylate
• the selected lipophilic xenobiotic is mixed with an alkanol and a suitable nonionic surfactant before addition to an actively growing microbial culture.
• the alcohol may be ethanol.
• Additional suitable alcohols include: methanol, isopropanol, 1-propanol, and other suitable alcohols well known in the art.
• the xenobiotic compound is mixed with a solvent before being added to a microorganism culture.
• the solvents may be sterilized prior to mixing with the xenobiotic compound.
• a surfactant is also added to the xenobiotic compound - solvent mixture.
• Suitable solvents of the present invention may be any solvent that does not inhibit the growth and metabolism of the microorganism.
• the solvent may be non- polar, polar aprotic, or polar protic.
• the solvents include the hydrocarbon series solvents, such as benzene, toluene, n-hexane, cyclohexane, etc.; ether series solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether, dimethoxyethane, ethylene glycol dimethyl ether, etc.; alcohols such as methanol, ethanol, 1-propanol, isopropanol, etc.; halogen-containing solvents such as methylene chloride, chloroform, 1,1,1-trichloroethane, etc.; and other solvents such as dimethylformamide, N-methylpyrrolidone, hexamethylphosphorotriamide, etc.
• the solvent is preferably not DMSO.
• PBS phosphate buffered saline
• TDM therapeutic dose monitoring
• PEG polyethylene glycol
• LC Liquid Chromatography
• a column having a stationary phase formed by chemically bonding a long-chain hydrocarbon group to a porous silica matrix a Waters Symmetry C8, 2.1X50mm, 3.5 ⁇ m analytical column (Waters cat# WAT 200624) with a guard column 2 x 20mm (Upchurch Scientific cat# C-130B) packed with Perisorb RP-8 (Upchurch Scientific cat# C- 601) was used.
• the solvent percentages and flow rates utilized in the LC program are given in Table 2:
• Table 4 shows ions and ion-specific instrument settings.
• ISA247 a 50:50 mixture of cis:trans ISA247.
• ISA247 and its metabolites were extracted from whole blood using tertbutyl-methyl-ether (or methyl tertbutyl ether, MTBE), dried and reconstituted into methanol.
• 2mL of MTBE catalog. No. 7001-2; Caledon
• MTBE methyl tertbutyl ether
• 2mL of MTBE catalog. No. 7001-2; Caledon
• the top MTBE layer was removed and concentrated under vacuum. That residue was reconstituted in 200 uL of methanol.
• Bile and urine extractions can be performed similarly, as can extractions from microsome preparations and biotransformation preparations.
• metabolites can be characterized using HPLC-MS, NMR, or other techniques known in the art.
• Figure 3 shows that results of LC-MS performed as described in General Materials and Methods.
• the ISA247 metabolites from whole blood include three main groups, the diols, hydroxylated and demethylated metabolites.
• Dog liver microsomes were prepared in the following manner: after removing the liver, it was flushed with 1.15% potassium chloride (KCl); diced into small pieces (approximately 25 g) and ground until major chunks were disintegrated in chilled grinding buffer (0.1 M phosphate buffer pH 7.4; 4° C; 1:1 ratio of buffer to liver) utilizing a Polytron Homogenizer at 15,000 rpm for 3 to 5 minutes, thus forming a homogenate, which contained membrane-bounded organelles, including liver microsomes. After decantation of supernatant from the particulate matter, the supernatant was centrifuged for 90 min. at 100,000 x g to yield a pellet and a supernatant.
• KCl potassium chloride
• Protein content was determined using the Lowry protein assay. The protein concentration of this microsomal preparation was approximately 23.2 mg/niL. To avoid enzyme activity loss, microsomes were stored in 4.0 or 6.0 mL aliquots at -8O 0 C to avoid freeze thaw cycling. [0067] The following ingredients were added stepwise into a 257 mL Erlenmeyer flask: 57.3 mg of NADP, 254 mg of Glucose-6-Phosphate, and 23.0 mg NADPH were added to 6.0 mL of Phosphate Buffer (adjusted to pH 7.4).
• Metabolites produced by this method were then extracted with an organic solvent, and further separated using high-pressure liquid chromatography (HPLC).
• HPLC high-pressure liquid chromatography
• the metabolites were further characterized by electrospray mass spectrometry (MS) and NMR.
• MS electrospray mass spectrometry
• NMR nuclear magnetic resonance
• This example illustrates a biotransformation system utilized microorganisms containing the microbial equivalent of human cytochrome P450 microsomal enzyme and a medium suitable for active growth of the microorganism.
• the parent compound which is poorly soluble in water, was mixed with ethanol and a surfactant prior to addition to the biotransformation system.
• ISA247 in ethanol was mixed with TWEEN® 40 and then added to a biotransformation system containing Saccharopolyspora erytheraea (ATCC 11635).
• Phase I Media were prepared with 10 g/L dextrin, lg/L glucose, 3 g/L beef extract, 10 g/L yeast extract, 5 g/L magnesium sulfate and 400 mg/L potassium phosphate. These ingredients were mixed in deionized water up to 1 liter, pH neutralized as needed to 7 with NaOH and 50 mL was aliquoted into each of two baffled 250 mL culture flasks. The medium was sterilized for 30 min. at 100° C. 5 mL of the media was aliquoted into a slant tube containing Saccharopolyspora erythraea.
• the cells were scraped off the surface of the slant and 2.5 mL of the suspension was placed in each flask.
• the flasks were placed on a Labline Incubator at 27° C and shaken at 250 rpm for 3 days (72 hrs).
• Saccharopolyspora erythraea was transferred to Phase II media from Phase I media by centrifuging the contents of a Phase I flask at 3300 rpm for 5 min. and decanting off the supernatant to obtain a pellet. 5 mL of Phase II media was added to the pellet and the tube was vortexed, then centrifuged at 3300 rpm for 4 min. Again the supernatant was decanted. The pellet was resuspended in Phase II media. The subsequent suspension was added to 50 mL of Phase II medium in a baffled culture flask.
• Phase II Media contained 10 g/L glucose, 1 g/L yeast extract, 1 g/L beef extract and 11.6 g/L of 3-N-morpholinopropanesulfonic acid (MOPS) buffer. These ingredients were mixed in deionized water to one liter; then 50 mL were dispensed into two baffled culture flasks (250 mL). After adjustment to pH to 7.0 with 5M NaOH, the medium was autoclaved for 30 min. at 100° C, then cooled. TWEEN® 40 was autoclaved before mixing with ISA247 and ethanol.
• MOPS 3-N-morpholinopropanesulfonic acid
• ISA247 (4mg of -50/50 mixture of E and Z isomers) was dissolved in 95% ethanol (0.1 ml), then mixed with 0.4 ml TWEEN® 40 (polyoxyethylene sorbitan monopalmitate; Cat. No. Pl 504. Sigma- Aldrich, St. Louis, MO) The parent compound-surfactant mixture was then added to Saccharopolyspora erythraea in the Phase II culture medium. A zero time sample was obtained and frozen. Each flask was then capped and placed on an Innova Incubator at 27° C and incubated for 120 hrs with shaking at 170 rpm. [0075] A second sample was obtained from the Phase II culture medium.
• the zero time sample and the second sample were extracted using tert-butyl-methyl ether (cat. No. 7001-2; Caledon).
• the extracted metabolites were reconstituted in methanol (HPLC grade) and analyzed by LC-MS as previously described. As shown in Figure 4, the metabolite profile obtained by this method is similar to that obtained from human whole blood (see Example 1).
• IM4n IS A247 Metabolite that is N-demethylated at amino acid-4
• IM9 ISA247 Metabolite that is hydroxylated at amino acid-9
• IM4 ISA247 Metabolite that is hydroxylated at amino acid-4
• Ml-c-l See table 1
• IMl-d-1 Table 1
• IMl-d-2 Table 1
• IMl-d-3 Table 1). Therefore, seven out of eight IS A247 metabolites revealed in human blood were produced in this biotransformation system.
• Tube 1 - PEG 400 polyethylene glycol 400; Carbowax - Fisher Scientific, FairLonn NJ;
• Tube 2 - castor oil (Wiler Fine Chemicals Ltd, London Ont);
• Tube 3 - isopropyl myristate (Wiler Fine Chemicals Ltd, London Ont.);
• Tube 4 - glycerine (BDH Fine Chemicals, Toronto Ont. Lot # 120343/73865);
• Tube 5- Cremophor® EL (Sigma Chemical, St Louis MO);
• Tube 6 - Labrasol® (Gatte Fosse, Cedex France);
• Tube 7- TWEEN® 40 Aldrich Chemical Company Inc., Milwaukee Wis.
• the parent compound-surfactant mixture was added to the actively growing culture of Saccharopolyspora and a zero time sample was taken. After incubation with shaking at 27°C for 5 days, samples were obtained, extracted, and the metabolites were quantified as described in Example 4. Area under the curve of HPLC peaks, similar to those shown in Figures 3 and 4, was measured as an indication of the quantity of metabolite present.
• the HPLC peaks corresponded to one N-demethylated metabolite, which was identified as IM4n; two hydroxylated metabolites which were identified as IM4 and IM9; one cyclic metabolite identified as IMl-c-1; and three diol metabolites, diols formed at the 1 amino acid of the ISA247 compound, identified as IMl-d- 1, IMl-d-2 and IMl-d-3 (See Table 1).
• the seven surfactants were not all equivalent in their activity in increasing the production of metabolites in the biotransformation preparation. As shown in Figure 5, the addition of glycerine or PEG 400 to the biotransformation preparation resulted in significant increases in the quantity of metabolites produced.
• a variety of microorganisms were evaluated for production of IS A247 metabolites from ISA247, including Curvularia lunata (University of Alberta Microfungal Collection and Herbarium (UAMH) 9191; ATCC 12017), Cunningham ella echinulata var. elegans (UAMH 7370; ATCC 36112), Curvularia echinulata var. blakesleena (UAMH 8718; ATCC 8688a), Cunninghamella ephinulata var.
• Curvularia lunata Universality of Alberta Microfungal Collection and Herbarium (UAMH) 9191; ATCC 12017
• Cunningham ella echinulata var. elegans UMH 7370; ATCC 36112
• Curvularia echinulata var. blakesleena UMH 8718; ATCC 8688a
• microorganisms were screened for metabolite conversion yield (amount of known ISA247 metabolites produced) as well as metabolic diversity (number of different ISA247 metabolites produced).
• the microorganisms were grown in Phase I and incubated with IS A247 in Phase II. After the addition of ISA247 to the fermentation media, samples were taken from the media and analyzed with LC-MS against a human standard IS A247 metabolite profile to identify and quantify the metabolites collected. After primary testing of each strain via 96 5 hour biotransformation cycles, the two strains with the highest combination of metabolite conversion and metabolic diversity were tested again in Phase III with different media compositions, in order to select improved media compositions.
• Each microorganism tested was maintained on culture-specific agar slants. All slants o were prepared one month in advance to avoid contamination. Prepared agar media were autoclaved at 123°C and partial pressure of 360 mmHg for 58 minutes, cooled slightly, and 6 mL was pipetted into sterile 16x125 mm culture tubes. After placing the agar into the tube, the tube was rested on an incline to create a slant, cooled until the agar set, labeled, and incubated at 27°C for 1-2 weeks.
• ATCC 11635 and ATCC 53771 were sporulated using ISP agar (0.4% yeast 5 extract, 1% malt extract, 0.4% dextrose and 2% granulated agar).
• ATCC 53828, UAMH 8717 and UAMH 8718 were sporulated using potato dextrose agar (PDA, 3.9% in distilled water).
• UAMH 4145, UAMH 7369, UAMH 7370 and UAMH 9191 were sporulated on cereal slants (10%% mixed cereal, dry, preferably pabulum for infants; 2% granulated agar; the cereal was mixed before and after sterilization to prevent clumping of the cereal and inadequate distribution 0 of agar in the slants). Following incubation for two weeks, each microorganism was inoculated onto the microorganism-specific agar and returned to 27°C. Once a full lawn of colonies was seen on the slants, the slants were preferably used immediately in Phase I or, if necessary, stored at 4 0 C.
• Phase I media containing ISP seed broth: 5 1% dextrin, 1% glucose, 0.27% beef extract, 1% yeast extract, 0.004% magnesium sulfate, and 0.036% potassium diphosphate, at pH 7.0
• ISP seed broth 5 1% dextrin, 1% glucose, 0.27% beef extract, 1% yeast extract, 0.004% magnesium sulfate, and 0.036% potassium diphosphate, at pH 7.0
• a source slant containing the ( microorganism to be tested.
• the colonies were removed, vortexed and then the resulting suspension was added to 50 mL of sterile Phase I media, contained in a sterile, 250-mL baffled culture flask. Each flask was incubated for 96 hours in to increase biomass before the addition of IS A247 in Phase II.
• Phase II To prepare biomass for transfer to Phase II, the cells were washed thoroughly to remove Phase I residue. Phase I contents were aseptically transferred into a 50-mL conical centrifuge tube, centrifuged at 3300 rpm for five minutes, and decanted to remove supernatant. The cells were washed with 5mL of excess Phase II media (3.65% MOPS, 0.31% yeast extract, 3.14% glucose and 0.31% beef extract, atpH 7.0) and centrifuged again for five minutes at 3300 rpm, after which the supernatant was decanted and 5mL of fresh Phase II media was added.
• Phase II media 3.65% MOPS, 0.31% yeast extract, 3.14% glucose and 0.31% beef extract, atpH 7.0
• Samples were thawed from storage at -80 0 C and 16x10 mm culture tubes were labeled to represent the samples to be analyzed.
• a 200 ⁇ L aliquot was removed from each 0.5 mL sample, and 25uL of a 1 mg/mL solution of CsA (Cyclosporine A) was added as an internal standard.
• 2mL of HPLC-grade methanol was added to each sample and the samples were capped and shaken for twenty minutes.
• the samples were centrifuged at 3300 rpm for lminute and 45seconds. The supernatant was decanted into clean, labeled 16x10 mm culture tubes and vacuum concentrated to remove organic solvent.
• the dried layer, containing both the metabolites and parent drug was re-constituted in 200 ⁇ L of HPLC-grade methanol, and the samples were quantitatively transferred to auto sampler vials. Samples were run for 15 minutes in deionized water with 0.01% acetic acid/sodium acetate, starting with a 12 minute gradient of increasing m-TBE (methyl tert-butyl ether) and HPLC grade methanol.
• m-TBE methyl tert-butyl ether
• Figure 6 is a graph of mass spec signal versus retention time for typical metabolites from a sample pooled from human participants. Table 6 summarizes the ion masses found, corresponding quantifiable ISA247 metabolites and approximate retention times. Ion masses quantified included 1223, 1237, 1239, 1253, 1255, 1267 and 1271. Note that two diols (Ml-d-1 and IMl-d-4) were detected here, whereas three diols (EVQ-d-1, IMl-d-2, and IMl-d-3) were detected in Example, 1.
• ATCC 11635 displayed the greatest percent conversion and the greatest metabolic diversity. Eight known human
• ISA247 metabolites were detected in ATCC 11635 samples.
• UAMH 4145 produced six of the eight metabolites.
• ATCC 53771 often used in the lab because of its inherent ability to generate large amounts of IM4n (6.66%), produces five of the eight human metabolites.
• ATCC 53828 produced four of the eight metabolites; although each of these metabolites was produced in small quantities, the rare metabolite 1239 was produced.
• UAMH 7369 and UAMH 7370 each produced four of the metabolites.
• UAMH 9191 and UAMH 8718 each produced six • metabolites.
• UAMH 8717 produced three metabolites.
• IMl -d-4 is defined as metabolites that are not produced in large amounts by ATCC 11635, e.g., IMl -d-4, 1239 and 1255.
• IMl -d-4 was present in ATCC 11635, UAMH 8717 and UAMH 9191, but was produced in the greatest quantity by ATCC 11635.
• the microbial strains ATCC 11635, ATCC 53771, ATCC 53828, UAMH 7370, UAMH 8717, UAMH 8718, UAMH 9191 all produced 1239.
• the microorganism that produced the greatest quantity was UAMH 8717.
• the metabolite corresponding to ion 1255 was manufactured by ATCC 11635, UAMH 4145, ATCC 53771, UAMH 8717, with the greatest conversion by ATCC 11635.
• FIG. 7 shows the results for ATCC 11635.
• Media 3 and Media 16 produced similar amounts of each metabolite except for IMl-d-1, IM 1 -d-4 and IM 1 -c- 1.
• IM 1 -d- 1 production decreased with Media 3 and was not present in detectable levels with Media 16.
• IMl -d-4 production decreased with both Media 3 and Media 16.
• IMl-c-1 production increased 10% with Media 3.
• Figure 8 is a graph of the effect of media composition on the production of ISA247 metabolites in ATCC 53771.
• IMl-d-1 was detected only when using ISP2 media and IM9 and IM4 were only detected when using Media 3 and Media 16. Most of the metabolites, with the exception of IMl-d-4 were increased in quantity by using Media 3 and Media 16. Therefore, the growth media of the microorganisms can be altered , to optimize the effect of biotransformation.
• Example 5 Beauvaria bassiana (UAMH 8717) only weakly produced ISA247 metabolites.
• DMSO dimethyl sulfoxide
• TWEEN® 40 glycerol

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