JP2008524992A - Biotransformation of cyclosporine compound ISA247 - Google Patents

Abstract

A method of producing a metabolite of a xenobiotic compound by biotransformation using a microorganism, wherein the xenobiotic compound is cyclosporin ISA247, and reaches the microorganism in a mixture of surfactants It is. The method can be scaled up to produce large amounts of metabolites by, for example, biotransformation in a reactor. The metabolites produced by the methods of the present invention can be used for antibody production as therapeutic dose monitoring or as a standard for pharmaceutical applications.
[Selection] Figure 1

Description

  The present invention relates to a method for preparing a metabolite of a compound, and more specifically to the adjustment of the metabolite by biotransformation.

  When administering a pharmaceutical product to a patient, it is often necessary to monitor the serum concentration of the drug to confirm that the patient is receiving a therapeutic amount of the drug. This is called therapeutic dose monitoring (TDM). TDM can measure the parent compound and / or metabolites of one or more of the parent compounds. A metabolite is formed when an enzyme, generally a liver enzyme, acts to decompose or modify the drug so that the drug is easily excreted from the body. If the parent compound is rapidly metabolized, it may be most convenient to measure the concentration of the metabolite for TDM purposes. An immunoassay is often used for such measurement.

  If the TDM assay that measures the metabolite is an immunoassay, the drug, or an isolated, purified metabolite of the drug, may be used to generate and / or select an antibody with the desired specificity for the use of the assay. Sometimes used. Alternatively, the purified metabolite may be used to limit antibodies specific for the parent compound, ie, antibodies that exhibit minimal cross-reactivity between the parent compound and the parent compound metabolite. . Therefore, in order to obtain a certain amount of metabolites suitable for producing antibodies for TDM, an efficient method for producing isolated metabolites is required.

  Metabolites may have uses that are unrelated to TDM. Metabolites may have pharmaceutically important activity. For example, metabolites exhibit beneficial characteristics such as improved pharmacokinetics, increased pharmacological activity, or increased bioavailability. The metabolite of the parent drug compound itself can be a useful treatment. For example, A77 1726 is an active metabolite of leflunomide, hydroxy-tert-butyramide is an active metabolite of the HIV therapeutic drug nelfinavir, and 4-OH-tamoxifen is an active metabolite of tamoxifen. When the metabolite exhibits activity, a method for efficiently producing a large amount of the metabolite is required. Alternatively, one or more of the metabolites may be toxic. Therefore, knowledge about the mode of drug metabolism, the resulting metabolites, and the activity of these metabolites is important in understanding drug activity. This information may be required before drug approval. In order to identify the metabolite and its characteristics, it is necessary to produce and isolate a sufficient amount of the metabolite.

  One method of producing metabolites is to administer the drug to mammals such as humans, collect body fluids such as blood, urine, bile, etc., and extract, purify, and isolate metabolites from these body fluids. . In general, biotransformation, which is the conversion of a drug to a metabolite of the drug, is achieved by the liver cytochrome P450 enzyme group (CYP450 or P450) in the liver of a human patient. The P450 enzyme family includes an estimated about 70 types of enzymes that act to enhance the solubility of the compound for excretion in bile or urine. In order to monitor the formation of metabolites by biotransformation, it is possible to label the parent compound so that the metabolites can be recognized. Alternatively, if the results are monitored by high pressure liquid chromatography separation and mass spectral analysis, drugs with similar structures may be analyzed simultaneously. The biotransformation method of the second parent compound uses cultured cells such as whole organs, tissue slices, or hepatocytes as the biotransformation system. In the third method, microsomes prepared from mammalian cells may be used. Since these approaches use animal isolates, there is a risk of introducing unwanted contaminants into the metabolites. These methods are difficult to scale up when larger quantities of one or more of the metabolites are required. Furthermore, in order to convert the parent compound into a metabolite, biotransformation using a microorganism may be used.

  If the xenobiotic is highly lipophilic, that is, very insoluble in the aqueous culture medium used for large-scale fermentation methods, it can be particularly difficult to produce large quantities of metabolites. Therefore, there is a need for a method that efficiently produces large amounts of insoluble xenobiotic metabolites.

  The present invention provides a method for producing a metabolite of a xenobiotic compound by biotransformation using a microorganism. The xenobiotic compound can also be made to the microorganism during mixing with a surfactant. It is also possible to produce large quantities of metabolites by scaling up the method and biotransforming it in a reactor, for example. The metabolites produced by the methods of the present invention can be used for antibody production, for example as a standard for therapeutic dose monitoring or pharmaceutical applications.

Accordingly, one aspect of the invention provides a method for producing at least one metabolite of a xenobiotic compound in a microorganism, the method comprising:
(A) providing a mixture of the xenobiotic compound and a surfactant;
(B) adding the mixture to the culture solution of the microorganism;
(C) culturing the culture solution for a time sufficient for the metabolite to form.

  The mixture includes the xenobiotic compound, a solvent, and the surfactant. Any solvent suitable for the xenobiotic compound can be used. For example, the solvent can be an alcohol such as ethanol. The solvent may have one or more substances. In some embodiments, the solvent comprises both alcohol and dimethyl sulfoxide (DMSO).

  The microorganism can be any microorganism that can metabolize the xenobiotic compound, and preferably has the same metabolic pathway of the xenobiotic compound as humans. In certain embodiments, the microorganism is Actinoplanes sp. , Streptomyces griseius, Streptomyces setoniii, Saccharopolyspora erythraea. The microorganism may be Cunningham ellachinulinata, Neurospora crassa, or Actinoplanes sp. It can also be.

  The surfactant can be any suitable surfactant and can be identified by one skilled in the art based on the teachings of the present disclosure. For example, the surfactant is polyethylene glycol (PEG) 400, castor oil, isopropyl myristate, glycerin, Cremophor (registered trademark) (polyoxy castor oil), Labrasol (registered trademark) (caprylocaproyl macrogol glyceride) , And TWEEN® 40.

  The xenobiotic compound is preferably a compound having low solubility in an aqueous solution. In some embodiments, the xenobiotic compound is selected from the group consisting of immunosuppressive and antimicrobial compounds, preferably cyclosporine compounds, more preferably ISA247 or cyclosporin A. The metabolite is preferably IM1-d-1, IM1-d-2, IM1-d-3, IM1-d-4, IM1-c-1, IM1-c-2, IM1-e-1, Selected from the group consisting of IM1-e-2, IM1-e-3, IM9, IM4, IM4n, IM6, IM46, IM69, and IM49.

  The method of the present invention can further optionally include a step of isolating the metabolite from the culture solution.

  In another aspect of the present invention, there is provided a method for identifying a microorganism suitable for use in a biotransformation system, a) comparing the structure of a metabolized compound with a known enzymatic activity, and b) said known Using the microorganism that expressed the identified enzyme in a biotransformation system, c) identifying the enzyme that expressed the enzyme activity of c), c) identifying the microorganism that expressed the identified enzyme, and d) And producing a metabolite of the compound. In certain embodiments, the microorganism can be identified by comparing the genomic sequence of various microorganisms with the sequence of the identified enzyme, thereby identifying at least one microorganism that expressed the enzyme.

  The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

  The present invention provides a method for producing a metabolite of a xenobiotic compound by biotransformation using a microorganism. In particular, the xenobiotic compound is mixed with a surfactant to reach the microorganism. Large scale metabolites can be produced by scaling up the method and biotransforming it in a reactor, for example. The metabolites produced by the present invention can be used for antibody production, for example, as therapeutic dose monitoring or as a standard in pharmaceutical applications.

  Many pharmaceutically active compounds are poorly soluble in aqueous solutions. For example, cyclosporine and certain other immunosuppressive agents (rapamycin, azathioprine, mizoribine, and FK506 (tacrolimus)) are known to be sparingly soluble in aqueous environments. The present inventors have used ISA247, which is a cyclosporin derivative, as a test compound, and have found that it is possible to successfully adjust a metabolite of a poorly soluble compound using microbial fermentation. Prior to describing the present invention in further detail, the terms used in the application documents are defined below unless otherwise indicated.

Definitions As used herein, the term “biotransformation” refers to the metabolic process of a compound by living cells, particularly microbial cells.

  A “xenobiotic compound” or “xenobiotic” is a compound that is not originally present in a microorganism. Xenobiotic compounds may be pharmaceutically active. The xenobiotic compound of the present invention is preferably not readily soluble in water. For example, the water solubility of the compound is 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 at 25 ° C. Hereinafter, it can be 0.06 mg / ml or less, 0.04 mg / ml or less, or 0.02 mg / ml or less.

  The “cyclosporine compound” is cyclosporine having an immunosuppressive action, or a derivative thereof. The terms are disclosed in naturally occurring cyclosporine, cyclosporine A-Z, ISA247, U.S. Pat. Nos. 4,108,985, 4,210,581, and 4,220,641. Synthetic and artificial dihydro- and iso-cyclosporine, U.S. Pat. Nos. 4,384,996, 4,703,033, 4,764,503, 4,771,122, 4,798 , 823, 4,885,276, 5,525,590, 5,643,870, 5,767,069, and international Cyclospots provided in published WO02069902, WO03033010, WO03030834, and WO04050687. Including emissions derivative compound.

ISA247 and its metabolites ISA247 (ISATX247 or ISA) and its ISA related families are illustrated in US Pat. Nos. 6,613,739 and 6,605,593. Like cyclosporin A, ISA247 is a cyclic undecapeptide consisting almost entirely of hydrophobic amino acids. Many of these amino acids are usually not found in mammalian proteins. FIG. 1 illustrates the structure of ISA247 and the 11 amino acid residues having the cyclic peptide ring of this molecule. As shown, the amino acid residues are numbered in a clockwise direction. As shown in FIG. 1, 7 amino acids out of 11 amino acids of the 11-membered cyclic amino acids are N-methylated. The remaining four protonated nitrogen atoms can form intermolecular hydrogen bonds with the carbonyl group, which contributes substantially to the hardness of the cyclosporine skeleton of CsA and ISA247. The solubility of CsA is about 0.04 mg / ml at 25 ° C. Due to low water solubility, the bioavailability of cyclosporin A is known to be 30% or less when administered orally to humans. ISA247 is similarly low in water solubility.

  ISA247 is a sarcosine residue (3-letter abbreviation is Sar, sarcosine is a methylated glycine residue, sometimes abbreviated as MeGly), D- and L-alanine (Ala) residues. One, an α-aminobutyric acid residue (Abu), a valine (Val) residue, an N-methylvaline (MeVal) residue, four N-methylleucine (MeLeu) residues, and (4R) -4-[( E) -2-Butenyl] -4, N-dimethyl-L-threonine (MeBmt), a β-hydroxylated amino acid that is a 9-carbon, including alkene, unique to cyclosporine. The chemical name of ISA247 is cyclo {{(E)-and (Z)-(2S, 3R, 4R) -3-hydroxy-4-methyl-2- (methylamino) -6,8-nonadenoyl} -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}. The empirical formula is C63H111N11O12. I. The molecular weight is about 1214.85.

  ISA247 is known to exist as two isomers, cis-ISA247 (or Z-ISA247) and trans-ISA247 (or E-ISA247). 2A and 2B show the trans and cis forms of ISA247. A mixture of the cis and trans forms of the ISA247 compound was found to be less toxic and more potent than CsA (see US Pat. Nos. 6,605,593 and 6,613,739). . Furthermore, ISA247 was found to be less toxic and more powerful than CsA as a mixture of cis and trans isomers when the mixture contained a high proportion of trans isomers. When referring to ISA247, one skilled in the art will understand that ISA247 is a mixture of cis and trans isomers, and that the mixture may contain many trans isomers of the compound. The isomeric compounds are present as a mixture and the cis: trans ratio ranges from 1:99 to 99: 1.

The ISA247 metabolite can be described as follows: A compound of formula 1, comprising:

R ¹ is selected from the following group.

Wherein R ² is selected from the group consisting of CH ₃ and H, R ³ is selected from the group consisting of CH 2 CH (CH 3) 2 and CH 2 C (CH ³ ) 2 OH, and R ⁴ is CH (CH 3) 2 and C (CH 3 ) Selected from the group consisting of 2OH.

  The structure of the ISA247 metabolite modified with amino acid 1 of the ISA247 compound is shown in Table 1. The rectangular part is amino acids 2-11 forming a cyclic part of the cyclosporine structure with the modified amino acid 1, see FIG. Table 1 is not a complete table of ISA247 metabolites modified with amino acid 1. For example, the amino acid 1 metabolite can include a 5, 6, 7, or 8 membered ring.

  ISA247 metabolites include N-demethylated metabolites, said N-demethylation being at least at the amide bond of an amino acid such as IM4n (ie ISA247 Metabolite, N-demethylation at amino acid-4), Occurs with one methylated nitrogen. N-demethylation occurs at amino acid-3 (IM3n), amino acid-4 (IM4n), amino acid-6 (IM6n), amino acid-9 (IM9n), amino acid-10 (IM10n), or amino acid-11 (IM11n) Can happen. An ISA247 metabolite also includes a hydroxylated metabolite, which is at least one methylleucine amino acid, such as amino acids 4, 6, 9, or 10 (IM4, IM6, IM9, or IM10), or a valine residue. Occurs at 5 (IM5) or methylvaline residue 11 (IM11). In IM46, both amino acids 4 and 6 are hydroxylated, and in IM49, both amino acids 4 and 9 are hydroxylated. N-demethylated and hydroxylated metabolites may be combined, and as shown in Table 1, metabolites in which amino acid-1 is changed may be combined with N-demethylated or hydroxylated. ISA247 metabolites also include metabolites that are glucuronides, sulfonides, glycosylated, and phosphorylated derivatives of the hydroxylated metabolite of ISA247. United States Patent Application No. (Attorney Docket No. 16593-009001, filed Dec. 19, 2005) assigned to a designated agent in co-pending provides ISA247 metabolites and uses thereof.

Preparation of ISA247 Metabolites Metabolites of ISA247 were first analyzed using human whole blood from subjects who received a 50:50 mixture of cis: trans ISA247 (Example 1). FIG. 3 is an HPLC scan showing the metabolic profile of metabolites isolated from this subject's whole blood. Using an organic extraction method on human whole blood, metabolites were extracted, dried, redissolved in methanol, and identified by a chromatographic method coupled with mass spectrometry. As shown in FIG. 3, in human whole blood, at least three diol metabolites, two hydroxylated metabolites, and three N-demethylated metabolites were detectable.

  A canine liver microsome sample was also used to produce the ISA247 metabolite (Example 2). The ISA247 metabolite can be produced by this method, but the yield was low and the cost was high. Therefore, using this approach, it is not practical to obtain meaningful amounts of ISA247 metabolites.

  Previous experience has not reported prior biotransformation methods to produce large amounts of CsA and ISA247 metabolites prior to the present invention, which is probably due to the lipophilic nature of these compounds. It is for having. While not wishing to be bound by theory, the problem is that hydrophobic compounds such as ISA247 adhere to the surface of filters, columns, and other instruments used to culture and process metabolites of the product. The present inventors believe that it has a tendency. Also, these very high lipophilic compounds do not dissolve in solution in the aqueous environment of microorganisms during culture. Cultured microorganisms may not be able to access these lipophilic compounds to metabolize. In some aspects, providing a drug to a microbial growth sample is no different from providing a drug to a mammal. Formulations that improve the bioavailability of the drug may be required.

  In humans, it is known that cytochrome P450 enzymes form metabolites from CsA. It has been found that cytochrome P450 enzymes also act on the formation of ISA247 metabolites. In particular, the cytochrome P450 enzyme CYP3A4 has been identified as an enzyme involved in the metabolism of cyclosporine and ISA247. In order to create a profile similar to the metabolic profile obtained in humans, the biotransformation system grows in a medium and culture conditions suitable for active growth and metabolism of microorganisms, and is a microorganism corresponding to human cytochrome P450 enzyme. It is necessary to use microorganisms having the following enzymes.

  The biotransformation method is exemplified by Smith et al. (Arch. Biochem. Biophys. (1974) 161: 551-558). Uracker and Schmid (Curr Opin Biotechnol. (2002) 13 (6): 557-64) point out that biotransformation can be performed using prokaryotic P450 monooxygenase enzymes. Venisity and Ciddi (Current Pharmaceutical Biotechnology (2003) 4 (3): 153-167) proposed to apply microorganisms to natural drugs to find new drugs.

By Sebekia benihana, the biotransformation of cyclosporin A dissolved in methanol to [γ-hydroxy-MeLeu] 4 cyclosporine (AM4) and [γ-hydroxy-MeLeu] ⁴ [γ-hydroxy-MeLeu] ⁶ cyclosporine (AM46) U.S. Pat. No. 6,255,100. However, when CsA is administered to humans, the main metabolites produced and monitored for TDM are AM1 (amino acid-1, metabolite of CsA hydroxylated with MeBmt), AM9 (MeLeu at amino acid position 9). Note that it is a hydroxylated metabolite) and AM4n (Mesu demethylated CsA metabolite at amino acid position 4) (LeGatt et al., Clin Biochem. (1994) 27 (1). ): 43-8). Therefore, Sebekia benihana exemplifies microorganisms that do not provide a metabolic profile that mimics the mammalian metabolic profile of CsA.

  Herein, it is proved that biotransformation can be utilized to produce a metabolite of ISA247. As shown in Example 3, by allowing a mixture containing ISA247 and surfactant TWEEN® 40 (polyoxyethylene sorbitan monopalmitate) to reach Saccharopolyspora erytheraea, the main ISA247 metabolite found in human blood All categories were produced. In particular, seven types of ISA247 metabolites were detected, including IM4n (ISA247 metabolite N-demethylated at amino acid 4), IM9 (ISA247 metabolite hydroxylated at amino acid 9), IM4 (water at amino acid 4 and water). Oxidized ISA247 metabolite), IM1-c-1 (see Table 1), IM1-d-1 (Table 1), IM1-d-2 (Table 1), and IM1-d-3 (Table 1) Met. The type, number and amount of ISA247 metabolites produced by microorganisms are different and the production can be optimized by changing the medium (Example 5). Furthermore, in certain microorganisms, the use of different surfactants or solvents may increase the amount of metabolites or improve the production profile. For example, when Saccharopolyspora erytheraea is used, the production of metabolites is increased by PEG 400 and glycerol (Example 4), but TWEEN® 40 significantly increases the types of metabolites produced by Beauvaria bassiana (Example 6).

As a result, one aspect of the present invention provides a method for producing at least one metabolite of a xenobiotic compound in a microorganism, the method comprising:
(A) providing a mixture of the xenobiotic compound and a surfactant;
(B) adding the mixture to the culture solution of the microorganism;
(C) culturing the culture solution for a time sufficient for the metabolite to form. The method may optionally further comprise the step of isolating the metabolite from the culture medium.

  In certain embodiments of the present invention, poorly soluble compounds as set forth herein, particularly cyclosporine (eg, ISA247 and CsA), macrolide lactone (eg, FK506), and triene macrolide (eg, rapamycin) An in vitro biotransformation system that produces large amounts of metabolites of immunosuppressive compounds such as Details of suitable xenobiotic compounds are discussed below.

  Furthermore, after the parent compound-surfactant mixture is added to the biological reaction mixture containing the microorganisms in the growth medium, the biological reaction can be allowed to proceed temporarily under conditions where the parent compound is metabolized. After the target time, the metabolite is extracted from the biological reaction mixture and separated and purified by chromatography such as high pressure liquid chromatography and mass spectral analysis (HPLC-MS). Nuclear magnetic resonance analysis can be used to confirm that individual metabolites have been isolated from each other and to confirm their structure. Individual metabolites identified as alternative chemical structures can be used as standards in subsequent assays.

  Purified metabolites may be used in TDM assays. For example, ISA247 can be administered to an organ transplant patient in an amount sufficient to achieve immunosuppression and suppress rejection of the transplanted organ. Collect blood from the patient at an appropriate time to ensure that the appropriate drug concentration is maintained in the patient and, therefore, to maintain the appropriate immunosuppressant concentration to prevent transplant rejection Sometimes. The blood concentration of ISA247 may be measured. In addition, the blood concentration of at least one metabolite may be monitored to confirm that the patient's body is metabolizing the drug in a predictable manner. If the patient's own metabolism is not functioning to excrete the drug from the patient's system, the blood concentration of the unmetabolized active drug may increase and the patient's administration may need to be changed. Quantification can be performed, for example, by immunoassay or HPLC-MS. Similarly, antibodies specific to ISA247 or one of its metabolites can be developed.

  In some embodiments of the invention, ISA247 in ethanol is mixed with glycerol and then added to a biotransformation system (such as ATCC 11635) containing Saccharopolyspora erytheraea. In another embodiment, PEG 400 is mixed with ISA247 in ethanol prior to adding ISA247 to the biotransformation system. In another embodiment, castor oil is mixed with ISA247 in ethanol before adding ISA247 to the biotransformation system. In another embodiment, isopropyl myristate is mixed with ISA247 in ethanol before adding ISA247 to the biotransformation system. In another embodiment, Cremophor® is mixed with ISA247 in ethanol before adding ISA247 to the biotransformation system. In another embodiment, Labrazole® is mixed with ISA247 in ethanol before adding ISA247 to the biotransformation system. In another embodiment, TWEEN® 40 is mixed with ISA247 in ethanol before adding ISA247 to the biotransformation system.

Additional Typical Xenobiotic Compounds Additional examples of drugs that are poorly soluble in aqueous solutions include analgesics / antipyretics (eg, aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine, propoxyphene hydrochloride, propoxyphene napsylate, hydrochloric acid) Meperidine, hydromorphone hydrochloride, morphine, oxycodone, codeine, dihydrocodeine hydrogen tartrate, pentazocine, hydrocodone hydrogen tartrate, levorphanol, diflunisal, trolamine salicyl salt, nalbuphine hydrochloride, mefenamic acid, butorphanol, choline salicylate, butalbital, phenyltoloxamine citrate , Diphenhydramine citrate, metotrimeprazine, cinnamedrine hydrochloride, and meprobamate), anti-asthmatic drugs (eg, ketotifen and Traxanox), antibiotics (eg, neomycin, streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline, and ciprofloxacin), antidepressants (eg, nefopam, oxypertin, doxepin, amoxapine) , Trazodone, amitriptyline, maprotiline, phenelzine, desipramine, nortriptyline, tranylcypromine, fluoxetine, doxepin, imipramine, imipramine pamoate, isocarboxazide, trimipramine, and protriptyline), antidiabetics (eg, biguanides and sulfonylurea derivatives) ), Antifungal agents (eg griseofulvin, ketoconazole, itraconazole, amphotericin B, nystatin, and potassium Dicidin), antihypertensive drugs (eg propranolol, propaphenone, oxyprenolol, nifedipine, reserpine, trimetaphane, phenoxybenzamide, pardiline hydrochloride, deserpidine, diazoxide, guanethidine monosulfate, minoxidil, resinamine, sodium nitroprusside, indojabok , Alseroxilone, and phentolamine), anti-inflammatory drugs (eg (non-steroidal) indomethacin, ketoprofen, flurbiprofen, naproxen, ibuprofen, ramifenazone, piroxicam, (steroidal) cortisone, dexamethasone, fluazacort, celecoxib, Rofecoxib, hydrocortisone, prednisolone, and prednisone), antineoplastic agents (eg, cyclophosphine) Mido, actinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, camptothecin and its derivatives, phenesterine paxeltaxel And its derivatives, vinblastine, vincristine, tamoxifen, and pipesulfan), anxiolytics (eg, lorazepam, prazepam, chlordiazepoxide, oxazepam, dipotassium chlorazepate, diazepam, hydroxyzine pamoate, hydroxyzine hydrochloride, alprazolam, droperidol, halazepam, Chlormezanone and Dantorole ), Anti-migraine drugs (eg, ergotamine, propranolol, isomethepene mucate, and dichlorfenazone), sedative / hypnotics (eg, bartotools such as pentobarbital, pentobarbital, and secobarbitur) Acid salts and benzodiazepines such as flurazepam hydrochloride, triazolam, and midazolam), anti-anginal agents (eg, β-adrenergic blockers, nifedipine, and calcium channel blockers such as diltiazem, and nitroglycerin, isosorbide nitrate, tetranitrate Pentaerythritol and nitrates such as erythritol tetranitrate), antipsychotics (eg, haloperidol, loxapine succinate, loxapine hydrochloride, thioridazine, thioridazine hydrochloride) Thiothixene, fluphenazine, fluphenazine decanoate, fluphenazine enanthate, trifluoperazine, chlorpromazine, perphenazine, lithium citrate, and prochlorperazine), antiarrhythmic drugs (eg, bretylium tosylate, esmolol, verapamil) , Omiodarone, encainide, digoxin, digitoxin, mexiletine, disopyramide phosphate, procainamide, quinidine sulfate, quinidine sulfate, polygalacturonic acid quinidine, flecainide acetate, tocainide, and lidocaine), anti-arthritic agents (eg, phenylbutazone, sulindac) , Penicillamine, salsalate, piroxicam, azathioprine, indomethacin, meclofenamate, gold sodium thiomalate, ketoprofen, auranov , Aurothioglucose, and tolmetin sodium), anti-gout drugs (eg, colchicine, and allopurinol), anticoagulants (eg, heparin, sodium heparin, and warfarin sodium), thrombolytic agents (eg, urokinase, strepto) Kinases and alteplases), antifibrinolytic agents (eg, aminocaproic acid), blood rheology agents (eg, pentoxifylline), antiplatelet agents (eg, aspirin), anticonvulsants (eg, valproic acid, divalprox) Sodium, phenytoin, sodium phenytoin, clonazepam, primidone, phenobarbitol, carbamazepine, amobarbital / sodium, methosuximide, metalbital, mefobarbital , Mephenytoin, fenceximide, parameterdione, ethotoin, phenacemide, secobarbitol sodium, chlorazepate dipotassium, trimethadione, antiparkinsonian drug (eg ethosuximide), antihistamine / itch agent (E.g., hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine maleate, cyproheptadine hydrochloride, terfenadine, clemastine fumarate, triprolysine, carbinoxamine, diphenylpyralin, phenindamine, azatadine, tripelenamine, dexchlorpheniramine maleate) ), Methodirazine, and), antibacterial drugs (eg, amikacin sulfate) Aztreonam, chloramphenicol, chloramphenicol palmitate, ciprofloxacin, clindamycin, clindamycin palmitate, clindamycin phosphate, metronidazole, metronidazole hydrochloride, gentamicin sulfate, lincomycin hydrochloride, tobramycin sulfate, Vancomycin hydrochloride, polymyxin B sulfate, colistimateate sodium, and colistin sulfate), antiviral agents (eg, interelon α, β, or γ, zidovudine, amantadine hydrochloride, ribavirin, and acyclovir), antibacterial agents (For example, cefazolin sodium, cefradine, cefaclor, cefapirin sodium, ceftizoxime sodium, cefoperazone sodium, cef Tetan disodium, cefuroxime e azotil, cefotaxime sodium, cefadroxyl monohydrate, cephalexin, cephalothin sodium, cephalexin hydrochloride monohydrate, cefamandole nafate, cefoxitin sodium, cefoniside Cephalosporin such as sodium, ceforanide, cefadroxyl, cephrazine, and cefuroxime sodium, ampicillin, amoxicillin, penicillin G benzathine, cyclacillin, ampicillin sodium, penicillin G potassium, penicillin V potassium, piperacillin sodium, oxacillin sodium, vacacicillin hydrochloride, bacanpicillin hydrochloride Phosphate sodium, ticarcillin disodium, azocillin sodium, carben Penicillin such as phosphoindanyl sodium, penicillin G procaine, methicillin sodium, and nafcillin sodium, erythromycin ethyl succinate, erythromycin, erythromycin estrate, erythromycin lactobionate, erythromycin stearate, and erythromycin ethyl succinate, and tetracycline hydrochloride , Doxycycline hycrate, and tetracycline such as minocycline hydrochloride, azithromycin, clarithromycin), antiinfectives (eg GM-CSF), bronchodilators (eg epinephrine hydrochloride, metaproterenol sulfate, terbutaline sulfate, isoetarine, isoetarine)・ Mesylate, isoetarine hydrochloride, vitorterol mesylate sympathomimetics such as bitolterolmesylate), isoproterenol hydrochloride, terbutaline sulfate, epinephrine hydrogen tartrate, metaproterenol sulfate, epinephrine sulfate, and epinephrine hydrogen tartrate, anticholinergics such as ipratropium bromide, aminophylline, dyphylline, sulfuric acid Metaproterenol, and xanthines such as aminophylline, mast cell stabilizers such as sodium cromoglycate, steroidal compounds and hormones (eg, danazol, testosterone cypionate, fluoxymesterone, ethyl testosterone, testosterone enateate, methyl Testosterone, fluoxymesterone, and testosterone scipione Androgens such as estrogen, estradiol, estropipete, estrogens such as conjugated estrogens, progestins such as methoxyprogesterone acetate, and norethindrone acetate, triamcinolone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, dexamethasone prednisone acetate, methylpredniso acetate Suspension, triamcinolone acetonide, methylprednisolone, sodium prednisolone phosphate, methylprednisolone sodium succinate, hydrocortisone sodium succinate, triamcinolone hexacetonide succinate, hydrocortisone, hydrocortisone cypionate, prednisolone, fludrocortisone acetate, tebutoacetate Acid prednisolone, Corticosteroids such as prednisolone acetate, sodium prednisolone phosphate, and hydrocortisone sodium succinate, and thyroid hormones such as sodium levothyroxine), hypoglycemic drugs (eg glyburide, chlorpropamide, tolbutamide, and tolazamide), lipid lowering drugs (Eg, clofibrate, dextrothyroxine sodium, probucol, pravastatin, atorvastatin, lovastatin, and niacin), anti-ulcer / reflux drugs (eg, famotidine, cimetidine, and ranitidine hydrochloride), antiemetics / Antiemetics (eg, meclizine hydrochloride, nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride, thiethylperazine, and scopolamine) And an oil-soluble vitamins. Metabolites of these poorly soluble compounds can be produced by the method of the present invention.

Microorganisms Suitable microorganisms for successful biotransformation can be selected based on the presence or absence of microbial enzymes such as cytochrome P450 enzymes that have the ability to metabolize the parent compound. Microorganisms that are considered useful for biotransformation include bacteria, fungi, actinomycetes, and the like that have cytochrome P450 activity. Organisms with these enzymes can be identified empirically by comparing metabolites found in blood or urine after administration of ISA247 with metabolites found using biotransformation or microbial transformation samples. it can. For example, CYP3A4 is a human P450 enzyme characterized by the ability to produce 6β-hydroxytestosterone by hydroxylating testosterone. The enzyme is inhibited by compounds such as clotrimazole and naringenin. This is induced by carbamazepine, phenobarbital, and rifampin. Microorganisms that express an enzyme with cytochrome P450 activity and grow in the growth medium should produce 6β-hydroxytestosterone when testosterone is introduced into the medium, and this production is influenced by known inhibitors and inducers. Should receive. Other substrates that are metabolized by CYP3A4 include, for example, acetaminophen, diazepam, theophylline, warfarin, taxol, and nifedipine. Similarly, when these compounds are introduced into a medium containing microorganisms, they should metabolize the substrate if the microorganisms express the enzyme.

  Known elucidated enzymes are those having known elucidated activities. By comparing the structure of a metabolic compound with a known enzyme activity, it is possible to identify an enzyme having the metabolic activity of the compound. It is also possible to screen microorganisms for the presence or absence of the identified enzyme. Thus, in one aspect of the present invention, a method for identifying a microorganism suitable for use in a biotransformation system is provided, the method comprising: a) comparing the structure of a metabolized compound with a known enzymatic activity. B) identifying an enzyme that has expressed the desired enzyme activity; c) identifying a microorganism that has expressed the identified enzyme; and d) expressing the identified enzyme in a biotransformation process. And producing a metabolite of the compound using the prepared microorganism. By using this method, microorganisms that are considered useful for producing metabolites of compounds can be identified.

  Alternatively, by using gene sequence data, a microorganism considered to be useful can be identified by comparing the gene sequence of the microorganism with the sequence of a known mammalian gene encoding a cytochrome enzyme such as CYP3A4. A microorganism having an appropriate gene sequence and grown under appropriate conditions should express the target enzyme. In addition to control compounds, compounds that inhibit or induce specific human P450 enzymes can be considered in both systems.

  Drugs known to be metabolized by certain cytochrome enzymes include (1) acetaminophen, aromatic amines, caffeine, estradiol, imipramine, phenacetin, theophylline, and warfarin that are degraded by CYP1A2. ) Amitriptyline degraded by CYP2D6, bufurolol, captopril, clozepine, debrisopine, flecainide, fluoxetine, haloperidol, metoprolol, mexiletine, spartetrol, promotine, Acetaminophen, diazepam, amiodarone, benzphetamine, carbamazepine degraded by CYP3A4 Carbemazepine), including cyclosporine, digitoxin, diltiazem, erythromycin, etoposide (etopiside), flutamide, imipramine, lidocaine, loratidine, nifedipine, midazolam, retinoic acid, steroids, tamoxifen, taxol, terfenadine, THC, verapamil, and warfarin.

  Microorganisms that express the CYP3A4 enzyme and are considered useful for biotransformation include Actinoplanes sp. (Eg, ATCC number 53771), Streptomyces grieseus (eg, ATCC 13273), Saccharopolyspora erythraea (eg, ATCC number 11635), and Streptomyces setonii (eg, ATCC number 39116). Other useful microorganisms capable of expressing cytochrome P450 enzymes include Amycolata autotrophica (eg, ATCC number 35204), Streptomyces californica (eg, ATCC number 15436), Saccharopolysora hirsute (eg, ATCC For example, ATCC number 55209), Streptomycines aureofaciens (eg, ATCC number 10762), Streptomyces rimosus (eg, ATCC number 28893), Bacillus subtilis (eg, ATCC number 5560), and Nocardia asteris ( For example, ATCC number 3318), Saccharomyces cerevisiae (for example, ATCC number 20137 or ATCC number 64667), Aspergillus nidulans (for example, ATCC number 32353), Cunninghamella echinulata var. elegans (e.g. ATCC number 36112), Rhizopus stolonifer (e.g. ATCC number 6227b), Candida apicola (e.g. ATCC number 96134), Coprinus cireneus (e.g. ATCC number MYA-727, MYA-726, MYA-28, MYA-28) 729, MYA-730, MYA-731).

  Selection of appropriate culture times, culture conditions, extraction and purification methods are well known to those skilled in the art. Growth of selected microorganisms is accomplished by one of ordinary skill in the art using appropriate growth media containing nutrients such as carbon and nitrogen, buffer systems, and trace elements, and using pH, temperature, and air supply conditions that promote growth. can do. Typical carbon sources include glucose, maltose, dextrin, starch, lactose, sucrose, molasses, soybean oil and the like. Suitable nitrogen sources include soybean meal, cottonseed meal, fish meal, yeast, yeast extract, peptone, rice bran, meat extract, ammonium nitrate, ammonium sulfate and the like. Inorganic salts such as phosphate, sodium chloride and calcium carbonate can also be added. Depending on the growth stage of the microorganism, different growth media may be used.

  Biotransformation of cyclosporine and cyclosporin derivatives typically used for the growth of microorganisms in biotransformation of microorganisms suitable for growth by Saccharopolysora erythraea (e.g. ATCC 11635), Saccharopolysora hirsutee (e.g. ATCC 20501), Amycolata autotrophica (e.g. ATCC 35204), Conditions and media are provided in Corconan, Methods in Enzymology 43: 487-498 (1975), US Pat. Nos. 5,124,258, 6,043,064, and 6,331,622. ing. Actinoplanes sp. The growth conditions (eg, ATCC No. 53771) are exemplified in US Pat. No. 5,270,187.

  Typical growth conditions for the biotransformation of macrolides into their hydroxylated and / or demethylated metabolites by microorganisms are exemplified in 1) U.S. Pat. No. 5,268,370, Actinomycete sp. Growth conditions disclosed for demethylation of L-679,934 (FK-506) to its metabolite L-683,519 using (Merck Culture Collection MA 6474; ATCC No. 53828), and 2) United States Actinoplanes sp. Using the growth conditions provided in US Pat. No. 5,202,258. Demethylation from L-679,934 to L-682,993 or L-683,590 to L-683,742 according to (ATCC No. 53771). Actinoplanes spp. ATCC 53771, Saccharopolyspora erthyreae ATCC 11653, Streptomyces lavandulae ATCC 55209, Stretomyces aureofaciens ATCC 10762, Streptomyces rimosus ATCC 28893, Bacillus subtillis ATCC 55060, and Nocardia asteroids ATCC with 3318, hydroxide metabolite of rapamycin (e.g., 24-OH rapamycin) And / or demethylated metabolites (eg 39-O-demethylated rapamycin) can be produced (Kuhnt, M., et al, 1997, Enzyme and Microbial Tec. hnology 21: 405-412).

Surfactants Surfactants suitable for use in the method embodiments of the present invention can withstand autoclaving prior to introduction into the microbial growth environment. Suitable surfactants are biocompatible surfactants such as, but not limited to, PEG 300, PEG 400, PEG 600 (BASF's Lutrol® E 300, Lutrol® E 400, Lutrol) Nonionic surfactants such as polyethylene glycols such as (R) E 600, Lutrol® F 127, and Lutrol® F 68), Labrasol® (Gatté Fosse, France) Caprylocaproyl macrogol-8 glycerides such as Cedex), Tween® 20, Tween® 21, Tween® 40, Tween® 80, Tween® 80K, Tween (Registered trademark) 81, and polyoxyethylene sorbitan fatty acid esters such as ween <(R)> 85 (obtained from ICI Americas Inc., Bridgewater, NJ, Aldrich Chemical Company, Inc. (Miwaukee, Wis.)), glycerin (BDH Fine Tone, BDH Fine Tone) Oil (Wiler Fine Chemicals Ltd, London, Ontario), Isopropyl myristate (Wiler Fine Chemicals Ltd, London, Ontario), Cremophor® EL (Sigma Chemical, 27 St. Louis, Missouri) Pluroni s including poloxamer, such as (registered trademark) L108 (BASF). Other usable surfactants include surfactants that can act as lubricants or emulsifiers such as tyloxapol [4- (1,1,3,3-tetramethylbutyl) phenol polymer and formaldehyde and oxirane], Polyethoxylated castor oils such as BASF's Cremophor® A25, Cremohol® A6, Cremophor® EL, Cremophor® ELP, Cremophor® RH, and Alkamuls EL620 of Rhone Poulenc Co , Polyethoxylated hydrogenated castor oil such as HCO-40, and polyethylene 9 castor oil.

  Other surfactants that can be used include polysorbate 20, polysorbate 60, and polysorbate 80, Cremophor® RH, poloxamer, Plonics L10, L31, L35, L42, L43, L44, L61, L62, L63, L72, L81, L101, L121, L122, PEG 20 almond glyceride, PEG 20 corn glyceride and the like. Suitable surfactants include alkyl glucoside, alkyl maltoside, alkyl thioglycoside, lauryl macrogol glycoside, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenol, polyoxyethylene glycol fatty acid ester, polyethylene glycol glycerol fatty acid ester, polyoxyethylene -Polyoxypropylene block copolymer, polyglycerol fatty acid ester, polyoxyethylene glyceride, polyoxyethylene sterol, polyoxyethylene vegetable oil, polyoxyethylene hydrogenated vegetable oil, polyoxyethylene alkyl ether, polyethylene glycol fatty acid ester, polyethylene glycol glycerol fatty acid Esters, polyoxyethylene sorbitan fatty acid esters, polio Siethylene-polyoxypropylene block copolymer, polyglycerol fatty acid ester, polyoxyethylene glyceride, polyoxyethylene vegetable oil, polyoxyethylene hydrogenated vegetable oil, PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 Stearate, PEG-32 distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, P G-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG -40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil PEG-6 caprate / caproic glyceride, PEG-8 caprate / caproic glyceride, polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phytosterol, PEG-30 soybean sterol, PEG-20 trioleate, P EG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, Tocopheryl PEG-100 succinate, PEG-24 cholesterol, polyglyceryl-10 oleate, sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG 10-100 nonylphenols, poloxamer PEG 15-100 octylphenols, PEG-35 castor oil, PEG-40 hydrogenated castor oil, PEG-60 corn oil, PEG-25 glyceryl trioleate, PEG-6 caprate / cap Also included are reaction mixtures of polyols, such as proacid glycerides, PEG-8 caprate / caproate glycerides, polysorbate 20, polysorbate 80, tocopheryl PEG-1000 succinate, and poloxamer PEG-24 cholesterol. In addition, almond oil, babas oil, borage oil, black currant seed oil, canola oil, coconut oil, corn oil, cottonseed oil, evening primrose oil, oil taken from grape seeds, peanut oil, mustard 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 oil and castor oil, semi-hardened Soybean oil, soybean oil, glyceryl tricaproate, glyceryl tricaprylate, glyceryl tricaprate, glyceryl triundecanoate, glyceryl trilaurate, glyceryl trioleate, glyceryl trilinoleate, glyceryl trilinolenate, glyceryl tricaprylate / Capre Oil such as glyceryl tricaprylate / caprate / laurate, glyceryl tricaprylate / caprate / linoleate, glyceryl tricaprylate / caprate / stearate, saturated polyglycolized glyceride, linoleic acid glyceride, caprylic acid / capric acid glyceride It can also be used. In addition, mixtures of surfactants and / or oils and / or alcohols can be used.

  In some embodiments of the invention, the selected lipophilic xenobiotic is mixed with the alkanol and a suitable nonionic surfactant prior to addition to the actively growing microbial culture. When the parent compound is mixed with alcohol, the alcohol can be ethanol. Further suitable alcohols include methanol, isopropanol, 1-propanol, and other suitable alcohols well known in the art.

Solvent In some embodiments of the invention, the xenobiotic compound is mixed with a solvent prior to addition to the microbial culture. The solvent may be sterilized before mixing with the xenobiotic compound. Optionally, a surfactant is also added to the xenobiotic compound-solvent mixture. The solvent suitable for the present invention can be any solvent that does not inhibit the growth and metabolism of the microorganism. The solvent can be non-polar, aprotic, or protic. For example, the solvent includes hydrocarbon solvents such as benzene, toluene, n-hexane and cyclohexane, ether solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether, dimethoxyethane, and ethylene glycol dimethyl ether. , Alcohols such as methanol, ethanol, 1-propanol, isopropanol, halogen-containing solvents such as methylene chloride, chloroform, 1,1,1-trichloroethane, other solvents such as dimethylformamide, N-methylpyrrolidone, hexamethylphosphorotriamide including. The solvent is preferably not DMSO.

  The following examples are provided to illustrate the invention and are not to be construed as limiting the scope of the invention in any way. The present invention has been particularly shown and described with reference to preferred embodiments thereof, but it should be understood that various changes may be made thereto without departing from the scope and scope of the invention as defined in the appended claims. Those skilled in the art will appreciate that changes in form and detail may be made.

Examples In the following examples, the following abbreviations have the following meanings. Abbreviations that are not defined have a generally accepted meaning.
° C = Celsius temperature hr = Time min = Minute sec or S = Second μM = Micromolar mM = Millimol M = Molarity ml = Milliliter μl = Microliter mg = Milligram μg = Microgram mol = Mol pmol = Picomol ATCC = American type Culture Collection PBS = phosphate buffered saline CSA = cyclosporin A
TDM = therapeutic dose monitoring LC = liquid chromatography MS = mass spectrometry PEG = polyethylene glycol General materials and methods Liquid chromatography (LC) conditions In liquid chromatography (LC or HPLC), long chain hydrocarbon groups are porous A column with a stationary phase formed by chemically binding to a silica substrate, ie, a Waters Symmetry C8, 2.1 × 50 mm, 3.5 μm analytical column (Waters cat # WAT 20000624) and Perisorb RP-8 (Upchurch) A guard column 2 × 20 mm (Upchurch Scientific cat # C-130B) packed with Scientific cat # C-601) was used. The proportions and flow rates of solvents used in the LC program are shown in Table 2.

Mass Spectrometry (MS) Conditions For mass spectrometry, an Applied Biosystems / MDS Sciex API 3000 (analysis software v1.2) machine was used. The runtime was 15 minutes, the injection volume was 5 μL, the guard column temperature and the analytical column temperature were 60 ° C. Manual settings are as follows. The turbo ion spray was set to 8000, the turbo ion spray horizontal setting was +4, and the turbo ion spray outer setting was set to 10. The Sciex machine was set with the parameters shown in Table 3.

  Table 4 shows the ion and ion specific instrument settings.

Preparation of ISA247 metabolites from whole blood Whole blood was collected from humans after administration of ISA247 (50:50 mixture of cis: trans ISA247). ISA247 and its metabolites were extracted from whole blood using tertbutyl-methyl-ether (or methyl tertbutylether, MTBE), dried and redissolved in ethanol. 2 mL of MTBE (cat. No. 7001-2; Caledon) was added to 200 uL of blood, shaken for 10 minutes, and centrifuged in a table top centrifuge for 2 minutes. The upper MTBE layer was removed and concentrated in vacuo. This residue was redissolved in 200 uL of methanol. Bile and urine can be extracted in the same manner as microsomal samples and biotransformation samples. Once extracted, the metabolite can be characterized by HPLC-MS, NMR, or other methods known in the art. FIG. 3 shows the results of LC-MS performed as described in General Materials and Methods. There are mainly three groups of ISA247 metabolites in whole blood: diols, hydroxylated and demethylated metabolites.

Production of ISA247 metabolites by canine liver microsome samples Preparation of canine microsomes Canine liver microsomes were prepared by the following method. After removing the liver, it was perfused with 1.15% potassium chloride (KCl), cut into small pieces (about 25 g), and a large mass cooled at 15,000 rpm for 3-5 minutes using a Polytron homogenizer. A homogenate is produced by grinding in a buffer solution (0.1 M phosphate buffer pH 7.4, 4 ° C., buffer: liver ratio 1: 1) until it breaks down, which includes membranes such as liver microsomes Connective organelles were included. After decanting the supernatant from the particulate matter, the supernatant was centrifuged at 100,000 × g for 90 minutes to obtain a pellet and supernatant. Protein content was determined by Lowry protein measurement. The protein concentration of this microsomal sample was approximately 23.2 mg / mL. To avoid loss of enzyme activity, microsomes were stored in 4.0 or 6.0 mL portions at -80 ° C. to avoid repeated freeze-thaw cycles.

  The following ingredients were gradually added to a 257 mL Erlenmeyer flask. 57.3 mg NADP, 254 mg glucose-6-phosphate, and 23.0 mg NADPH were added to 6.0 mL phosphate buffer (adjusted to pH 7.4). Next, 2.0 mL of 5.0 mM MgCl and 6.0 mL of glucose-6-phosphate dehydrogenase (10 units / mL, CALBIOCHEM, available from San Diego, Calif., Cat. No. 346774) to the solution. Added. Finally, 10 mL phosphate buffer (pH 7.4) was added. After adding 6 mL of canine liver microsomes prepared as described above to the flask, ISA247 was added and cultured at 37 ° C. for 2 hours at 250 rpm in an incubator / shaker with controlled environment. At 2 hours, 500 μL of 2M HCl was added to stop the reaction.

  Next, the metabolite produced by this method was extracted with an organic solvent, and further separated by high pressure liquid chromatography (HPLC). The metabolites were further characterized by electrospray mass spectrometry (MS) and NMR. The metabolite profile obtained (data not shown) was comparable to human whole blood. However, the canine microsomal diol was much less.

Production of ISA247 metabolites by biotransformation using Saccharopolyspora erythraea In this example, biotransformation systems using microorganisms containing enzymes of microorganisms corresponding to human cytochrome P450 microsomal enzymes, and suitable for active growth of said microorganisms The culture medium will be described. Prior to addition to the biotransformation system, a poorly water soluble parent compound was mixed with ethanol and a surfactant. In this example, an ethanol solution of ISA247 was mixed with TWEEN® 40 and then added to a biotransformation system containing Saccharopolyspora erytheraea (ATCC 11635).

  The starting culture was prepared as follows. Fifteen ISP2 slope culture tubes (16 × 26 mm, 6 ml each) containing 4 g / L yeast extract, 10 g / L malt extract, 4 g / L dextrose, and 2 g / L agar were prepared. These ingredients were mixed in up to 1 liter of demineralized water and neutralized to pH 7 with NaOH as needed. The medium was sterilized at 100 ° C. for 30 minutes. The test tube was stored at 4 ° C. until use. Each slant medium was seeded with Saccharopolvspora erythraea (ATCC No. 11635). The seeded slope medium was cultured at room temperature for 3 weeks under sterile conditions.

  The preculture was transferred to phase I medium. Phase I medium was prepared with 10 g / L dextrin, 1 g / 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 up to 1 liter of demineralized water, neutralized to pH 7 with NaOH as needed, and placed in 50 mL in each of two baffled 250 mL culture flasks. The medium was sterilized at 100 ° C. for 30 minutes. 5 mL of the medium was placed in a slant medium test tube containing Saccharopolyspora erythraea. Cells on the surface of the slant medium were scraped off, and 2.5 mL of the suspension was placed in each flask. The flask was placed in a Labline incubator at 27 ° C. and shaken at 250 rpm for 3 days (72 hours).

  The contents of the phase I flask were centrifuged at 3300 rpm for 5 minutes, and the supernatant was decanted to transfer the Saccharopolyspora erythraea from the phase I medium to the phase II medium to obtain a pellet. 5 mL of Phase II medium was added to the pellet and the tube was vortexed and then centrifuged at 3300 rpm for 4 minutes. The supernatant was decanted again. The pellet was resuspended in Phase II medium. The suspension was added to 50 mL of phase II medium in a baffled culture flask.

  Phase II medium contained 10 g / L glucose, 1 g / L yeast extract, 1 g / L beef extract, and 11.6 g / L 3-N-morpholinopropane sulfonic acid (MOPS) buffer. These components were mixed in up to 1 liter of deionized water and then 50 mL was distributed between two baffled culture flasks (250 mL). After adjusting the pH to 7.0 with 5M NaOH, the medium was autoclaved at 100 ° C. for 30 minutes and then cooled. TWEEN® 40 was autoclaved prior to mixing with ISA247 and ethanol.

  ISA247 (4 mg of about 50/50 mixture of E and Z isomers) is dissolved in 95% ethanol (0.1 ml) and then 0.4 ml TWEEN® 40 (polyoxyethylene sorbitan monopalmitate, Cat No. P1504, Sigma-Aldrich, St. Louis, MO). The parent compound-surfactant mixture was then added to Saccharopolyspora erythraea in the Phase II medium. A zero hour sample was obtained and frozen. The cap of each flask was closed, placed in an Innova incubator at 27 ° C., and cultured for 120 hours while shaking at 170 rpm.

  The second sample was obtained from the phase II medium. The zero hour sample and the second sample were extracted with tert-butyl-methyl ether (cat. No. 7001-2, Caledon). The extracted metabolite was redissolved in methanol (HPLC grade) and analyzed by LC-MS as described above. As shown in FIG. 4, the metabolite profile obtained by this method is equivalent to the metabolite profile obtained from human whole blood (see Example 1). When the biotransformation mixture was analyzed, it was found that there were 7 types of metabolic compounds, IM4n (ISA247 metabolite N-demethylated at amino acid-4), IM9 (hydroxylated at amino acid-9) ISA247 metabolite), IM4 (ISA247 metabolite hydroxylated with amino acid-4), IM1-c-1 (see Table 1), IM1-d-1 (Table 1), IM1-d-2 (Table 1) , IM1-d-3 (Table 1). Therefore, 7 out of 8 types of metabolites of ISA247 revealed in human blood were produced in this biotransformation system.

Effect of changing the surfactant in biotransformation using Saccharopolyspora erythraea In order to examine the effect of various surfactants, the phase II culture medium of Saccharopolyspora erythraea (ATCC No. 11635) actively proliferating is as described above. It was adjusted. Seven test tubes containing ISA247 (56 mg, 50/50 mixture of E and Z isomers) in ethanol (0.33 ml) were prepared. Surfactant (0.67 ml per test tube) was added to each test tube as follows.
Test tube 1-PEG 400 (polyethylene glycol 400, Carbowax-Fisher Scientific, FairLon, NJ);
Test tube 2-castor oil (Wiler Fine Chemicals Ltd, London, Ontario);
Test tubes 3-Isopropyl myristate (Wiler Fine Chemicals Ltd, London, Ontario);
Test tube 4-glycerin (BDH Fine Chemicals, Toronto, Ontario, lot number 120343/73865);
Test tube 5-Cremohol® EL (Sigma Chemical, St Louis, MO);
Test tube 6-Labrazol® (Gatta Fosse, Cedex, France); and Test tube 7-TWEEN® 4 (Aldrich Chemical Company Inc., Milwaukee, Wis.).

  The parent compound-surfactant mixture was added to the actively growing Saccharopolyspora broth and a zero hour sample was taken. After culturing at 27 ° C. for 5 days, a sample was obtained and extracted, and the metabolite was quantified as shown in Example 4. The area under the curve of the HPLC peak equivalent to that shown in FIGS. 3 and 4 was measured as an indicator of the amount of metabolites present. The HPLC peak consists of one N-demethylated metabolite identified as IM4n, two hydroxylated metabolites identified as IM4 and IM9, one cyclic metabolite identified as IM1-c-1, and Corresponding to three diol metabolites, namely diols formed with one amino acid of said ISA247 compound identified as IM1-d-1, IM1-d-2, and IM1-d-3 (see Table 1) . The seven types of surfactants were not all equivalent in activity to enhance metabolite production of the biotransformation sample. As shown in FIG. 5, when glycerin or PEG 400 was added to the biotransformation sample, the amount of metabolites produced was greatly increased. However, when castor oil, isopropyl myristate, Cremophor (registered trademark), Labrazol (registered trademark), and TWEEN (registered trademark) 40 are all added, the in vivo Metabolite production in the converted sample was increased (not shown).

Biotransformation with various microorganisms Various microorganisms have been evaluated for the production of ISA247 metabolites from ISA247, including the Culvularia luna (University of Alberta Microfungal Collection and Herbarium (UAMH) 9191, ATCC 120 elegans (UAMH 7370, ATCC 36112), Curvularia echinulata var. blackesleena (UAMH 8718, ATCC 8688a), Cunninghamella echinula var. elegans (UAMH 7369, ATCC 26269), Beauvaria bassiana (UAMH 8717, ATCC 7159), Actinomycetes (ATCC 53828), Actinoplanes sp. (ATCC 53771), Cunninghamella echinulata (UAMH 4144, ATCC 36190), Cunninghamella echinulata (UAMH 7368, ATCC 9246), Cunninghamella bainier (ECC), and U.S.A.

  These microorganisms were screened for metabolite conversion yield (amount of known ISA247 metabolite produced) and metabolic diversity (number of different ISA247 metabolites produced). The microorganisms were grown in phase I and cultured with ISA247 in phase II. After ISA247 was added to the fermentation medium, samples were taken from the medium and analyzed by LC-MS against the human standard ISA247 metabolite profile to identify and quantify the recovered metabolites. In order to select an improved medium composition after the primary examination of each strain in a 96-hour biotransformation cycle, two strains having the highest combination of metabolite conversion and metabolic diversity were selected. Reexamined in Phase III medium.

Phase I / Phase II Method Each microorganism examined was managed in a culture medium-specific agar slant medium. To avoid contamination, all slope media was conditioned one month ago. The prepared agar medium was autoclaved at 123 ° C. and a partial pressure of 360 mmHg for 58 minutes, slightly cooled, and accurately weighed in 6 mL of a sterilized 16 × 125 mm culture tube. After the agar was put into the culture tube, the culture tube was placed on a slope to make a slope medium, cooled and labeled until the agar solidified, and cultured at 27 ° C. for 1-2 weeks. ATCC 11635 and ATCC 53771 were sporulated with ISP agar (0.4% yeast extract, 1% malt extract, 0.4% dextrose, and 2% granular agar). ATCC 53828, UAMH 8717, and UAMH 8718 were sporulated with potato dextrose agar (PDA, 3.9% in distilled water). UAMH 4145, UAMH 7369, UAMH 7370, and UAMH 9191 are cereal slope media (10% mixed cereal, dried, preferably infant nutrition, 2% granular agar. The cereal is mixed before and after sterilization, and the cereal is Agglomerate and prevent inadequate distribution of the agar on the slant medium). After culturing for 2 weeks, each microorganism was inoculated on microorganism-specific agar and returned to 27 ° C. After the slant medium was completely covered with colonies, the slant medium was used immediately in phase I if possible, or stored at 4 ° C. if necessary.

  Thereafter, 2 mL of sterile phase I medium (containing ISP seed culture. 1% dextrin, 1% glucose, 0.27% beef extract, 1% yeast extract, 0.004% magnesium sulfate, and 0.036% Potassium phosphate, pH 7.0) was added to the raw slant medium containing the microorganisms to be studied. After removing the colonies using a sterile inoculation loop and vortexing, the resulting suspension was added to 50 mL of sterile phase I medium contained in a sterile 250 mL baffled culture flask. Each flask was incubated for 96 hours to increase biomass before adding ISA247 in Phase II.

  In order to adjust the biomass (biomass) transferred to Phase II, the cells were washed thoroughly to remove Phase I residues. The contents of Phase I were aseptically transferred to a 50 mL centrifuge tube, centrifuged at 3300 rpm for 5 minutes, decanted and the supernatant removed. The cells were washed with 5 mL of excess Phase II medium (3.65% MOPS, 0.31% yeast extract, 3.14% glucose, and 0.31% beef extract, pH 7.0) and again at 3300 rpm for 5 Centrifugation was then carried out, after which the supernatant was decanted and 5 mL of freshly prepared phase II medium was added. The resulting mixture was vortexed well and quantitatively transferred to 50 mL sterile phase II medium in a 250 mL baffled culture flask under aseptic conditions. A portion of ISA247 (0.5 mL, 56 mg / mL ISA247 (mainly trans) 33.75% ethanol (95%): 66.25% glycerol solution) was added to the medium and the mixture was vigorously shaken. did. Aliquots were taken (0.5 mL, taken at 12 hour intervals during 96 hours of Phase II fermentation) and stored at −80 ° C. until LC-MS analysis. The fermentation was completed after 96 hours.

Phase III In Phase III, medium C (2% glucose, 2% starch, 0.5% yeast extract, 2% soy protein, 0.32% CaCO ₃ , 0 using ATCC 53771 and ATCC 11635 as seed cultures Incubate for 96 hours in 25% NaCl) and then aseptically medium 3 (2% glycerol, 0.5% peptone, 0.5% yeast extract, 0.2% beef extract,. _{1% (NH 4) 2 HPO} 4, 0.1% CaCO 3, 0.3% NaCl, 0.03% MgSO 4 -7H 2 O, pH 7.0) or medium 16 (2% glucose, 1% glycerol 1% peptone, 1% meat extract, 2% soy protein, 0.5% CaCO ₃ , 0.5% NaCl, pH 7.0). Care was taken when collecting samples for LC-MS due to the consistency of the viscosity of the fermentation medium.

Metabolite analysis by LC-MS Samples stored at −80 ° C. were thawed and labeled on a 16 × 10 mm culture tube to indicate the sample to be analyzed. 200 μL was removed from each 0.5 mL sample, and 25 uL of 1 mg / mL CsA (cyclosporin A) solution was added as an internal standard. 2 mL of HPLC-grade methanol was added to each sample, the sample was capped and shaken for 20 minutes. The sample was centrifuged at 3300 rpm for 1 minute 45 seconds. The supernatant was decanted into a clean and labeled 16 × 10 mm culture tube and concentrated in vacuo to remove the organic solvent. Dried, the layer containing the metabolite and parent drug was redissolved in 200 μL HPLC-grade methanol and the sample was quantitatively transferred to an autosampler vial. Samples were measured with deionized water and 0.01% acetic acid / sodium acetate for 15 minutes and a gradient with increasing amounts of m-TBE (methyl tert-butyl ether) and HPLC-grade methanol was initiated for 12 minutes.

  FIG. 6 is a graph comparing mass spectrometry signal versus retention time for a typical metabolite of a sample pooled from human participants. Table 6 summarizes the observed ion mass, the corresponding quantifiable ISA247 metabolite, and the approximate retention time. Quantified ion masses were 1223, 1237, 1239, 1253, 1255, 1267, 1271, and the like. Two diols (IM1-d-1 and IM1-d-4) were detected here, whereas in Example 1, three diols (IM1-d-1, IM1-d-2, and IM1-d-3) were detected. Note that) was detected. The reason for this difference is that the trans form of ISA247 is metabolized to IM1-d-1 and IM1-d-4, whereas the cis form of ISA247 is metabolized to IM1-d-2 and IM1-d-3. is there. The metabolic profile was different from the diol because the parent compound used in Example 1 was a 50:50 mixture of cis: trans and the parent compound in this example was primarily trans-ISA247.

The relative conversion rate (%) was calculated from the ratio of the area under the peak curve (AUC) of each detected quantifiable metabolite and the area under the curve of the internal CsA standard by the following formula.
Conversion rate (%) = (metabolite AUC) / (CsA internal standard AUC) × 100
Results The metabolic diversity after 96 hours of biotransformation is summarized in Table 7. A check mark indicates that a quantifiable amount of metabolite has been produced. Table 8 shows the amount of each metabolite produced by each examined microorganism.

  Therefore, among the microorganisms examined in this experiment, ATCC 11635 showed the greatest conversion rate and metabolic diversity. In the ATCC 11635 sample, eight known human ISA247 metabolites were detected. UAMH 4145 produced 6 out of 8 metabolites. Due to its inherent ability to generate large amounts of IM4n (6.66%), ATCC 53771, often used in the laboratory, produced 5 out of 8 human metabolites. ATCC 53828 produced 4 out of 8 metabolites, each of which produced a small amount, but a rare metabolite 1239 was produced. UAMH 7369 and UAMH 7370 each produced four types of metabolites. UAMH 9191 and UAMH 8718 each produced 6 metabolites. UAMH 8717 produced three types of metabolites.

  Another factor considered in the selection of microorganisms was that ISA247 has “rare” metabolites. “Rare” is defined as metabolites that ATCC 11635 does not produce in large quantities, such as IM1-d-4, 1239, and 1255. IM1-d-4 is present in ATCC 11635, UAMH 8717, and UAMH 9191, but was most produced by ATCC 11635. Microbial strains ATCC 11635, ATCC 53771, ATCC 53828, UAMH 7370, UAMH 8717, UAMH 8718, and UAMH 9191 all produced 1239. The most abundant microorganism was UAMH 8717. Metabolites corresponding to ion 1255 were produced by ATCC 11635, UAMH 4145, ATCC 53771, UAMH 8717, and ATCC 11635 had the highest conversion rate.

  In phase III of this experiment, ATCC 11635 and ATCC 53771 were cultured in medium 3 and medium 16, and the effects of the medium were compared. FIG. 7 shows the results of ATCC 11635. Medium 3 and medium 16 produced the same amount of each metabolite except IM1-d-1, IM1-d-4, and IM1-c-1. IM1-d-1 production was reduced in medium 3 and was not present at detectable levels in medium 16. IM1-d-4 production was reduced in both medium 3 and medium 16. IM1-c-1 production increased by 10% in medium 3. FIG. 8 is a graph showing the effect of medium composition on the production of ISA247 metabolites in ATCC 53771. IM1-d-1 was detected only when ISP2 medium was used, and IM9 and IM4 were detected only when medium 3 and medium 16 were used. Except for IM1-d-4, most of the metabolites were increased by using medium 3 and medium 16. Therefore, the action of biotransformation can be optimized by changing the growth medium of the microorganism.

Effects of surfactants and solvents on biotransformation using Beauvaglia bassiana In Example 5, only Beauvaglia bassiana (UAMH 8717) produced only a slight ISA247 metabolite. To examine whether the production profile can be altered by surfactants or solvents, dimethyl sulfoxide (DMSO) and TWEEN® 40 were compared to glycerol (used in Example 5). 0.5 mL of 56 mg / mL ISA247 solution (33.75% in ethanol (95%), 66.25% DMSO or TWEEN® 40 instead of glycerol) was added to 50 mL of medium. The same phase I and phase II media and the methods of the examples were used.

  As shown in FIG. 9, when glycerol is used as a surfactant, IM1-d-4, 1239, and 1255 are formed, and in DMSO, IM1-d-1, IM4n, IM4 are formed, and TWEEN® 40 is formed. Then, IM1-d-1, IM1-d-4, 1255, IM4n, IM9, and IM4 were formed. The amount of conversion has also changed dramatically. As a result, the biotransformation results can be optimized by changing the solvent or surfactant used to reach the parent compound, and those skilled in the art will be able to optimize the specific solvent, surfactant, medium. To increase the production of a specific metabolite included in the combination of metabolites.

  All documents, patents, and patent application documents cited in this specification are the same as if each individual document, patent application document, or patent was specifically and individually indicated to be incorporated as a reference. Are incorporated herein by reference in their entirety.

  A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the scope and spirit of the invention.

FIG. 1 shows the structure of ISA247. The amino acid residues of ISA247 are indicated by numbers. The Greek letter indicates the carbon position of amino acid 1. Figures 2A and 2B provide the structures of the trans (E-) and cis (Z-) isomers of the ISA247 molecule, respectively. FIG. 3 is an HPLC scan showing the profile of ISA247 metabolites isolated from human whole blood of subjects who received a 50:50 mixture of cis: trans ISA247. FIG. 4 is an HPLC scan showing the profile of an ISA247 metabolite isolated by the biotransformation method described in Example 4. FIG. 5 is a graph showing the effect of different surfactants on the production of ISA247 metabolites in a biotransformation system. FIG. 6 shows the LC-MS profile of an ISA247 metabolite isolated from human whole blood of a subject who was administered an ISA247 formulation containing mainly the trans isomer of ISA247. FIGS. 7 and 8 respectively show Actinoplanes sp. 2 compares the effects of medium 3 and medium 16 on the production of ISA247 metabolites by (ATCC 53771) and Saccharopolyspora erythraea (ATCC 11635). 7 and 8 show Actinoplanes sp. FIG. 3 compares the effects of medium 3 and medium 16 on the production of ISA247 metabolites by (ATCC 53771) and Saccharopolyspora erythraea (ATCC 11635), respectively. FIG. 9 shows the effect of different solvents and detergents on the production of ISA247 metabolites by Beauvaria bassiana.

Claims (18)

A method for producing at least one metabolite of a xenobiotic compound in a microorganism comprising:
(A) providing a mixture of the xenobiotic compound and a surfactant;
(B) adding the mixture to the culture solution of the microorganism;
(C) culturing the culture solution for a time sufficient for the metabolite to form.   The method of claim 1, wherein the mixture comprises the xenobiotic compound, a solvent, and the surfactant.   The method of claim 2, wherein the solvent is an alcohol.   4. The method of claim 3, wherein the alcohol is ethanol.   The method according to any one of claims 1 to 4, wherein the solvent has both alcohol and DMSO.   6. The method according to any one of claims 1 to 5, wherein the microorganism is Actinoplanes sp. , Streptomyces griseus, Streptomyces setonii, and Saccharopolyspora erythraea.   6. The method according to any one of claims 1 to 5, wherein the microorganism is Cunningham ellachinulinata.   The method according to any one of claims 1 to 5, wherein the microorganism is Neurospora crassa.   6. The method according to any one of claims 1 to 5, wherein the microorganism is Actinoplanes sp.   10. The method according to claim 1, wherein the surfactant is from PEG 400, castor oil, isopropyl myristate, glycerin, Cremophor (registered trademark), Labrasol (registered trademark), TWEEN (registered trademark) 40. It is selected from the group consisting of:   11. The method according to any one of claims 1 to 10, wherein the xenobiotic compound is selected from the group consisting of immunosuppressants and antibacterial compounds.   12. The method according to any one of claims 1 to 11, wherein the xenobiotic compound is a cyclosporine compound.   13. The method of claim 12, wherein the cyclosporine compound is ISA247.   13. The method of claim 12, wherein the cyclosporine compound is cyclosporin A.   15. The method of any one of claims 1-14, wherein the metabolite is IM1-d-1, IM1-d-2, IM1-d-3, IM1-d-4, IM1-c-1, IM1- c-2, IM1-e-1, IM1-e-2, IM1-e-3, IM9, IM4, IM4n, IM6, IM46, IM69, and IM49.   16. The method according to any one of claims 1 to 15, wherein the microorganism is Saccharopolyspora erythraea (ATCC 11635). 17. A method according to any of claims 1 to 16, further comprising:
A step of isolating the metabolite from the culture solution.   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, and b) an enzyme expressing said known enzyme activity. A step of identifying, c) a step of identifying a microorganism that expresses the identified enzyme, and d) a metabolite of the compound using the microorganism that expressed the identified enzyme in a biotransformation system. A method comprising:

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