KR20070101867A - Methods for the biotransformation of the clyclosporin compound isa247 - Google Patents

Abstract

A method of producing metabolites of a xenobiotic compound by biotransformation using a microorganism, wherein the xenobiotic compound is cyclosprorm ISA247, which 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 antibody production, as standards in therapeutic dose monitoring, or in pharmaceutical applications.

Description

METHODS FOR THE BIOTRANSFORMATION OF THE CLYCLOSPORIN COMPOUND ISA247}

The present invention relates to a process for the preparation of metabolites of compounds, and more particularly to a process for preparing such metabolites by biotransformation.

When administering medication to a patient, it is necessary to monitor the serum concentration of the drug to ensure that the patient is receiving the effective therapeutic dose of the drug. This is called therapeutic dose monitoring (TDM). TDM measures the parent compound and / or one or more metabolites of the parent compound. Metabolites are formed when enzymes, usually liver enzymes, act to degrade or modify the drug so that the drug can be removed more easily from the human body. When the parent compound is metabolized quickly, it is most convenient to measure the metabolite concentration for the purpose of TDM. Such assays are often used for immunoassays.

If the TDM assay for measuring metabolites is an immunoassay, the drug or its isolated, purified metabolites may be used to generate and / or select antibodies with the specificity required for the assay. have. Alternatively, purified metabolites may be used to specify antibodies specific for the parent compound, ie, antibodies that exhibit minimal cross-reactivity between the parent compound and the metabolite of the parent compound. Therefore, there is a need for a method for effectively producing isolated metabolites in order to quantitatively secure metabolites suitable for use in the production of antibodies suitable for TDM.

Metabolites also have uses that are independent of TDM. Metabolites may have pharmaceutically important activities. For example, metabolites may exhibit beneficial properties such as improved pharmacokinetics, increased pharmacologic activity or improved bioavailability. Metabolites of parent drug compounds may themselves be useful therapeutic agents. For example, A77 1726 is an active metabolite of leflunomide; Hydroxy-tert-butylamide is an active metabolite of Nelfinavier, the HIV drug; 4-OH-tamoxifen is an active metabolite of tamoxifen. If the metabolite is active, an effective method of producing the metabolite in large quantities may be desired. One or more metabolites may be toxic. Thus, knowledge of how drugs are metabolized, and therefore the metabolites and the activity of these metabolites, plays an important role in understanding the activity of the drugs. This information may also be required before the drug is approved. To identify metabolites and their properties, metabolites must be produced and separated in sufficient quantities.

One method of preparing metabolites is to administer the drug to a mammal, such as a human, and then collect blood, urine, bile or other body fluids, and then extract and purify the metabolite from the body fluids. In human patients, the bioconversion of the drug to metabolites of the drug usually takes place in the liver by the cytochrome P450 enzyme (CYP450 or P450) of the liver. The P450 family of enzymes is estimated to contain over 70 enzymes that are responsible for dissolving the compounds into bile or urine. To monitor metabolite formation by bioconversion, the parent compound can be tagged to recognize the metabolite. Alternatively, a drug having a similar structure may be analyzed while monitoring the results by high pressure liquid chromatography separation and mass spectrometry. The second method for bioconversion of the parent compound should use cultured cells such as hepatocytes, tissue slices, or whole organs as bioconversion systems. The third method includes using microsomes prepared from mammalian cells. Because this approach uses animal body isolates, there is a risk that the metabolites may be contaminated unexpectedly. This method is difficult to scale up if more than one metabolite is required in large quantities. In addition, bioconversion may be used to convert the parent compound into metabolites using microorganisms.

If the xenobiotic agent is highly lipophilic or highly insoluble in the aqueous medium used in large scale fermentation, mass production of metabolites can be particularly difficult. Therefore, there is a need for a method for efficiently producing large amounts of metabolites of insoluble foreign matter.

Summary of the Invention

The present invention provides a method for producing a metabolite of a foreign body compound by bioconversion using a microorganism. The bioforeign compound is delivered to the microorganism in a mixture with a surfactant. This method can be scaled up to produce large amounts of metabolites, for example by bioconversion in reactors. Metabolites produced by the methods of the invention can be used, for example, in the production of antibodies or in standard or pharmaceutical applications in therapeutic dose monitoring.

Thus, one aspect of the present invention involves the following steps:

(a) providing a mixture of a bioforeign compound and a surfactant;

(b) adding the mixture to the microbial culture; And

(c) providing a method for producing at least one metabolite of a bioforeign compound, comprising incubating the culture for a period sufficient to produce the metabolite:

The mixture may include a bioforeign compound, a solvent and a surfactant.

Any solvent may be used as long as it is a solvent suitable for a bioforeign compound. For example, an alcohol such as ethanol can be used as the solvent. The solvent may be plural kinds. In some embodiments, the solvent includes both alcohol and dimethyl sulfoxide (DMSO).

The microorganism may be any microorganism capable of metabolizing a bioforeign compound, and preferably, a microorganism whose metabolic pathway for the bioforeign compound is the same as that of humans. In certain embodiments, the microorganism evil Martino Plastic Ness species (Actinoplanes sp.), Streptomyces draw three-house (Streptomyces griseus ), Streptomyces setonii ) and Saccharopolyspora erythraea . Or the chinul Cunningham Ella Rata (Cunningham Among ellaechinulata), Castello La nero larger Lhasa (Nerospora crassa), or evil Tino Playa Ness species (Actinoplanes sp) can also be selected microorganisms.

(폴리옥실 카스터유), 라브라솔

(카프릴카프로일 마크로골글리세라이드) 및 트윈

40으로 구성된 군중에서 선택될 수 있다.The surfactant may be any one selected by those skilled in the art based on the current level of knowledge. For example, the surfactant may be polyethylene glycol (PEG) 400, castor oil, isopropyl myristate, glycerin, cremophor

(Polyoxyl castor oil), Labrasol

(Caprylcaproyl macrogolglycerides) and twin

Can be chosen from a crowd of 40.

The bioforeign compound is preferably a compound having low solubility in aqueous solution. In some embodiments, the bioforeign compound is selected from the group consisting of immunosuppressants and antibacterial compounds, preferably cyclosporin compounds, more preferably ISA247 or cyclosporin A. Metabolites are preferably Ml-d-1, IMl-d-2, IMl-d-3, IMl-d-4, Ml-c-1, IMl-c-2, IMl-e-1, IMl- It is preferably selected from the group consisting of e-2, IMl-e-3, IM9, IM4, IM4n, IM6, M46, M69, and IM49.

The method of the present invention may further comprise the step of separating the metabolite from the culture as needed.

Another aspect of the present invention provides a method for preparing a metabolite comprising a) comparing the structure of a compound to be metabolized with known enzyme activity; b) identifying an enzyme that expresses known enzyme activity; c) identifying microorganisms expressing the identified enzymes; And d) using a microorganism expressing the identified enzyme in a bioconversion system to make a metabolite of the compound, thereby providing a method for identifying a microorganism suitable for use in a bioconversion system. In certain embodiments, microorganisms are identified by a method of identifying one or more microorganisms expressing the enzymes by comparing the genome sequences of various microorganisms with sequences of enzymes already identified.

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 following description, drawings, and claims.

The present invention provides a method for producing a metabolite of a foreign foreign compound by bioconversion using a microorganism. More specifically, the bioforeign compound is delivered to the microorganism in a mixture with a surfactant. The process of the invention can be scaled up to produce large amounts of metabolites, for example by bioconversion in reactors. Metabolites produced by the methods of the present invention can be used, for example, in standard or pharmaceutical applications in antibody production, therapeutic dose monitoring.

Many pharmaceutically active compounds have poor solubility in aqueous solutions. For example, cyclosporin and certain other immunosuppressive agents (rapamycin, azathioprine, missorivine and FK506 (tacrolimus)) are known to exhibit very poor solubility in aqueous environments. We have found that microbial fermentation can be used successfully to prepare metabolites of poorly soluble compounds using the cyclosporin derivative IS A247 as a test compound. Prior to describing the present invention in more detail, unless otherwise stated, it is to be understood that the terms used in the present invention have the following definitions.

Justice

As used herein, the term "bioconversion" refers to the process of metabolizing a compound by living cells, in particular microbial cells.

"Xenobiotic compound" or "xenobiotic" refers to a compound that is not natural to any microorganism. The bioforeign compound may be pharmaceutically active. It is preferable that the bioforeign compound of the present invention is not easily dissolved in water. For example, the solubility of the compounds of the present invention in water is at most 1 mg / ml, at most 0.75 mg / ml, at most 0.5 mg / ml, at most 0.25 mg / ml, at most 0.1 mg / ml, at most 0.08 mg / ml at 25 ° C. , 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 a derivative thereof having immunosuppressive activity. The term includes synthetic and artificial dihydro- and iso-cyclosporins as described in naturally occurring cyclosporins, cyclosporin A-Z, ISA247, US Pat. Nos. 4,108,985, 4,210,581 and 4,220,641; US Patent 4,384,996; 4,703,033; 4,764,503; 4,771,122; 4,798,823; 4,885,276; 5,525,590; 5,643,870; Inducible cyclosporin as disclosed in 5,767,069; And WO02069902; WO03033010; WO03030834; And cyclosporin derivative compounds as disclosed in WO04050687.

ISA247  And his Metabolite

ISA247 (ISATX247 or ISA) and its ISA related population members are shown in US Pat. Nos. 6,613,739 and 6,605,593. Like cyclosporin A, ISA247 is a cyclic undecapeptide that is almost all composed of hydrophobic amino acids. Many of these amino acids are usually not found in mammalian proteins. 1 depicts the structure of ISA247 and the eleven amino acid residues having the cyclic peptide ring of this molecule. As shown in the figure, amino acid residues are numbered clockwise. As shown in FIG. 1, seven of the 121 amino acids of the 11-membered amino acid ring are N-methylated. The other four quantized nitrogen atoms can form intermolecular hydrogen bonds with the carbonyl group, which is a substantial cause of the rigidity of the cyclosporin backbone of both CsA and ISA247. The solubility of CsA is about 0.04 mg / ml at 25 ° C. Due to this low water solubility, the bioavailability of cyclosporin A is known to be 30% or less upon oral administration to humans. ISA247 similarly has low water solubility.

ISA247 is a sarcosine residue (its three letter abbreviation is Sar; sarcosine is also abbreviated MeGly as a methylated glycine residue), one D- and L-alanine (Ala) residue, one α-amino butyric acid residue (Abu), valine (Val) residues, N-methyl valine (MeVal) residues, four N-methyl leucine (MeLeu) residues, and alkene-containing 9-carbons, (4R) -4-β-hydroxylated amino acid (4R)- It contains β-hydroxylated amino acids specific for cyclosporine called 4-[(E) -2-butenyl] -4, N-dimethyl-L-threonine (MeBmt). The chemical name of ISA247 is cyclo {{(E)-and (Z)-(2S, 3R, 4R) -3-hydroxy-4-methyl-2- (methylamino) -6,8-nonenoenoyl}- L-2-Aminobutyryl-N-methyl-glysil-N-methyl-L-leusil-L-valyl-N-methyl-L-leucil-L-alanyl-D-alanyl-N-methyl-L -Leucine-N-methyl-L-leucine-N-methyl-L-valyl}. The empirical formula for this compound is C63H11 INl 1012.I. The molecular weight of this compound is about 1214.85.

ISA247 is known to exist in two isomeric forms, cis-ISA247 (or Z-ISA247) and trans-ISA247 (or E-ISA247). 2A and 2B show the trans and cis morphology 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 US Pat. Nos. 6,605,593 and 6,613,739). In addition, ISA247 was found to be more toxic and more potent than CsA when a higher proportion of trans isomers resulted in a mixture of trans and cis forms. When referred to as ISA247, to those skilled in the art, ISA247 is understood to be a mixture of cis isomers and trans isomers, which are understood as mixtures richer in the trans isomeric form of these compounds. This isomeric compound may exist as a mixture of cis: trans ratios of 1:99 to cis: trans ratios of 99: 1.

The ISA247 metabolite can be described as follows. Compound of Formula 1:

Wherein R ¹ is

Is selected from,

R ² is CH ₃ And H; R ³ is selected from CH ₂ CH (CH ₃ ) ₂ and CH ₂ C (CH ₃ ) ₂ OH; R ⁴ is selected from CH (CH ₃ ) ₂ and C (CH ₃ ) ₂ OH.

The structure of the amino acid-1 modified ISA247 metabolite of the ISA247 compound is shown in Table 1 below. The boxes below show amino acids 2-11 together with the modified amino acid-1 forming a ring portion of the cyclosporin structure. See FIG. 1. Table 1 is not the only list of amino acid-1 modified ISA247 metabolites. For example, the metabolite of amino acid 1 may comprise a 5, 6, 7 or 8 membered ring.

ISA247 metabolites include one or more methylated nitrogens of amide bonds of amino acids, such as, for example, IM4n (or I SA247 M etabolite, N -demethylation at amino acid-4; ie, N-demethylated ISA247 metabolite from amino acid-4). N-demethylated metabolites in which N-demethylation has occurred. 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) have. ISA247 metabolites also include one or more methyl leucine amino acids, such as amino acids 4, 6, 9, or 10 (IM4, IM6, IM9 or IMlO), EH is hydrated at valine residue 5 (IM5) or methyl valine residue 11 (IM11). It also includes hydroxylated metabolites where hydroxylation occurs. IM46 is hydroxylated at both amino acid 4 and amino acid 6. Likewise IM49 is hydroxylated at both amino acid 4 and amino acid 9. As shown in Table 1, combinations of N-demethylation and hydroxylated metabolites as well as combinations of metabolites in which N-demethylation or hydroxylation alternately occur at amino acid-1 can occur. ISA247 metabolites also include metabolites which are glucuronide, sulfonide, glycosylated and phosphorylated derivatives of the hydroxylated metabolites of ISA247. Another application of this patent pending in the United States Patent Office (US Application No. 16593-009001, filed Dec. 19, 2005) discloses ISA247 metabolite and its use.

ISA247 Metabolites  How to make

Metabolites of ISA247 were first analyzed using whole blood from human subjects receiving a mixture containing cis: trans ISA247 in a 50:50 ratio (Example 1). FIG. 3 is an HPLC scan showing the metabolite profile of metabolite isolated from whole blood of this subject. Using organic extraction for human whole blood, metabolites were extracted, dried and reconstituted in methanol and identified by the combination of chromatography techniques and mass spectrophotometry. As shown in FIG. 3, at least three diols, two hydroxylated and three N-demethylated metabolites could be detected in human whole blood.

ISA247 metabolite was also prepared using the microsomal preparation of the dog's liver (Example 2). ISA247 metabolites can be prepared in this way, but they are low in yield and expensive. Thus, this approach is not a practical way to obtain significant amounts of ISA247 metabolites.

To the best of our knowledge, conventional bioconversion methods prior to the present invention have not reported substantial production of metabolites of ISA247 and CsA, presumably due to the lipophilic properties of these compounds. Without being bound by a particular theory, the inventors believe that hydrophobic compounds, such as ISA247, tend to stick to the surfaces of filters, columns, and other hardware used to culture and process the metabolites that are products. In addition, these highly lipophilic compounds do not become solutions in the aqueous environment of the microorganisms in culture. Cultured microorganisms will not have access to these lipophilic compounds to metabolize them. In some aspects, providing a drug to a microbial growth preparation is no different from providing a drug to a mammal. Formulations that increase the bioavailability of the drug may be needed.

In humans, cytochrome P450 enzymes are known to form metabolites from CsA. Cytochrome P450 enzymes have also been known to form ISA247 metabolites. In particular, the cytochrome P450 enzyme CYP3A4 has been identified as an enzyme responsible for the metabolism of cyclosporine and ISA247. To produce metabolic profiles similar to those obtained from humans, biotransformation systems must utilize microorganisms with microbial equivalents of human cytochrome P450 enzymes grown under medium and under culture conditions suitable for the active growth and metabolism of microorganisms.

Bioconversion methods are illustrated in Smith et al. (Arch. Biochem. Biophys. (1974) 161: 551-558). Woollaker and Schmidt (Curr Opin Biotechnol. (2002) 13 (6): 557-64) suggest that bioconversion can be performed using the prokaryotic P450 monooxygenase enzyme. Beniceti and CD (Current Pharmaceutical Biotechnology (2003) 4 (3): 153-167) have proposed the application of microorganisms to natural drugs to find new drugs.

US Pat. No. 6,255,100 discloses cyclosporin A dissolved in methanol using Sebekia benihana to [γ-hydroxy-MeLeu] ⁴ -cyclosporin (AM4) and [γ-hydroxy-MeLeu] ^4- [γ -Hydroxy-MeLeu] ⁶ cyclosporin (AM46) is disclosed. However, when CsA is administered to humans, most of the metabolites produced and monitored for TDM are Am1 (a metabolite of CsA hydroxylated at amino acid-1, MeBmt), Am9 (hydroxyl at MeLeu at amino acid position 9). It should be borne in mind that the metabolites to be misfired) and Am4n (metabolites of CsA demethylated at MeLeu in amino acid position 4) (LeGatt et al., Clin BIochem. (1994) 27 (1) "43-8). Sebekia benihana is an example of a microorganism that does not provide a metabolic profile that mimics the metabolic profile of mammals of CsA.

We have demonstrated that bioconversion methods can be used to produce metabolites of ISA247. As described in Example 3, a mixture containing ISA247 and surfactant Tween® 40 (polyoxyethylene sorbitan monopalmitate) was added to Saccharopolyspora. erythraea ) produced the major categories of ISA247 metabolites found in human blood. In particular, seven ISA247 metabolites were detected: IM4n (ISA247 metabolite N-demethylated at amino acid-4), IM9 (ISA247 hydroxylated at amino acid-9), IM4 (hydroxylated at amino acid-4) ISA247 metabolites), IMl-cl (see Table 1), IMl-d-1 (Table 1), IMl-d-2 (Table 1) and IMl-d-3 (Table 1). Different microorganisms produce different kinds of ISA247 metabolites in different numbers and amounts, the production of which can be optimized by changing the medium (Example 5). In addition, for a given microorganism, the use of different surfactants or solvents can increase the production of metabolites or improve the production profile. For example, PEG 400 and glycerol increased the production of metabolites when using Saccharopolyspora erythraea , whereas Tween® 40 was Beauvaria. hassiana ) significantly increased the variety of metabolites produced (Example 6).

Thus, one aspect of the present invention is:

(a) providing a mixture of a bioforeign compound and a surfactant;

(b) adding the mixture to a microbial culture; And

(c) providing a method for producing at least one metabolite of a bioforeign compound in a microorganism, comprising incubating the culture for a sufficient time until the metabolite is produced. The method may further comprise separating the metabolite from the culture as needed.

Certain embodiments of the present invention are directed to metabolites of low solubility compounds, in particular cyclosporins (eg ISA247 and CsA), macrolide lactones (eg FK506) and triene macrolides (eg rapamycin) as described above. ) in vivo (in mass production for the metabolites of immunosuppressive compounds such as in vitro ) provides a bioconversion system. Suitable bioforeign compounds are discussed in detail below.

The parent compound-surfactant mixture is added to the bioreaction mixture containing the microorganisms in the growth medium and the bioreaction occurs under conditions that allow the parent compound to metabolize for a period of time. After the required time has passed, the metabolite is extracted from the bioreaction mixture and separated and purified, for example by chromatography such as high pressure liquid chromatography and mass spectral analysis (HPLC-MS). Nuclear magnetic resonance analysis can be used to verify that individual metabolites are separated from each other and to verify structure. Individual metabolites that have proven to be separate chemicals can be used as standards in subsequent analysis.

Purified metabolites can be used in TDM assays. For example, ISA247 may be administered to transplant recipients at a dosage sufficient to achieve immunosuppression and prevent rejection of the transplanted organ. Blood samples may be taken from patients at regular intervals to ensure that patients maintain adequate levels of drug and thereby maintain adequate immunosuppressive levels to prevent rejection of transplanted organs. Blood levels of ISA247 can be measured. Furthermore, blood levels of at least one metabolite may be monitored to determine if the patient's body metabolizes the drug in a predictable manner. If the patient's own metabolic activity fails to extinguish the drug from the patient's body, it may be necessary to increase the blood concentration of the unmetabolized active drug, thus changing the patient's drug administration regimen. For example, it can be quantified by immunoassay or HPLC-MS. Similarly, antibodies to ISA247 or its metabolites can be made.

와 혼합한다. 또 다른 구체예에서는 ISA247를 생물전환 시스템에 첨가하기에 앞서서 에탄올 중의 ISA247를 라브라솔

과 혼합한다. 또 다른 구체예에서는, 또 다른 구체예에서는 ISA247를 생물전환 시스템에 첨가하기에 앞서서 에탄올 중의 ISA247를 트윈

과 혼합한다.In some embodiments of the present invention, ISA247 in ethanol is mixed with glycerol and then Saccharopolyspora erythraea ) (eg, ATCC 11635). In another embodiment, ISA247 in ethanol is mixed with PEG400 prior to adding ISA247 to the bioconversion system. In another embodiment, ISA247 in ethanol is mixed with castor oil prior to adding ISA247 to the bioconversion system. In another embodiment, ISA247 in ethanol is mixed with isopropyl myristate prior to adding ISA247 to the bioconversion system. In another embodiment, Cremophore ISA247 in ethanol prior to adding ISA247 to the bioconversion system

Mix with In another embodiment, ISA247 in ethanol is added to Labrasol prior to adding ISA247 to the bioconversion system.

Mix with In another embodiment, another embodiment tweens ISA247 in ethanol prior to adding ISA247 to the bioconversion system.

Mix with

Biological foreign substance  Another example of a compound

Other examples of drugs with low solubility in aqueous solutions include: analgesics / antipyretics (eg, aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine, propoxyphen hydrochloride, propoxyphene naphsyl) Laterate, meperidine hydrochloride, hydromorphone hydrochloride, morphine, oxycodone, codeine, dihydrocodeine bitartrate, pentazosin, hydrocodone bitartrate, levorpanol, diflunisal, trolamine salicylate, Nalbuphine hydrochloride, mefenamic acid, butorpanol, choline salicylate, butalbital, phenyltoloxamine citrate, diphenhydramine citrate, methotrimreprazine, cinnamned hydrochloride and meprobamate); Anti-asthmatic agents (eg, ketotifen and traxanox); Antibiotics (eg neomycin, streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline and ciprofloxacin); Antidepressants (e.g., nepopam, oxyfertin, doxepin, amoxapine, trazodone, amitriptyline, maprotiline, phenelzin, decipramine, nortriptyline, tranilcipromin, fluoxetine, doxepin, Imipramine, imipramine pamoate, isocarboxazide, trimipramine, and protriptiline); Antidiabetic agents (eg, biguanides and sulfonylurea derivatives); Antifungal agents (eg, griseofulvin, ketoconazole, itraconazole, amphotericin B, nystatin and candicidine); Antihypertensive agents (e.g., propanolol, propofenone, oxyprenolol, nifedipine, reserpin, trimetaphan, phenoxybenzamine, pargiline hydrochloride, deserpidine, diazoxide, guanethidine monosulfate, Minoxidil, recinnamin, sodium nitroprusside, ranwolpia serpentina, alseroxylon, and phentolamine); Anti-inflammatory agents such as (nonsteroidal) indomethacin, ketoprofen, flubiprofen, naproxen, ibuprofen, ramipenazone, pyricampam, (steroidal) cortisone, dexamethasone, fluzacote, celecoxib, Rofecoxib, hydrocortisone, prednisolone, and prednisone); Anti-neoplastic agents (e.g. cycloporamide, actinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU), methyl CCNU, Cisplatin, Etoposide, Camptothecin and Derivatives thereof, Fennesterine, Paclitaxel and Derivatives thereof, Docetaxel and Derivatives thereof, Vinblastine, Vencristine, Nacoxifen and Pifosulfan); Anti-anxiety agents (e.g., lorazepam, prazepam, chlordiazepoxide, oxazepam, chlorazate dipotassium, diazepam, hydroxyzin pamoate, piroxyzin hydrochloride, alprazolam, dropperidol, halazepam, chlor Mezzanone and dantrolene); Antimigraine agents (eg, ergotamine, propanolol, isomeptene mucate, and dichloralfenazone); Sedatives / sleeping agents (eg barbiturates such as pentobarbital, pentobarbitan and secobarbital; and benzodiazepines, such as flulazepam hydrochloride, triazolam and midazolam); Antianginal agents (eg, beta-adrenergic blockers; calcium channel blockers such as diltiazem and nifedipine; nitrates such as nitroglycerin, isosorbide dinitrate, pentaerythritol tetranitrate and erythritol tetranitrate) ; Antipsychotics (e.g., halohelidol, roxaphin succinate, roxaphine hydrochloride, thiolidazine, thiolidazine hydrochloride, thioticene, flufenazine, flufenazine decanoate, flufenazine enanthate, Trifluoroperazine, chlorpromazine, perphenazine, lithium citrate and prochlorperazine; Antiarrhythmic agents (e.g., pretyl ring tosylate, esmolol, verapamil, amidodarone, encainide, digoxin, digtoxin, mexyltin, disopyramid phosphate, procainamide, quinidine sulfate, quinidine gluconate, quinidine poly Galacturonate, flkanide acetate, tocainide and lidocaine); Anti-arthritis agents (e.g., phenylbutazone, sulindac, penicylamine, salsarate, pyroxicam, azathioprine, indomethacin, meclofenamate, gold sodium thiomalate, ketoprofen, oranopine, Orothioglucose, and tolmetin sodium); Antigout agents (eg, colchicine, and allopurinol); Anticoagulants (eg, heparin, heparin sodium and warfarin sodium); Thrombolytics (eg urokinase, streptokinase and alteplase); Antifibrinolytic agents (eg, aminocapric acid); Hemoheological agents (eg, pentoxifylline); Antiplatelet agents (eg, aspirin); Anticonvulsants (e.g., valproic acid, divalproex sodium, phenytoin, phenytoin sodium, clonazepam, primidone, phenobarbitol, carbamazepine, amobarbital sodium, metsuccimid, metavirtal, mepo Barbital, mefenitoin, pensuccimid, paramethadione, etotoin, phenacemid, secobarbitol sodium, chlorazate dipotassium and trimethadione); Anti-Parkinson's agents (eg, ethosuccimid); Antihistamines / anti-itch agents (e.g., hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine maleate, ciproheptadine hydrochloride, terpenadine, clemastine fumarate, triprolidine, carbinoxamine, di Phenylpyraline, phenindamin, azatadine, tripelennamin, dexchlorpheniramine maleate, metdilazine, and); Antimicrobial agents (e.g. amikacin sulphate, aztrenam, chloramphenicol, chloramphenicol palmitate, ciprofloxacin, clindamycin, clindamycin palmitate, clindamycin phosphate, metronidazole, metronidazole hydrochloride, gentamicin sulfate, lincomycin sulfate, tobramycin hydrochloride Vancomycin hydrochloride, polymyxin B sulfate, colistimitate sodium and colistin sulfate); Antiviral agents (eg, interferon alpha, beta or gamma, zidobudine, amantadine hydrochloride, ribavirin and acyclovir); Antimicrobial agents (e.g., cephalosporins, e.g., cefazoline sodium, cepradine, cefaclo, sodium cefapirine, ceftioxime sodium, cephaperazone sodium, cetethetan disodium, cepuroxime azotyl, cefotaxime sodium Cephadroxy monohydrate, cephalexin, cephalotin sodium, cephalexin hydrochloride monohydrate, cephamandol naphate, cephacithin sodium, cenisidide sodium, celarinide, cepadroxyl, cepradine and Ceproxyl sodium; penicillins such as ampicillin, amoxicillin, penicillin G benzatin, cyclacillin, ampicillin sodium, penicillin G potassium, penicillin potassium, piperacillin sodium, oxacillin sodium, bacampicillin hydrochloride, xaxacillin Sodium, Ticacillin Disodium, Azlocillin Sodium, Carbenicillin Indanyl Sodium, Penicillin G Procaine, Methicillin Sodium and Others Sodium psilin; erythromycins such as erythromycin ethyl succinate, erythromycin, erythromycin estoleate, erythromycin lactobionate, erythromycin stearate, and erythromycin ethyl succinate; and tetracyclines such as Tetracycline hydrochloride, doxycycline hydrate, and minocycline hydrochloride, azithromycin, clarithromycin); Anti-inflammatory agents (eg, GM-CSF); Bronchodilators (eg, sympathomimetic, e.g. epinephrine hydrochloride, metaproterenol sulfate, terbutalin sulfate, isotarin, isoetherin mesylate, isotarin hydrochloride, bitolterolmethylate, isoprotere Nal hydrochloride, terbutalin sulfate, epinephrine bitartrate, metaproterenol sulfate, epinephrine and epinephrine bitartrate;

Anticholinergic agents such as ifpratropium bromide; Xanthines such as aminophylline, diphylline, metaproterenol sulfate and aminophylline; Mast cell stabilizers such as, for example, chromoline sodium; Steroidal compounds and hormones (e.g., androgens such as danazol, testosterone cypionate, fluoxymesterone, ethyltestosterone, testosterone enateate, methyltestosterone, fluoxymesterone and testosterone cypionate; estradiol, estrophy Fates, and estrogens such as conjugated estrogens; progesterones such as methoxyprogesterone acetate, and noethynedrone acetate; Prednisolone acetate suspension, triamcinolone acetonide, methylprednisolone, prednisolone sodium phosphate, methylprednisolone sodium succinate, hydrocortisone Sodium succinate, triamcinolone hexacetonide, hydrocortisone, hydrocortisone cypionate, prednisolone, fludrocortisone acetate, paramethasone acetate, prednisolone tebutate, prednisolone acetate, prednisolone sodium phosphate and hydrocortisone sodium succinate sodium and sodium succinate; Thyroid hormones such as); Hypoglycemic agents (eg, glyburide, chlorporamide, tolbutamide and tolazamide); Hypolipidemic agents (eg, clofibrate, dextrothyroxine sodium, probucol, pravastatin, atorvastatin, lovastatin and niacin); Antiulcers / antireflux agents (eg, famotidine, cimetidine and ranitidine hydrochloride); Motion sickness / antiemetic agents (eg, mecrizine hydrochloride, nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride, thiethylperazine and scopolamine); And fat soluble vitamins. Low solubility metabolites such as these can be treated by the methods of the present invention.

microbe

Based on the presence of microbial enzymes such as cytochrome P450 enzymes that are capable of metabolizing the parent compound, microorganisms suitable for successful bioconversion can be selected. Microorganisms useful for the bioconversion method include bacteria, fungi, and actinomycetes having cytochrome P450 activity. Microorganisms with these enzymes can be identified experimentally by comparing metabolites found in blood or urine after administration of ISA247 with those obtained by bioconversion or microbial conversion. For example, CYP3A4 is a human P450 enzyme characterized by the ability to produce 6β-hydroxytestosterone by hydroxylating testosterone. This enzyme is inhibited by compounds such as clotrimazole and naringenin. This enzyme is induced by carbamazipine, phenobarbital and rifampin. Microorganisms in culture in growth medium, which express enzymes having cytochrome P450 activity, produce 6β-hydroxytestosterone when testosterone is introduced into the medium, and this production is affected by known inhibitors and triggers. Other substrates metabolized by CYP3A4 include, for example, acetaminophen, diazepam, terrophylline, warfarin, taxol and nifedipine. Likewise, when these compounds are introduced into a medium containing microorganisms, when the microorganisms express their enzymes, they metabolize their substrates.

Known characterized enzymes have known characterized activity. By comparing the structure of the compounds to be metabolized with VII with known activity, enzymes with activity to metabolize the compounds can be identified. It is also possible to screen microorganisms with identified enzymes. Accordingly, one aspect of the present invention provides a method of identifying a microorganism suitable for use in a bioconversion system, the method comprising: a) comparing the structure of a compound to be metabolized with known enzyme activity; b) identifying an enzyme that expresses the desired enzymatic activity; c) identifying microorganisms expressing the identified enzymes; And d) metabolizing the compound using a microorganism expressing the identified enzyme in a bioconversion process. By using this method, it is possible to identify microorganisms that may be useful for producing metabolites of certain compounds.

On the other hand, genetic sequence data can also be used to identify potentially useful organisms by comparing the genome sequences of certain organisms with known mammalian gene sequences encoding cytochrome enzymes such as CYP3A4, for example. Microorganisms with appropriate gene sequences, cultured under appropriate conditions, express target enzymes. In addition to the reference compounds, compounds that inhibit or induce specific human P450 enzymes can be tested in both systems.

Drugs known to be metabolized by specific cytochrome enzymes include: (1) acetaminophen, aromatic amines, caffeine, estradiol, imipramine, phenacetin, theophylline and warfarin, degraded by CYP1A2; (2) Amitriptyline, Bufurol, Captropill, Clozepine, Desrisopurine, Flecainide, Fluoxetine, Haloperidol, Metoprolol, Mexyletine, Spartane, Timolol, C.O. And codeine; (3) acetaminophen, diazepam, amiodarone, benzpetamine, carmezapine, cyclosporine, digitoxin, diltiazem, erythromycin, etoposide, flutamide, imipramine, lidocaine, loratidine degraded by CYP3A4 Nifedipine, midazolam, retinic acid, steroids, taboxifen, taxol, terpenadine, THC, verapamil and warfarin.

Examples of microorganisms expressing the CYP3A4 enzyme and useful microorganisms in biotransformation are: Actinoplanes sp : for example, ATCC No. 53771), Streptomyces griseus : for example ATCC 13273), Saccharopolyspora erythraea : for example, ATCC No. 11635), and Streptomyces setonii : for example, ATCC No. 39116). Other useful microorganisms expressing the cytochrome P450 enzyme include Amycolata ( Amycolata). autotrophica ; For example, ATCC No. 35204), Streptomyces californica ; For example, ATCC No. 15436), Saccharopolysora hirsute ; For example, ATCC No. 20501), Streptomyces lavandulae : for example, ATCC No. 55209, Stretomyces aureofaciens ; For example, ATCC No. 10762), Streptomyces rimosus (e.g. ATCC No. 28893), Bacillus subtilis subtillis ; For example, ATCC No. 55060), and Nocardia steroid ( Nocardia asteroids : for example, ATCC No. 3318), Saccharomyces cerevisiae ; For example, ATCC No. 20137 or ATCC No. 64 667), Aspergillus nidul lance (Aspergillus nidulans ; For example, ATCC No. 32353 Cimninghamella echinulata var . elegans ; For example, ATCC No. 36112, Rhizopus stolonifer ; For example, ATCC No. 6227b), Candida Bahia Cola (Candida apicola ; For example, ATCC No. 96134), co-Taunus-free Cyreneus (Coprinus cireneus; for example, ATCC No. MYA-727, MYA-726, MYA-728, MYA-729, MYA-730, MYA-731).

] Selection of appropriate incubation periods, culture conditions, extraction and purification methods is well known to those skilled in the art. Cultivation of the selected microorganisms can be accomplished by those skilled in the art, for example, by using suitable growth media containing nutrients such as carbon and nitrogen, buffers, and trace elements, and aeration conditions conducive to proper pH, temperature and culture. Examples of carbon sources include glucose, maltose, dextrin, starch, lactose, sucrose, molasses, soybean oil and the like. Suitable nitrogen sources include soybean wheat, cottonseed oil, fish oil, yeast, yeast extract, peptone, rice bran, meat extract, ammonium nitrate, ammonium sulfate and the like. Inorganic salts such as phosphate, sodium chloride, calcium carbonate and the like can also be added. Other growth media may also be used depending on the growth stage of the microorganism.

Saccharopolyspora erythraea : for example ATCC 11635, Saccharopolyspora hirsute ; ATCC 20501, for example Amycolota Autotrophica autotrophica ; For example, by ATCC 35204) exemplary conditions and media for the growth of microorganisms suitable for use in the bioconversion of cyclosporin and cyclosporin derivatives are described in Corconan, Methods in Enzymology 43: 487-498 (1975), US Pat. No. 5,124,258; 6,043,064; And 6,331,622. Culture conditions for Actinoplanes species (such as ATCC No. 53771) are described in US Pat. No. 5,270,187.

Examples of culture conditions for bioconverting macrolides to their hydroxylated and / or demethylated metabolites by microorganisms include: 1) Actinomycete sp ., As exemplified in US Pat. No. 5,268,370. Culture conditions as disclosed for the demethylation of L679,934 (FK-506) with its metabolite L-683,519 using Merck Culture Collection MA 6474; ATCC No. 53828) and 2) US Pat. No. 5,202,258 disclosed using the culture conditions Ill Martino Plastic Ness species as: the a L-679,934 by (Actinoplanes sp. ATCC No. 53771) with L-682,993 or L-683,5990 there is a condition to demethylation with L-683,742 . Ill Martino Plastic Ness (Actinoplanes spp. ATCC 53771), Saccharomyces to a police Fora Erie Tra (Saccharopolyspora erthyreae ATCC 11653), ( Streptomyces in Streptomyces Laban dulra lavandulae ATCC 55209, Stretomyces aureofaciens ATCC 10762, Streptomyces rimosus ATCC 28893, Bacillus subtilis subtillis ATCC 55060) and Nocardia Asteroids ( Nocardia asteroids ATCC 3318) can be used to prepare hydroxylated metabolites of rapamycin (such as 24-OH rapamycin) and / or demethylated metabolites (such as 39-O-demethylrapamycin) (Kuhnt, M. et al., 1997, Enzyme and Microbial Technology 21: 405-412.

Surfactants

20, 트윈

21, 트윈

40, 트윈

80, 트윈

80K, 트윈

81 및 트윈

85과 같은 폴리옥시에틸렌 소르비탄 지방산 에스테르 (뉴저지 브릿지워터 소재 ICI Americas Inc., 위스콘신 밀워키 소재 Aldrich Chemical Company Inc.로부터 구득); 글리세린 (온타리오 토론토 소재BDH Fine Chemicals); 카스터유 (온타리오 런던 소재 Wiler Fine Chemicals Ltd); 이소프로필 미리스테이트 (온타리오 런던 소재 Wiler Fine Chemicals Ltd, London Ont.); 크레모포어

EL (미주리 세인트루이스 소재 Sigma Chemical, St Louis MO); 및 플루로닉스

F127 및 플루로닉스

L108 (BASF)과 같은 폴록사머와 같은 비이온계 계면활성제를 들 수 있으나 이에 한정되지 않는다. 그 밖에 사용가능한 계면활성제로는 윤활제나 유화제로 작용할 수 있는 것들, 예컨대 틸록사폴 [포름알데히드와 옥시란과의 4-(l,l,3,3-테트라메틸부틸)페놀 폴리머]; BASF사의 크레모포어

A6, 크레모포어

EL, 크레모포어

ELP, 크레모포어

RH 및 Rhone Poulenc Co의 알카물스 EL620; HCO-40과 같은 폴리에톡실화된 수소첨가 카스터유; 및 폴리에틸렌 9 카스터유를 들 수 있다.Surfactants suitable for use in embodiments of the methods of the invention are those that can withstand the autoclave treatment prior to their introduction into the microbial growth environment. Suitable surfactants include 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) as biocompatible surfactants; Caprylocaproyl macrogol-8 glycerides such as labrasol (Gatte Fosse, Cedex, France); Twin

20, twin

21, twin

40, twin

80, twin

80K, twin

81 and twin

Polyoxyethylene sorbitan fatty acid esters such as 85 (ICI Americas Inc., Bridgewater, NJ, obtained from Aldrich Chemical Company Inc., Milwaukee, Wis.); Glycerin (BDH Fine Chemicals, Toronto, Ontario); Castor oil (Wiler Fine Chemicals Ltd, London, Ontario); Isopropyl myristate (Wiler Fine Chemicals Ltd, London Ont., London, Ontario); Cremophor

EL (Sigma Chemical, St Louis MO, St. Louis, MO); And pluronics

F127 and Pluronix

Nonionic surfactants such as poloxamers such as L108 (BASF). Other surfactants that can be used include those which can act as lubricants or emulsifiers such as tyloxapol [4- (l, l, 3,3-tetramethylbutyl) phenol polymer of formaldehyde with oxirane]; BASF's Cremophor

A6, Cremophor

EL, Cremophor

ELP, Cremophor

Alkamuls EL620 from RH and Rhone Poulenc Co; Polyethoxylated hydrogenated castor oil such as HCO-40; And polyethylene 9 castor oil.

RH; 폴록사머; 플루오닉스 LlO, L31, L35, L42, L43, L44, L61, L62, L63, L72, L81, LlOl, L121, L122; PEG 20 아몬드 글리세라이드; PEG 20 옥수수 글리세라이드; 등을 들 수 있다. 적절한 계면활성제로는 알킬글루코사이드; 알킬말토사이드; 알킬티오글루코사이드; 라우릴 마크로골 글리세라이드; 폴리옥시에틸렌 알킬 에테르; 폴리옥시에틸렌 알킬페놀; 폴리에틸렌 글리콜 지방산 에스테르; 폴리에틸렌 글리콜 글리세롤 지방산 에스테르; 폴리옥시에틸렌-폴리옥시프로필렌 블록 코폴리머; 폴리글리세롤 지방산 에스테르; 폴리옥시에틸렌 글리세라이드; 폴리옥시에틸렌 스테롤; 폴리옥시에틸렌 식물성유; 폴리옥시에틸렌 수소첨가된 식물성유; 폴리옥시에틸렌 알킬에테르; 폴리에틸렌 글리콜 지방산 에스테르; 폴리에틸렌 글리콜 글리세롤 지방산 에스테르; 폴리옥시에틸렌 소르비탄 지방산 에스테르; 폴리옥시에틸렌-폴리옥시프로필렌 블록 코폴리머; 폴리글리세롤 지방산 에스테르; 폴리옥시에틸렌 글리세라이드; 폴리옥시에틸렌 식물성유; 폴리옥시에틸렌 수소첨가된 식물성유; PEG-10 라우레이트, PEG-12 라우레이트, PEG-20 라우레이트, PEG-32 라우레이트, PEG-32 디라우레이트, PEG-12 올리에이트, PEG-15 올리에이트, PEG-20 올리에이트, PEG-20 디올리에이트, PEG-32 올리에이트, PEG-200 올리에이트, PEG-400 올리에이트, PEG-15 스테아레이트, PEG-32 디스테아레이트, PEG-40 스테아레이트, PEG-100 스테아레이트, PEG-20 디라우레이트, PEG-25 글리세릴 트리올리에이트, PEG-32 디올리에이트, PEG-20 글리세릴 라우레이트, PEG-30 글리세릴 라우레이트, PEG-20 글리세릴 스테아레이트, PEG-20 글리세릴 올리에이트, PEG-30 글리세릴 올리에이트, PEG-30 글리세릴 라우레이트, PEG-40 글리세릴 라우레이트, PEG-40 팜커넬유, PEG-50 수소첨가된 카스터유, PEG-40 카스터유, PEG-35 카스터유, PEG-60 카스터유, PEG-40 수소첨가된 카스터유, PEG-60 수소첨가된 카스터유, PEG-60 옥수수유, PEG-6 카프레이트/카프릴레이트 글리세라이드, PEG-8 카프레이트/카프릴레이트 글리세라이드, 폴리글리세릴-10 라우레이트, PEG-30 콜레스테롤, PEG-25 피토 스테롤, PEG-30 대두 스테롤, PEG-20 트리올리에이트, PEG-40 소르비탄 올리에이트, PEG-80 소르비탄 라우레이트, 폴리소르베이트 20, 폴리소르베이트 80, POE-9 라우릴 에테르, POE-23 라우릴 에테르, POE-IO 올레일 에테르 POE-20 올레일 에테르, POE-20 스테아릴 에테르, 토코페릴 PEG-100 숙시네이트, PEG-24 콜레스테롤, 폴리글리세릴-10 올리에이트, 수크로스 모노스테아레이트, 수크로스 모노라우레이트, 수크로스 모노팔미테이트, PEG 10-100 노닐 페놀 시리즈, PEG 15-100 옥틸 페놀 시리즈, 폴록사머; PEG-35 카스터유, PEG-40 수소첨가된 카스터유, PEG-60 옥수수유, PEG-25 글리세릴 트리올리에이트, PEG-6 카프레이트/카프릴레이트 글리세라이드, PEG-8 카프레이트/카프릴레이트 글리세라이드, 폴리소르베이트 20, 폴리소르베이트 80, 토코페릴 PEG-1000 숙시네이트 및 PEG-24 콜레스테롤, 폴록사머와 같은 폴리올의 반응 혼합물을 들 수 있다. 이에 더해, 아몬드유; 바바수유; 보라지유; 블랙커런트씨유; 카놀라유; 코코넛유; 옥수수유; 면실유; 달맞이꽃유; 포도씨유; 땅콩유; 겨자씨유; 올리브유; 팜유; 팜커널유; 피넛유; 채종유; 홍화유; 참기름; 상어간유; 대두유; 해바라기유; 수소첨가된 카스터유; 수소첨가된 코코넛유; 수소첨가된 팜유; 수소첨가된 대두유; 수소첨가된 식물성유; 수소첨가된 면실유 및 카스터유; 특히 수소첨가된 대두유; 대두유; 글리세릴 트리카프로에이트 트리카프로에이트; 글리세릴 트리카프릴레이트; 글리세릴 트리카프레이트; 글리세릴 트리운데카노에이트; 글리세릴 트리라우레이트; 글리세릴 트리올리에이트; 글리세릴 트리리놀리에이트; 글리세릴 트리리놀리네이트; 글리세릴 트리카프릴레이트/카프레이트/스테아레이트; 포화된 폴리글리콜화 글리세라이드; 리놀레산 글리세라이드; 카프릴산/카프르산 글리세라이드를 사용할 수 있다. 나아가, 계면활성제 및/또는 오일 및/또는 알코올의 혼합물도 이용가능하다.Other surfactants that can be used include: polysorbate 20, polysorbate 60 and polysorbate 80; Cremophor

RH; Poloxamer; Fluoronix L10, L31, L35, L42, L43, L44, L61, L62, L63, L72, L81, L10, L121, L122; PEG 20 almond glycerides; PEG 20 corn glycerides; Etc. can be mentioned. Suitable surfactants include alkylglucosides; Alkyl maltosides; Alkylthioglucosides; Lauryl macrogol glycerides; Polyoxyethylene alkyl ethers; Polyoxyethylene alkylphenols; Polyethylene glycol fatty acid 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 oil; Polyoxyethylene alkyl ethers; Polyethylene glycol fatty acid 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 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, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glycerol Ryl 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 capre / Caprylate glyceride, PEG-8 caprate / caprylate glyceride, polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phytosterol, PEG-30 soy sterol, PEG-20 triol 8, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-IO 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 nonyl phenol series, PEG 15-100 octyl phenol series, poloxamer; PEG-35 castor oil, PEG-40 hydrogenated castor oil, PEG-60 corn oil, PEG-25 glyceryl trioleate, PEG-6 caprate / caprylate glyceride, PEG-8 caprate / capryl And reaction mixtures of polyols such as late glycerides, polysorbate 20, polysorbate 80, tocopheryl PEG-1000 succinate and PEG-24 cholesterol, poloxamer. In addition, almond oil; Babass feeding; Borage oil; Blackcurrant seed oil; Canola oil; Coconut oil; Corn oil; Cottonseed oil; Evening primrose oil; Grape seed oil; Peanut 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 oils; Hydrogenated cottonseed oil and castor oil; Especially hydrogenated soybean oil; Soybean oil; Glyceryl tricaproate tricaproate; Glyceryl tricaprylate; Glyceryl tricaprate; Glyceryl trioundecanoate; Glyceryl trilaurate; Glyceryl trioleate; Glyceryl trilinoleate; Glyceryl trilinolinate; Glyceryl tricaprylate / caprate / stearate; Saturated polyglycolated glycerides; Linoleic acid glycerides; Caprylic / capric glycerides can be used. Furthermore, mixtures of surfactants and / or oils and / or alcohols are also available.

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

menstruum

In some embodiments of the invention, the bioforeign compound is mixed with a solvent prior to addition to the microbial culture. Such solvents may be sterilized prior to mixing with the bioforeign compound. If desired, surfactants may also be added to the bioforeign compound-solvent mixture. Any solvent suitable for use in the present invention may be any solvent as long as it does not inhibit the growth and metabolism of microorganisms. The solvent may be nonpolar, polar aprotic or polar protic. For example, solvents include hydrocarbon solvents such as benzene, toluene, n-hexane, cyclohexane, and the like; Ether solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether, dimethoxyethane, ethylene glycol dimethyl ether and the like; Alcohols such as methanol, ethanol, 1-propanol, isopropanol and the like; Halogen-containing solvents such as methylene chloride, chloroform, 1,1,1-trichloroethane and the like; And other solvents such as dimethylformamide, N-methylpyrrolidone, hexamethylphosphorotriamide and the like. The solvent is preferably not DMSO.

The following examples are merely for illustrating the present invention, but the scope of the present invention is not limited thereto in any way. Although the present invention is embodied and described with reference to some preferred embodiments as follows, those skilled in the art can make various modifications in form or detail without departing from the spirit and scope of the invention as defined in the appended claims. Will understand.

1 is a diagram showing the structure of ISA247. The amino acid residues of ISA247 are numbered. The Greek letter indicates the carbon position of amino acid 1.

2A and 2B refer to the trans (E-) and cis (Z-) isomers of ISA247 molecules, respectively.

FIG. 3 is an HPLC scan showing the profile of ISA247 metabolite isolated from human whole blood of a subject receiving a 50:50 mixture of cis: trans ISA247.

4 is an HPLC scan showing the profile of ISA247 metabolite isolated from the bioconversion method described in Example 4. FIG.

FIG. 5 is a graph illustrating the effects of different surfactants on ISA247 metabolite production in a bioconversion system.

FIG. 6 depicts LC-MS profiles of ISA247 metabolites isolated from human whole blood of subjects injected with an ISA247 formulation containing predominantly the trans-isomer of ISA247.

7 and 8 shows that medium 3 and medium 16 are Actinoplanes sp (ATCC 53771) and Saccharopolyspora ( Saccharopolyspora), respectively. erythraea : ATCC 11635) to compare the effect on the production of ISA247 metabolite.

Figure 9 demonstrates the effect of various solvents and surfactants on the production of ISA247 metabolite by Beauvaria bassiana .

In the following examples, the abbreviations described next have the following meanings. Abbreviations not specifically defined have a commonly used meaning.

℃ = Celsius

hr = hour

min = minutes

sec or s = seconds

μM = micromolar

mM = millimolar

M = mole

ml = milliliters

Μl = microliters

mg = milligrams

Μg = microgram

mol = mole

pmol = picomolar

ATCC = Amerian Type Culture Collection

PBS = phosphate buffered saline

CSA = cyclosporin A

TDM = therapeutic drug concentration monitoring

LC = liquid chromatography

MS = mass spectrophotometry

PEG = polyethylene glycol

Common Materials and Methods

Liquid chromatography ( LC ) Condition

For liquid chromatography (LC or HPLC), a column with a normal phase formed by chemically bonding long chain hydrocarbon groups to a porous silica matrix, i.e. guard column 2 filled with Perisorb RP-8 (Upchurch Scientific cat # C-601) Waters Symmetry C8, 2.1 × 50 mm 3.5 analytical column (Waters cat # WAT 200624) equipped with x 20 mm (Upchurch Scientific cat # C-130B) was used. Solvent percentages and flow rates used in the LC program are summarized in Table 2 below.

 Time (min) 0.2% GAA + 10 ^-5 M Na Acetate (%) MeOH: MtBE (9: 1) (%) Flow rate (mL / min)  0.00  55  45  0.5  5.00  45  55  0.5  10.00  5  95  0.5  12.00  5  95  0.5  12.01  55  45  0.5  15.00  55  45  0.5

Mass spectrum ( MS ) Condition

For mass spectrophotometry, Applied Biosystems / MDX Cyx API3000 (Analyst software v.1.2) instrument was used. The run time was 15 minutes, the injection volume was 5 microliters, the guard column temperature and the analytical column temperature were 60 ° C. The manual setting method was as follows: the turbo ion spray was 8000, the turbo ion spray horizontal setting was positive 4, and the turbo ion spray side setting was 1.0. The Cyx instrument was set to the parameters shown in Table 3.

  MS setting:   Scan type:   Multiple Response Monitoring (MRM)   polarity:   positivity   Period period:   15.00 minutes   Period Cycle:   1.32 seconds   cycle #   692 improved MS  setting:   Resolution Q1:   lowness   Q3:   lowness   Strength titer:   0   Fixed time:   50 milliseconds   Pose Time:   30 milliseconds  Parameter setting:   Ion source:   Turbo ion spray   Inhaler gas:  12   Curtain gas:   8   Collision gas:   12   Ion spray voltage:   5000 V   Temperature:   550 ℃ Compound setting:   Declustering Dislocation:   60 V   Focusing Dislocation:   400 V   Collision energy:   90 V

Table 4 shows the settings of the ion and ion-specific equipment.

 Q1 mass (amu)  Q3 mass (amu)  Time in milliseconds  1222.8  1098.7  100  1236.8  1112.7  100  1252.8  1128.7  100  1252.8  1224.7  100  1270.8  1112.7  100  1254.8  1130.7  100  1268.8  1128.7  100  1268.8  1144.7  100  1238.8  1114.7  100  1268.8  1240.8  100

Example  One

From whole blood  ISA247 Metabolites  How to make

Whole blood was drawn from the human body after administering ISA247 (50:50 mixture of cis: trans ISA247247). ISA247 and its metabolites were extracted from whole blood using terbutyl-methyl-ether (or methyl terbutyl ether, MTBE), dried and then reconstituted in methanol. 2 mL of MTBE (cat No. 7001-2; Caledon) was added to 200 μL of blood, shaken for 10 minutes and concentrated in vacuo. The residue was reconstituted in 200 μL of methanol. Bile and urine extracts can be treated as well, as are extracts from microsome preparations and bioconversion preparations. Once extracted, metabolites can be characterized using HPLC-MS, NMR and other techniques well known in the art. 3 shows the results of LC-MS described in General Materials and Methods. ISA247 metabolites from whole blood include three major groups: diols, hydroxylated metabolites and demethylated metabolites.

Example  2

Dog liver Microsome On preparation  By ISA247 Metabolite  Manufacturing method

Preparation of Dog Microsomes

Microsomes of dog livers were prepared in the following manner: The livers were extracted and rinsed with 1.5% potassium chloride (KCl); Compact into small sections (approx. 25 g) and use buffer at 1: 1 ratio for cold grinding buffer (0.1 M phosphate buffer pH 7.4; 4 ° C; using polytron homogenizer at 15,000 rpm for 3 to 5 minutes) ) Until it disintegrates and produced a homogenate containing the membrane-bound organelles containing the liver microsomes. After decanting the supernatant from the particulate matter, the supernatant was centrifuged at 100,000 x g for 90 minutes to obtain pellets and the supernatant. Protein content was determined using Lowry protein assay. The protein concentration of this microsome preparation was 23.2 mg / mL. To prevent loss of enzyme activity, the microsomes are stored in 4.0 or 6.0 mL aliquots at -80 ° C to avoid freeze thaw cycles.

The following components were added in turn to an 257 mL dose of Erlenmeyer flask: 57.3 mg of NADP, 254 mg of glucose-6-phosphate, and 23.0 mg of NADPH were added to 6.0 mL of 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, purchased from CALBIOCHEM, San Diego, Calif., Cat. No. 346774) were added to the solution. Finally, 10 mL of phosphate buffer (pH 7.4) was added. 6 mL of the microsomal liver prepared as described above was put in a flask, ISA247 was added thereto, and then incubated at 37 ° C. for 2 hours at a speed of 250 rpm in an incubator / shaker controlled by EK. After 2 hours, the reaction was stopped by adding 500 μl of 2M HCl.

The metabolite produced by this method was then extracted with an organic solvent and then further separated using high pressure liquid chromatography (HPLC). Metabolites were further characterized by electrospray mass spectra (MS) and NMR. The metabolite profile (data not shown) thus obtained was similar to that obtained from human whole blood. However, diols were much less in dog microsomes.

Example  3

Saccharopolis Fora Eritrea  ( Saccharopolyspora erythrae ) Port of Use  By biotransformation ISA247 Metabolite  Produce

40과 혼합한 다음 사카로폴리스포라 에리트라에 (Saccharopolyspora erythrae):ATCC 11635)를 함유하는 생물전환 시스템에 첨가하였다. This example describes a bioconversion system containing microorganisms containing microbial equivalents of human cytochrome P450 microsomal enzymes and media suitable for the active growth of microorganisms. Prior to addition to the bioconversion system, the poorly soluble parent compound is mixed with ethanol and then with a surfactant. In this example, twin ISA247 in ethanol

It was mixed with 40 and added to a bioconversion system containing Saccharopolyspora erythrae : ATCC 11635.

Starting cultures were prepared as follows: 15 (16 x 16) ISP2 medium slants containing 4 g / L yeast extract, 10 g / L malt extract, 4 g / L dextrose and 2 g / L agar. 26 mm; 6 ml each). These components were mixed with deionized water to 1 liter and, if necessary, the pH was made neutral to 7 with NaOH. The medium was sterilized at 100 ° C. for 30 minutes. The tube was stored at 4 ° C. until use. As Saccharomyces each of the slants (Saccharopolyspora the Erie Tribe Police Fora erythraea : ATCC 11635). Inoculated slants were incubated for 3 weeks at room temperature under sterile conditions.

Precultures were transferred to Phase I medium. Phase I medium was prepared using 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 to 1 liter with deionized water, the pH was adjusted to pH 7 with NaOH as needed and 50 mL was dispensed into each of the 250 mL culture flasks with two baffles. The medium was sterilized at 100 ° C. for 30 minutes. 5 mL of medium was dispensed into a slant tube containing Saccharopolyspora erythraea (ATCC 11635). Cells were stripped from the slant surface and 2.5 mL of suspension was placed in each flask. These flasks were placed in a 27 ° C. labline incubator and shaken at 250 rpm for 3 days (72 hours).

At 3300 rpm to a saccharide by centrifuging the contents of the flask of claim I 5 bungan (Saccharopolyspora the police Fora Erie trad erythraea : ATCC 11635) was transferred from Phase I medium to Phase II medium and pelleted by decanting the supernatant. 5 mL of Phase IIII medium was added to the pellet, vortexed in vitro, and separated for 4 minutes at 3300 rpm. The supernatant was decanted once again. The pellet was resuspended in phase II medium. The supernatant was then added to 50 mL of Phase II medium in a culture flask equipped with baffles.

40을 ISA247 및 에탄올과 혼합기 전에 오토클라브로 처리하였다.Phase II medium contained 10 g / L glucose 1 g / L yeast extract, 1 g / L beef extract and 11.6 g / L 3-morpholinopropanesulfonic acid (MOPS) buffer. These components were mixed with deionized water until 1 liter; 50 mL was placed in a culture flask (250 mL) equipped with two baffles. After pH was adjusted to 7 using 5 M NaOH, the medium was autoclaved at 100 ° C. for 30 minutes and then cooled. Twin

40 was autoclaved before mixing with ISA247 and ethanol.

40 (폴리옥시에틸렌 소르비탄 모노팔미테이트; Cat No. P1504: 미주리주 세인트루이스 소재 Sigma-Aldrich사) 0.4 ml와 혼합하였다. 모화합물-계면활성제 혼합물을 제II상 배양 배지 중의 사카로폴리스포라 에리트라에 (Saccharopolyspora erythraea)에 첨가하였다. 제로타임 시료를 채취해서 동결시켰다. 이어서 각각의 플라스크를 덮은 다음 27℃의 이노바 인큐베이터에 넣고 170 rpm으로 진탕시키면서 120 시간 동안 인큐베이트시켰다.ISA247 (4 mg of ~ 50/50 mixture of E and Z isomers) was dissolved in 95% ethanol (0.1 ml) and then Tween

0.4 ml of 40 (polyoxyethylene sorbitan monopalmitate; Cat No. P1504: Sigma-Aldrich, St. Louis, MO). Parent compound-surfactant mixture in Indianapolis Fora Erie Tra as saccharide in the culture medium of claim II (Saccharopolyspora erythraea ). Zero time samples were taken and frozen. Each flask was then covered and incubated for 120 hours in an innovator at 27 ° C. with shaking at 170 rpm.

A second sample was taken from phase II culture medium. The zero time sample and the second sample were extracted using tert-butyl-methyl-ether (cat No. 7001-2; Caledon), and the T extracted extracted metabolite was reconstituted in methanol (HPLC grade) as described above. Analysis by LC-MS. As shown in Figure 4, the metabolite profile obtained by this method is similar to that obtained from human whole blood (see Example 1 0. Analyzing the bioconversion mixture, the following seven metabolites The mixture was found to be present: IM4n (N-demethylated ISA247 metabolite at amino acid-4), IM9 (ISA247 metabolite hydroxylated at amino acid-9), IM4 (ISA247 hydroxylated at amino acid-4) Metabolites), IM1-c-1 (see Table 1), IM1-d-1 (see Table 1), IM1-d-2 (see Table 1) and IM1-d-3 (see Table 1). Seven of the eight ISA247 metabolites found in human blood were produced in this bioconversion system.

Example  4

Saccharopolis Fora Eritrea  ( Saccharopolyspora erythraea Effect of Changes of Surfactant on Bioconversion Using

With, on the first saccharide being actively growing as described above II To test the effect of various surfactants on Indianapolis Fora Erie Tra (Saccharopolyspora erythraea : ATCC 11635). Seven test tubes were prepared, each containing ISA247 (56 mg; 50/50 mixture of E and Z isomers) in ethanol (0.33 ml). Surfactant (0.67 ml / test tube) was added to each test tube as follows:

Test Tube 1-PEG 400 (polyethylene glycol 400; Fisher Scientific, Fairon, Carbowax, NJ)

Test Tube 2-Caster Oil (from Wiler FIne Chemicals Ltd., London, Ontario)

Test Tube 3-Isopropyl Myristate (from Wiler FIne Chemicals Ltd., London, Ontario)

Test tube 4-glycerin (BDH Fine Chemicals, Lot # 120343/73865, Turonto, Ontario);

EL (미주리주 세인트루이스 소재 Sigma Chemical 제품);Test Tube 5-Cremophor

EL (Sigma Chemical, St. Louis, MO);

(프랑스 세덱스 소재 Gatte Fosse 제품); 및Test Tube 6-Labrasol

(Manufactured by Gatte Fosse, Cedex, France); And

40 (위스콘신 밀워키 소재 Aldrich Chemical Company Inc. 제품).Test Tube 7-Twin

40 (product of Aldrich Chemical Company Inc., Milwaukee, Wisconsin).

, 라브라솔

및 트윈

40은 모두 계면활성제 없는 프레퍼레이션 (나타내지 않음)에 있어서의 대사산물 생산보다, 생물전환 프레퍼레이션에서 대사산물 생산량을 증가시켰다. The parent compound-surfactant mixture was added to the actively growing saccharopolypora culture medium and zero time samples were taken. After incubation with shaking at 27 ° C. for 5 days, the extracts were extracted and the metabolites were quantified as described in Example 4. The area under the HPLC peak curve was similar to those shown in FIGS. 3 and 4 and was measured as a measure of the amount of metabolite present. HPLC peaks were N-demethylated metabolite identified as IM4n; Cyclic metabolites identified as IM1-c-1; And three diol metabolites, diols formed in one amino acid of the ISA247 compound, identified as IM1-d-1, IM1-d-2 and IM1-d-3 (see Table 1). The seven surfactants were not all equal in activity to increase metabolite production in bioconversion preparations. As shown in Figure 5, when glycerin or PEG 400 was added to the bioconversion preparation, the production of metabolites increased significantly. However, castor oil, isopropyl myristate, cremophore

, Labrasol

And twin

All 40 increased metabolite production in bioconversion preparations rather than metabolite production in surfactant free preparations (not shown).

Example  5

Several  Bioconversion using microorganisms

Curvularia lunata : University of Alberta Microfungal Collection and Herbarium (UAMH) 9191; ATCC 12017), Cunninghamella Variant of Cunninghamella Echinulata echinulata var . elegans : UAMH7370; ATCC 36112), Culbularia Echinulata Variant Blaquesrina ( Curvularia) echinulata var. blakesleena : UAMH 8718; ATCC 8688a), Cunninghamella echinulata var . elegans : UAMH 7369; ATCC 26269, Beauvaria Bassiana bassiana : UAMH 8717; ATCC 7159), Actinomycetes (ATCC 53828), Actinoplanes sp . ATCC 53771), Cunninghamella echinulata : UAMH 4144; ATCC36190), Cunninghamella echinulata : UAMH 7368; ATCC 9246, Cunninghamella baniere ( echinulata ) : UAMH 4145; ATCC9244) and Saccharopolyspora erythraea (ATCC 11635) and other microorganisms were evaluated for the production of ISA247 metabolites from ISA247.

These microorganisms were screened for metabolite conversion yields (quantities of known ISA247 metabolites produced) and metabolite diversity (various kinds of ISA247 metabolites produced). Microorganisms were cultured in phase I and incubated with ISA247 in phase II. After adding ISA247 to the fermentation medium, samples were taken from the medium and analyzed using LC-MS against human standard ISA247 metabolite profiles to identify and quantify the collected metabolites. To screen for improved media composition, each strain was first tested through a 96 hour bioconversion cycle, followed by two strains with the highest combination of metabolite conversion and metabolite diversity in Phase III with different media compositions.

Phase I / S II Phase method

Each microbe tested was maintained on culture specific agar slants. All slants were prepared one month earlier to prevent contamination. The prepared agar medium was autoclaved at 123 ° C. and subjected to a partial pressure of 380 mm Hg for 58 minutes, cooled slowly, and 6 mL was taken and pipetted into a 16 × 125 mm sterile culture test tube. After the agar was placed in the test tube, the test tube was placed in an inclined state to make a slant, cooled until the agar solidified, and then incubated at 27 ° C. for 1-2 weeks. Spores of ATCC 11635 and ATCC 53771 were made using ISP agar (0.4% yeast extract, 1% malt extract, 0.4% dextrose and 2% granulated agar). ATCC 53828, UAMH 8717 and UAMH 8718 produced spores using potato dextrose agar (PDA, 3.9% in distilled water). UAMH 4145, UAMH 7369, UAMH 7370 and UAMH 9191 are suitable for grain slant (10 %% mixed grain, dry, preferably baby nutrition; 2% granular agar; prevents agglomeration of grains and improper distribution of agar in the slant) Spores were produced by mixing the grains before and after sterilization to prevent them from becoming. After incubation for two weeks, each microorganism was inoculated onto a microorganism-specific agar and kept at 27 ° C. Once the colonies were completely covered on the slant, the slant was preferably used immediately on phase I or stored at 4 ° C. if necessary.

Subsequently, 2 mL of sterile Phase I medium (containing ISP seed broth: 1%, dextrin, 1% glucose, 0.27% beef extract, 1% yeast extract, 0.004% magnesium sulfate and 0.036% potassium diphosphate) , pH 7.0) was added to the source slant containing the microorganisms to be tested. Using a sterile inoculation loop, colonies were removed, vortexed and this suspension obtained was added to 50 mL of sterile Phase I medium contained in a sterile 250 mL volume of bafflelin culture flask. Each flask was incubated for 96 hours to increase biomass before adding ISA247 on phase II.

To prepare the biomass for transfer to phase II, the cells were washed thoroughly to remove phase I residues. Phase I contents were aseptically transferred to 50 mL conical centrifuge tubes, centrifuged at 3300 rpm for 5 minutes and then decanted to allow for supernatant to be removed. 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 then centrifuged again at 3300 rpm for 5 minutes before decanting the supernatant. And 5 mL of fresh Phase II medium was added. The resulting mixture was vigorously vortexed and aseptically transferred to 50 mL of sterile Phase II medium in a culture flask equipped with a 250 mL baffle. A portion of ISA247 (0.5 mL, 33.75% ethanol (95%): 56 mg / mL ISA247, predominantly in trans form in 66.25% glycerol) was added to the medium and the mixture was shaken vigorously. Sample aliquots were taken (0.5 mL, 12 hour increments over 96 hours of Phase II fermentation) and stored at −80 ° C. until LC-MS analysis was performed. After 96 hours fermentation was complete.

My III Prize

In phase III, ATCC 53771 and ATCC 11635 strains were incubated for 96 hours in medium C (2% glucose, 2% starch, 0.5% yeast extract, 2% soy protein, 0.32% CaCO ₃ , 0.25% NaCl) as seed broth. Medium 3 (2% glycerol, 0.5% peptone, 0.5% yeast extract, 0.2% beef extract, 0.1% (NH ₄ ) ₂ HPO, 0.1% CaCO ₃ , 0.3% NaCl, 0.03% MgSO ₄ as a separate replication attempt. -7H ₂ O, pH 7.0) or aseptically in medium 16 (2% glucose, 1% glycerin, 1% peptone, 1% meat extract, 2% soy protein, 0.5% CaCO ₃ , 0.5% NaCl, pH 7.0) Added. Since the fermentation medium is very viscous, great care was taken when sampling for LC-MS analysis.

Table 5: Example  Badges and Agar Summary Table 5 Used  ATCC 11635  ATCC 53771  ATCC 53828  UAMH 4145  UAMH 7369  UAMH 7370  UAMH 8717  UAMH 8718  UAMH 9191     badge  ISP 2  ISP 2  ISP 2  ISP 2  ISP 2  ISP 2  ISP 2  ISP 2  ISP 2  Badge 3  Badge 3  ATCC 11635  ATCC 53771  ATCC 53828  UAMH 4145  UAMH 7369  UAMH 7370  UAMH 8717  UAMH 8718  UAMH 9191  Badge 16  Badge 16  Agar  ISP  ISP  PDA  CER  CER  CER  PDA  PDA  CER

LC - MS On by Metabolite  analysis

Samples stored at −80 ° C. were thawed and labeled in a 16 × 10 mm culture test tube to indicate samples to be analyzed. A 200 μL aliquot was taken from each 0.5 mL of sample and 25 μL of a 1 mg / mL solution of CsA (cyclosporin A) was added as internal standard. 2 mL of HPLC-gradient methanol was added to each sample and the sample capped and shaken for 20 minutes. Samples were centrifuged at 3300 rpm for 1 minute 45 seconds. The supernatant was decanted into clean 16 x 10 mm incubation test tubes labeled and concentrated in vacuo to remove organic solvents. The dried layer containing both metabolite and parent drug was reconstituted in 200 μL HPLC-grade methanol and quantitatively transferred into an auto sampler vial. The sample was treated for 15 minutes in deionized water with 0.01% acetic acid / sodium acetate and the analysis was initiated with a 12 minute gradient with increasing m-TBE (methyl tertiary butyl ether) and HPLC grade methanol.

6 summarizes typical metabolite values, corresponding quantifiable ISA247 metabolites and approximate retention times from samples collected from human participants. Quantified ion masses included 1223, 1237, 1239, 1253, 1255, 1267, and 1271. Note that in Example 1 three diols (IM1-d-1, IM1-d-2 and IM1-d-3) were detected while here two diols (IM1-d-1 and IM1-d-4) were detected. do. This difference is because the trans form of ISA247 is metabolized to IM1-d-1 and IM1-d-4, while the cis form of ISA247 is metabolized to IM1-d-2 and IM1-d-3. Since the parent compound used in Example 1 is a 50:50 mixture of cis and trans, and the parent compound in this example was mainly trans-ISA247, the metabolite profile with respect to diol is different.

 Ion mass  Metabolites from ISA247  Approximate residence time (min)  1223  IM4n  9.789  1237  ISA247  10.206  1239  1239  8.340  1253  IM1-c-1; IM9; Im4  8/575; 8.939; 9.440  1255  1255  8.899  1267  CSA (internal standard)  10.678  1271  IM1-d-1; IM1-d-4  7.535; 8.166

The relative% conversion from the area under the curve (AUC) and the area under the internal CsA standard curve of each quantifiable metabolite detected was calculated using the following equation:

% Conversion rate = ( Metabolite AUC ) / ( CsA  Internal standard AUC ) x 100

result

Metabolic diversity after 96 hours of bioconversion is summarized in Table 7. A check mark indicates that the metabolite was produced in quantifiable quantities. Table 8 shows the amount of each metabolite produced in each microorganism tested.

Thus, among all the microorganisms tested in this experiment, ATCC 11635 showed the highest percentage of conversion and the largest metabolic diversity. Eight known human ISA247 metabolites were detected in ATCC 11635 samples. UAMH 4145 produced six of eight metabolites. Due to its unique ability to mass produce IM4n (6.66%), ATCC 53771, which is frequently used in experiments, produced five of eight human metabolites. ATCC 53828 produced four of eight metabolites; Although each of these metabolites was produced in small quantities, 1239 rare metabolites were produced. UAMH 7369 and UAMH 7370 produced four metabolites, respectively. UAMH 9191 and UAMH 8718 produced six metabolites. UAMH 8717 produced three metabolites.

Another factor to consider in microbial selection is the presence of the "rare" metabolite of ISA247. "Rare" is defined as being a metabolite that is not produced in large quantities by ATCC 11635, such as, for example, IM1-d-4, 1239 and 1255. IM1-d-4 was present in ATCC 11635, UAMH 8717 and UAMH 9191, but was produced in the highest amount by ATCC 11635. The microbial strains ATCC 11635, ATCC 53771, ATCC 53828, UAMN 7370, UAMH 8717, UAMH 8718 and UAMH 9191 produced 1239. The highest microbe produced was UAMH 8717. Metabolites corresponding to ion 1255 were produced by ATCC 11635, UAMH 4145, ATCC 53771, UAMH 8717 and ATCC 11635 exhibited maximum conversion.

In the Phase III experiment of this experiment, ATCC 11635 and ATCC 53771 were incubated in medium 3 and medium 16 and the influence of the medium was compared. 7 shows the results of ATCC 11635. Medium 3 and medium 16 produced similar amounts of each metabolite except IM1-d-1, IM1-d-4 and IM1-c-1. IM1-d-1 production decreased for medium 3 and was not present at detectable concentration in medium 16. IM1-d-4 production was reduced in both media 3 and media 16. IM1-c-1 production was increased by 10% in medium 3. 8 is a graph showing the effect of media composition on the production of ISA247 metabolite in ATCC 53771. IM1-d-1 was detected only when using ISP2 medium and IM9 and IM4 were detected only when using medium 3 and medium 16. Most of the metabolites except IM1-d-4 were increased in amounts using medium 3 and medium 16. Thus, the growth medium of microorganisms can be altered to optimize the bioconversion effect.

Example  6

Beauvaria Bassiana  ( Beauvaria bassiana Biotransformation using on  Effect of surfactants and solvents in

40을 사용하여 비교하였다. 56 mg/mL의 ISA247 용액 (글리세롤 대신 33.75% 에탄올 (95%):66.25% DMSO 또는 트윈

40 중) 0.5 mL를 50 mL의 배지에 첨가하였다. 동일한 제I상 및 제II상 배지와 실시예의 과정은 다음과 같았다.In Example 5, Beauvaria bassiana : UAMH 8718) produced only a small amount of the ISA247 metabolite. To see if surfactants and solvents could change the production profile, dimethyl sulfoxide (DMSO) and tween instead of glycerol (used in Example 5)

40 was used for comparison. 56 mg / mL ISA247 solution (33.75% ethanol (95%) instead of glycerol: 66.25% DMSO or Tween

0.5 mL of 40) was added to 50 mL of medium. The procedures of the same phase I and phase II media and the examples were as follows.

40을 사용한 경우에는 IM1-d-1, IM1-d-4, 1255, IM4n, IM9 및IM4가 생산되었다. 전환량 역시 극적으로 변하였다. 결과적으로, 생물전환의 결과는 모화합물을 전달하기 위해 사용되는 용매나 계면활성제의 종류를 변화시킴으로써 최적화할 수 있으며, 당업자라면 대사산물의 조합의 주어진 대사산물의 생산량을 증가시키기 위해 특정 용매, 계면활성제 및 배지를 선택할 수 있다.As shown in Figure 9, IM1-d-4, 1239 and 1255 were produced when using glycerol as a surfactant; With DMSO, IM1-d-1, IM4n, and IM4 were produced and twin

When 40 was used, IM1-d-1, IM1-d-4, 1255, IM4n, IM9 and IM4 were produced. The amount of conversion also changed dramatically. As a result, the results of bioconversion can be optimized by varying the type of solvent or surfactant used to deliver the parent compound, and those skilled in the art will recognize that a particular solvent, interface can be used to increase the yield of a given metabolite of a combination of metabolites. Active agents and media can be selected.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety, as if individually and specifically indicated that the entire contents are incorporated herein by reference.

The present invention has been described with reference to some embodiments of the present invention. However, various other modifications are possible without departing from the spirit and scope of the invention.

Claims (18)

The following steps, namely: (a) providing a mixture of a bioforeign compound and a surfactant; (b) adding the mixture to the microbial culture; And (c) incubating said culture for a period sufficient to produce said metabolite, wherein said method comprises producing at least one metabolite of said bioforeign compound. The method of claim 1, wherein the mixture contains a bioforeign compound, a solvent, and a surfactant. The method of claim 2, wherein the solvent is an alcohol. The method of claim 3, wherein the alcohol is ethanol. 5. The method of claim 1, wherein the solvent contains both alcohol and DMSO. 6. 6. The microorganism of claim 1, wherein the microorganism is an Actinoplanes strain. sp . ), Streptomyces griseus ), Streptomyces setonii ) and Saccharopolyspora erthyraea . 6. The microorganism of claim 1, wherein the microorganism is Cunninghamella. echinulata ). The method according to any one of claims 1 to 5, wherein the microorganism is Nerospora. crassa ). 6. The microorganism of claim 1, wherein the microorganism is an Actinoplanes strain. sp .). The method of claim 1, wherein the surfactant is PEG 400, castor oil, isopropyl myristate, glycerin, cremophor

, Labrasol

, And twin

And from the group consisting of 40. The method of any one of claims 1 to 10, wherein the bioforeign compound is selected from the group consisting of immunosuppressive agents and antibacterial compounds. The method of claim 11, wherein the bioforeign compound is a cyclosporin compound. The method of claim 12, wherein the cyclosporin compound is ISA247. The method of claim 12, wherein the cyclosporin compound is cyclosporin A. The metabolite of claim 1, 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 microorganism of claim 1, wherein the microorganism is Saccharopolyspora. erthyraea : ATCC 11635). The method of claim 1, further comprising the step of separating the metabolite from the culture. A method of identifying a microorganism suitable for use in a bioconversion system, comprising: a) comparing the structure of a compound to be metabolized with known enzyme activity; b) identifying an enzyme that expresses known enzymatic activity; c) identifying microorganisms expressing the identified enzymes; And d) using a microorganism expressing the identified enzyme in a bioconversion system to make a metabolite of said compound.

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