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WO1998024316A1 - Composes et extrait d'agrume diriges contre l'effet de premier passage - Google Patents

Composes et extrait d'agrume diriges contre l'effet de premier passage Download PDF

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WO1998024316A1
WO1998024316A1 PCT/US1997/023033 US9723033W WO9824316A1 WO 1998024316 A1 WO1998024316 A1 WO 1998024316A1 US 9723033 W US9723033 W US 9723033W WO 9824316 A1 WO9824316 A1 WO 9824316A1
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optionally substituted
optionally
citrus
furocoumarin
compound
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James W. Harris
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones

Definitions

  • the present invention relates to anti-first-pass effect compounds.
  • the invention also relates to a citrus (most preferably lime and grapefruit) extract, oil, concentrate juice, byproduct, etc. having reduced levels as compared to naturally-occurring levels of phototoxic furocoumarins and/or containing enhanced amounts over naturally-occuring amounts of one or more invention anti-first-pass effect compounds.
  • the invention compounds and extracts are provided as a dietary supplement or as a medical food or as some other type of food product, or as a drug, pharmaceutical or drug preparation, or in some other physical form, and that, in addition to any other function they have, if any, function as an inhibitor of the first-pass effect of orally-administered drugs.
  • the preferred embodiment of the invention relates to the particular chemical species responsible for the herein observed inhibition of the first-pass effect.
  • the "first-pass effect" of drugs given orally refers to the process of drug degradation during a drug's transition from initial ingestion to circulation in the blood stream. Often discussed in terms of “bioavailability", it is not uncommon for a drug that is administered to a patient orally to be given in a 5-fold or greater amount than ultimately necessary due to the degradation that occurs in the patient's body after intake.
  • the impact of the first- pass effect can be demonstrated with the case of the antihistamine terfenadine, wherein 99.5% of a tablet given by mouth is quickly changed to metabolites; hence, the bioavailability of terfenadine is approximately 0.5% (D.
  • cyclosporin A administered to organ transplant patients, has a median oral bioavailability of approximately 30% and a bioavailability range of approximately 8-92% among patients. Because of this large interindividual variation in cyclosporin bioavailability, frequent monitoring of blood concentrations during therapy initiation is necessary.
  • the inhibition of a particular xenobiotic metabolism as a mechanism of action generally, as well as the inhibition of the first-pass effect with chemical agents specifically, is well known in the art and has been for some time.
  • Examples include the treatment of methanol (wood alcohol) poisoning with ethanol and the inhibition of the first-pass effect of cyclosporin with ketoconazole. See, for example, First, R.M. et al., The Lancet, 1198, November 18, 1989, incorporated herein by reference.
  • the drugs fluconazole, ritonavir, itraconazole, miconazole, erythromycin and troleandomycin have been identified as inhibitors of the first- pass effect.
  • These compounds are antiviral, antimicrobial, or antifungal agents. Because of the heightened current awareness of the fact that overuse of such agents can result in resistant microbial strains, because some of the most effective inhibitors are antimicrobials, and because transplant and HIV-infected patients have compromised immune systems, the use of these inhibitors of the first-pass effect has significant drawbacks and, for example, in the case of ketoconazole, the purposeful co-administration of this inhibitor with drugs susceptible to the first-pass effect has not become widespread.
  • grapefruit juice in particular and citrus products in general are known to contain phototoxic furocoumarin derivatives including psoralen, xanthotoxin and bergapten. While these compounds are useful for the controlled, clinical treatment of selected dermatological diseases including vitiligo, psoriasis and mycosis fungoides, they are also known to be toxic, in particular, phototoxic.
  • the structure-activity relationship for the phototoxicity of furocouma ⁇ ns has been clearly delineated from human studies (for example, L. Musajo et al, Herba Hungarica, 1971 , Tom. 10, No. 2-3, pp. 79-94), and these studies show that photosensitizing activity is removed by ring hydroxylation or by lengthening the alkyl- chain length of ether substituents.
  • Figure 1 ' shows the first-pass effect inhibiting ability of invention citrus-derived substances as compared to ketoconazole.
  • Figures 2', 3', 4' and 5' show the first-pass inhibiting ability of various materials.
  • Figures 6' and 7' show the proton and carbon NMR spectrum of Compound 4, respectively.
  • Figure 8' shows the carbon NMR spectrum of Compound 12.
  • Figure 9' shows the UV absorption spectrum of Compounds 4 and 12.
  • Figure 10' shows the first-pass inhibiting ability of Compound 12 and its degradation products.
  • Figure 1 1' shows the first-pass inhibiting ability of Compound 4 and its degradation products.
  • Figures 12'- 15' show various mass spectral analyses of Compounds 4 and 12.
  • a citrus-based (preferably lime- or grapefruit-based or derived) composition that inhibits the first-pass effect and that, optionally, is free of or contains a reduced amount (as compared to a naturally occurring amount) of phototoxic and, optionally, non-first-pass inhibiting low molecular weight furocoumarins.
  • Another object of the present invention is to provide a citrus-, preferably lime- or grapefruit-based composition that is effective against the first-pass effect.
  • Another object of the present invention is to provide a citris-, preferably lime- or grapefruit-based composition that contains no, or reduced amounts as compared to naturally occurring amounts, of phototoxic low molecular weight furocoumarins.
  • Another object of the present invention is to provide a citrus-, preferably lime- or grapefruit-based composition with consistent and reliable first-pass inhibiting activity.
  • Another object of the present invention is to provide the above-described compositions as a component of products that provide active ingredients, therapeutic agents, drugs, etc. or other substances that are subject to the first-pass effect in humans.
  • Another object of the present invention is to provide first-pass effect inhibiting compounds, also called bioenhancers and inhibitors herein.
  • Another object of the present invention is to provide mixtures of one or more invention first-pass effect inhibiting compounds with various therapeutic agents, active agents, drugs or other substances (hereinafter referred to as "drugs") that are subject to the first-pass effect.
  • Another object of the present invention is the provision of a method for inhibiting the first-pass effect in patients taking drugs having a first-pass effect.
  • Another object of the present invention is a method for preparing the above-described compositions, compounds and mixtures.
  • Another object of the present invention is a method for preparing a citrus-based composition containing no or reduced amounts as compared to naturally occurring amounts of phototoxic and non-first-pass inhibiting furocoumarins preferably using reagents that the U.S. Food and Drug Administration regards may be used for food or drug manufacturing including GRAS materials.
  • non-first-pass inhibiting includes first-pass activity provided by 2000 nM bergamottin and imperatorin according to Protocol C herein. See Figure 2'.
  • Another object of the invention is to provide and use first-pass effective compounds (dimers and trimers) and compositions in isolated form and/or pyrogen-free form and/or sterile form and/or substantially pure form and/or pharmaceutical form and/or chemically pure form. These forms are different from the invention dimers and trimers as they naturally occur in citrus and citrus products such as juice, commercially available juice concentrates, etc.
  • Another object of the invention is to provide a method of inhibiting the first-pass effect by administration of the above compounds and compositions to humans.
  • the citrus-based compositions of the present invention contain at least one first-pass effective citrus-derived extract, concentrate, peel, juice, oil, by-product, etc., (hereinafter referred to as the citrus-derived substance) and may be provided by any combination of these forms and may be derived from more than one citrus fruit.
  • Useful citrus fruits herein include grapefruit, lemon, lime and any citrus fruit containing a first-pass effect inhibiting compound or mixture of compounds. Prior work in the field indicates that a common type of orange (Citrus sinensis) does not inhibit the first-pass effect.
  • Citrus fruits that contain one or more substances that inhibit the first-pass effect are included in the invention, including all cross breeds, etc. and are referred to herein as "first-pass citrus".
  • a preferred citrus fruit useful in the present invention is grapefruit.
  • Another is lime.
  • First-pass effective materials described herein are materials that prevent or retard the degradation of orally administered drugs in the body.
  • the first-pass effective materials of the invention including invention compounds, increase drug bioavailability by at least 1%, preferably by more than 5% and most preferably by more than 15% including 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, etc. percent as measured by the Area Under the Curve (AUC) method.
  • AUC Area Under the Curve
  • Preferred citrus-derived substances of the invention include cold-pressed citrus oil, particularly cold-pressed grapefruit, lime, lemon, etc., oil, and citrus by-products including tailings from citrus packing/juice plants.
  • Cold pressed citrus oils including cold-pressed orange (except Citrus sinensis), grapefruit, lime and lemon oil, are commodities and are described, for example, in the Food Chemicals Codex, Fourth Edition. National Academy Press, Washington, D.C. 1996, incorporated herein by reference.
  • Other citrus-derived substances useful herein include the various other citrus oils (distilled, essential, desert type, etc.), bitter cold-pressed oils, etc. Geographical origin of the invention citrus providing the citrus-derived substance is unimportant herein.
  • Citrus juices or peel (rind) may also be used, as well as any first-pass effective solid, semi-solid or liquid portion of a first-pass citrus. Mixtures may be used.
  • the citrus-derived substance of the present invention compositions may make up the entire citrus-based composition or may be only a part thereof. Food grade or pharmaceutically acceptable diluents, excipients, carriers, etc., may be added, if desired.
  • the citrus-derived substance of the present invention composition is preferably treated so as to reduce the amount of phototoxic and, optionally, non-first-pass effective, furocoumarins naturally present therein.
  • these furocoumarins are completely removed, meaning that they are removed to an extent such that their presence is undectable by liquid and, preferably, gas chromatography.
  • the invention for removing phototoxic low molecular weight furocoumarins from invention citrus-derived components preferably comprises optional removal of volatile components (components removed after 12-24 h at a pressure of 10 "2 -10 "3 Torr) and extraction with mixtures of at least one C,-C 10 alcohol (preferably ethanol) and water.
  • C,-C 10 alcohol preferably ethanol
  • the extraction mixture of alcohol and water is discarded and what is left is useful herein.
  • C 2 -C 5 alcohols are also preferred as are C 2 and C 3 and C 4 alcohols.
  • the alcohol (ethanol) may either be 100% alcohol are may be conveniently supplied and used in commonly available alcohol-water dilutions (e.g., 95% ethanol/5% water, etc.). In all cases the alcohol (ethanol) reagent is preferably U.S. P. grade or better.
  • the water used herein for extracting the invention citrus- derived substance (component) is preferably distilled water, and is also preferably U.S. P. grade or better. Any combination of solvents or single solvent may be used herein for extraction.
  • the solvent(s) are preferably FDA acceptable for food and drug manufacturing.
  • the present invention method for removing phototoxic low molecular weight furocoumarins may include successive extractions with alcohol (ethanol)/water mixtures, and the successive alcohol (ethanol)/water mixtures used may be either of the same volume ratio or different volume ratios.
  • Preferred alcohol (ethanol): water volume ratios range from 1 : 10- 10: 1, are more preferably 1 : 1 ( ⁇ 3%, 5%, 8% or 10%) and may be 45-60% alcohol (ethanol) on a volume/volume basis, and include 2: 1, 3:1, 1 :2, 1 :3, etc. as well as 55/45, 60/40, 65/35, 70/30. 45/55, 40/60, 35/65, 30/70, etc.
  • the extractions may be accomplished by any method known in the art including liquid-liquid extraction, liquid-solid extraction, etc.
  • the raw material used to prepare the invention citrus-derived extract is, for example, an oil
  • the alcohol (ethanol) ⁇ vater mixture used for extraction can be simply added thereto, shaken therewith, and separated naturally or with the help of a centrifuge. Repeated extraction is helpful, as are continuous extraction methods such as countercurrent extraction, etc.
  • the invention method for removing phototoxic low molecular weight furocoumarins significantly diminishes, and preferably completely removes beyond the detection limits of liquid and, preferably, gas chromatography, methoxy-substituted linear and angular furocoumarins including xanthotoxin (8-methoxypsoralen), bergapten (5- methoxypsoralen), isobergapten, isopimpinellin, etc., and unsubstituted linear and angular furocoumarins (psoralen, angelicin, etc.).
  • Furocoumarins that have been determined herein to be ineffective first-pass effect furocoumarins may also be removed, if desired.
  • These compounds include bergamottin, psoralen, angelicin, isopimpinellin and imperatorin.
  • invention citrus-derived substance, invention compositions, invention mixtures, invention pharmaceutical compositions, etc. preferably contain at least one first-pass effective dimer of formula I:
  • S-A-L-B (I) where S is a side chain, A is a coumarin or furocoumarin, L is a linker and B is a furocoumarin, wherein, in general one or more of the hydrogen atoms in the above formula may be replaced by one or any combination of two or more of hydro xy, halogen, linear or branched C,-C 40 hydrocarbon, C,-C 40 linear or branched ether (-OR where R is linear or branched hydrocarbon), C,-C 40 alkylhydroxy (-ROH where R is linear or branched hydrocarbon and OH is bonded to a primary, secondary, or tertiary carbon), etc.
  • hydrocarbon means branched and linear alkyl and branched and linear alkenyl.
  • Alkenyl is any hydrocarbon with at least one double bond but including multiple conjugated and nonconjugated double bonds.
  • Compounds according to formula I, optionally substituted as above, wherein one or more of the ring double bonds is hydrogenated are also included herein as are all salts and stereoisomers.
  • the dimers described above and described more particularly below may be synthesized by any general technique known in the art, and their synthesis is within the skill of the ordinary artisan in this field. They can also be isolated from a citrus-derived substance as shown herein.
  • the present invention citrus-derived substance, mixtures, pharmaceutical compositions, etc. preferably contain at least one dimer according to formula I above. Mixtures may be used.
  • S in formula I above is preferably a high carbon number ether substituent which may be depicted chemically as -OR where R is a C C 40 hydrocarbon group which is optionally substituted with, e.g., OH and/or interrupted by an ether oxygen -0-, preferably a C 3 -C 40 optionally hydroxyl, etc. substituted and/or ether interrupted hydrocarbon group including terpenoid chains, etc., containing 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. carbon atoms.
  • S may more generally be depicted as H or -YR where Y is O, CO, COO, HCOH, sulfur, NH, NR or CH 2 and R is defined as above. It is highly preferred that the side chain S have a molecular weight of 153 excluding the linkage group or atom to A (e.g., Y).
  • the side chain S (e.g., -OR, -YR) also may optionally be terminated by an oxygen containing functionality such as an epoxide, hydroxyl, etc. This terminal function is included in the preferred 153 molecular weight. S preferably contains oxygen.
  • a in formula I is a coumarin:
  • A is a furocoumarin, it is one of three common naturally occurring furocoumarins:
  • the furan ring of the furocoumarins are preferably not substituted and are not bonded to S or L at the 5-membered furan ring.
  • the A coumarins and furocoumarins may be optionally substituted as described above and preferably with, e.g., C
  • B in formula I is a furocoumarin, as described above for A, and may be the same or different from A.
  • L is a linker preferably depicted as -Y-T-Y- where T is preferably a C,-C 40 hydrocarbon optionally substituted with OH, optionally interrupted by an ether function -O- or CO, COO, etc.
  • T is preferably terpenoid (see, e.g., Von Sydow, E., Acta Chemica Scandinavica, 17 (1963) 2504-2512, incorporated herein by reference) and preferably has a molecular weight of 153.
  • Y in -Y-T-Y- is O, CO, COO, HCOH, sulfur, NH, NR or CH 2 and preferably contains oxygen.
  • the two most preferred dimers according to formula I have molecular weights of 708 and 668.
  • the 708 dimer has two furocoumarin rings.
  • the 668 dimer has a furocoumarin ring and a coumarin ring.
  • the 668 dimer is referred to as fraction 4, compound 4, dimer 4, product 4 or Sample 4 herein.
  • the 708 dimer is referred to as fraction 12, compound 12, product 12, Sample 12 or dimer 12 herein.
  • the UV, ,3 C NMR and ⁇ NMR spectra of these two preferred dimers are included in the Figures.
  • trimer includes at least one furocoumarin.
  • the furan ring of at least one of the furocoumarins is preferably unsubstituted and unbonded to L or S.
  • a and L are as defined above for invention dimers.
  • the A groups in the trimer may be the same or different from one another.
  • compositions of the invention contain, preferably, more of the invention compounds, inhibitors, dimers, trimers, etc., than naturally present in citrus products. Dosages are determinable by those of ordinary skill in the art and depend upon the extent to which a, e.g., active agent (drug) is subject to the first-pass effect, etc.
  • the invention citrus- derived substance, compositions, mixtures and dimers may optionally be part of or added to a citrus-based composition or other edible material which is preferably a taste-masking flavor, juice, etc.
  • the citrus-derived substance, mixtures, compositions and dimers of the invention inhibit the first-pass effect of drugs taken orally by humans and other animals.
  • a "first-pass effective amount" of an invention material is any amount which reduces the first-pass effect of any substance by any amount (e.g., 1%, 5%, 10%), etc.: see above where the AUC method is described, including all values and ranges between these values) as compared to the case where no invention material is administered.
  • a "first-pass effective" invention citrus-derived substance, mixture, composition or dimer is a material that inhibits the observed first-pass effect of at least one drug, preferably the first-pass effect caused by the cytochrome P450 system. This is also referred to herein as anti-first-pass activity.
  • Administration is preferably co-administration, meaning just before, just after, or with drug, active agent, therapeutic agent, medical food subject to the first-pass effect, etc. "Just before” and “just after” include all times where the invention material provides a benefit by inhibiting the first-pass effect.
  • Preferred forms of the invention comprise the invention citrus-derived substance, mixture, composition or dimer inside of, e.g., a gel capsule, or co-formulated with food-grade or pharmaceutically-acceptable binders, diluents, etc.
  • Dosage forms salt or base, tablet or gum, etc.
  • binders, salt forms, excipients, etc. useful are found in, e.g., U.S. Patent Nos. 5,576,448, 5,576,446, 5,576,437, 5,576,439, 5,576,438, 5,576,337, 5,576,339 and 5,576,336, all incorporated herein by reference.
  • the invention citrus-derived substance, mixtures, compositions and dimers are preferably provided in an amount that provides consistent, reliable potency from batch to batch regardless of the form in which it is provided.
  • drug as used herein is defined as a chemical capable of administration to an organism which modifies or alters the organism's physiology. More preferably the word “drug” as used herein is defined as any substance intended for use in the treatment or prevention of disease, particularly for humans. Drug includes synthetic and naturally occurring toxins and bioaffecting substances as well as recognized pharmaceuticals, such as those listed in "The Physicians Desk Reference,” 47th edition, 1993, pages 101-321 ; “Goodman and Gilman's The Pharmacological Basis of Therapeutics” 8th Edition (1990), pages 84-1614 and 1655-1715; and "The United States Pharmacopeia, The National Formulary", USP XXII NF XVII (1990), the compounds of these references being herein incorporated by reference.
  • the term drug also includes compounds that have the indicated properties that are not yet discovered or available in the U.S.
  • the term drug includes proactive, activated and metabolized forms of drugs.
  • the present invention can be used with drugs consisting of charged, uncharged, hydrophilic, zwitter-ionic, or hydrophobic species, as well as any combination of these physical characteristics.
  • a hydrophobic drug is defined as a drug which in its non-ionized form is more soluble in lipid or fat than in water.
  • a hydrophobic drug is defined as a drug more soluble in octanol than in water. See U.S. Patent No. 5,567,592, incorporated herein by reference.
  • the present invention citrus-derived substance and invention dimers may be co- formulated with drugs, preferably drugs that are subject to the first-pass effect.
  • the drug has an oral bioavailability of 90%) or less, more preferably 50%o or less.
  • examples include, in addition to those incorporated by reference above, ritonavir, saquinavir, indinavir, L-deprenyl, tacrolimus, cyclosporin A (Sandimmune®), cyclosporin A (Neoral®), nelfinavir, VX-478/141 W94, felodipine, nifedipine and sumatriptan.
  • Such co-formulations include the invention citrus-derived substance and/or one or more dimers in amounts mentioned above with, typically, lesser amounts than currently necessary of drug active ingredients that are subject to the first-pass effect. Binders, diluents, etc. acceptable for pharmaceutical use can also be added.
  • One of ordinary skill in the art is capable of determining the dosage of the invention citrus-derived substance and/or dimer and drug active ingredient based on simple testing procedures well known in the art and including pharmacological experiments which determine the amount of drug in the blood stream over a given time period after administration.
  • citrus-derived components of the invention including cold-pressed citrus oils should preferably be assessed before they are further used in the production of, e.g., compositions of dietary supplements.
  • One strategy consists of sample preparation (Protocol A, this document), followed by chromatography (Protocol B, this document), and ending with comparisons to historical standards. Such assessment can provide consistent batches.
  • Protocol A Preparation of citrus oils for administration to humans by removal of toxic, low molecular weight furocoumarins.
  • a volume of cold-pressed citrus oil (Food Chemicals Codex grade) was transferred to a container, and all volatile materials were removed.
  • the preferred method uses Speed Vac concentrators (Savant Instruments: process requires 12-24 h and pressures of 10 "2 -10 "3 torr, and the system is run without added heat) because this method is gentle and expedient.
  • the nonvolatile product yield is generally 0.04 to 0.1 times the initial volume and is a viscous liquid.
  • Protocol B Chromatography methods for processed citrus oils
  • Protocol A The product of the above Protocol A is not suitable for any high pressure liquid chromatography because of the substantial presence of materials that are not soluble in the preferred mobile phase systems.
  • sample preparation protocol that follows is used prior to chromatography.
  • One volume of the product of Protocol A is mixed with four volumes of acetonitrile, the container is capped, the solution is mixed vigorously, the container is centrifuged (5 min at setting 35), and the top acetonitrile layer is filtered through a 0.22 micron teflon membrane.
  • the filtered solution is stored in a closed container at -20C for 2 days or more and then is passed through filter paper while cold to remove a copious precipitate. The precipitation and filtration step is repeated once.
  • the volume of the acetonitrile solution is noted, and the acetonitrile is removed using a Speed Vac apparatus. The residue is dissolved in half the original volume of acetonitrile, taking care not to disturb any crystalline precipitate, and the solution may now be used for HPLC assessment.
  • the chromatographic column has dimensions of 250 mm length x 4.6 mm internal diameter, is packed with C18 bonded to 4 micron silica particles (9% carbon load; ODS-L80, YMC, Inc.), is protected with a 23 mm length x 4 mm internal diameter column containing the same material and with a 0.5 micron filter, and is maintained at 40 +/- 0.2 C.
  • the flow rate is maintained at 1.0 mL/min during the 22 min run cycle.
  • the column eluate from each 25 uL injection is monitored at 400 +/- 200 nm and at 310 +/- 2 nm and is fractionated using a robotic collector (Gilson).
  • Linear gradients are used for elution and are formed by mixing mobile phase A composed of water with mobile phase B composed of acetonitrile (instrument: Hewlett Packard).
  • the elution time, in minutes, as well as the percentage of acetonitrile present in the mixed mobile phase are as follows: 0,10; 5, 10; 30, 80; 40, 80; 41 , 95; 50, 95; 53, 10; 60, 10.
  • the chromatographic column has dimensions of 150 mm length x 2.0 mm internal diameter, is packed with C18 bonded to 4 micron silica particles (14% carbon load; ODS-M80, YMC, Inc.), is protected with a 2 mm internal diameter column packed with a proprietary material (Prism, Keystone Scientific, Inc.) and with a PTFE filter, and is maintained at 35 +/- 0.2 C. The flow rate is maintained at 0.20 mL/min during the 60 min run cycle.
  • the column eluate from each 10 uL injection is monitored for absorbance at 400 +/- 200 nm and at 310 +/- 2 nm and for fluorescence with excitation at 229 nm, emission at 450 nm, and bandpass filtration at 370 nm.
  • Protocol C Assessment of human cytochrome P450-mediated biotransformation.
  • reaction buffer is composed of 0.10 M sodium phosphate, 1.0 mM ethylenediaminetetraacetic acid, and 5.0 mM magnesium chloride, pH 7.4 (all reagents: Fisher Scientific).
  • Inhibitory chemicals used were ketoconazole (Research Diagnostics, Inc.), miconazole, bergapten, xanthotoxin (previous three from Sigma), bergamotin, imperatorin, isopimpinellin, psoralen, angelicin (previous five from Indofine Chemical Company, Inc.), and fractions or precipitates resulting from Protocols A and B above.
  • final inhibitor concentrations were expressed in molarity by calculation from the weighed material or by interpolation from HPLC calibration curves prepared with reference materials; otherwise, concentrations are expressed as weight per volume. Reaction tubes are placed on ice in preparation for the manipulations that follow.
  • Sufficient reaction buffer is added so that the final volume of each tube will be 500 uL, 5 uL of a 100-fold concentrate for generating reduced nicotinamide adenine dinucleotide phosphate is added (such that completed reaction mixture contains 1.0 mM nicotinamide adenine dinucleotide phosphate, 1 U/mL glucose-6-phosphate dehydrogenase, and 10 mM glucose-6- phosphate; all from Sigma), and then human hepatic S9 (Anatomic Gift Foundation) is thawed and added in sufficient amounts to cause readily detectable amounts of metabolites to be formed in control reactions (amount necessary varies among individuals, but 10 uL is typical).
  • Reactions are pre-incubated for 3 min at 37 C in a Dubnoff-type water bath, the reaction mixture is completed by the addition of 10 uL of 100 uM terfenadine (Sigma) dissolved in 1 : 1 acetonitrile: water and by gentle mixing, the samples are incubated for 15 min at 37 C, and the reaction is stopped by placing the tube on ice and adding 2.5 mL of 300 nM terfenadine-related compound A (internal standard; U.S. Pharmacopeia) dissolved in acetonitrile.
  • the samples prepared above are readied for HPLC assessment using the protocol that follows. Each tube is vortex mixed and centrifuged for 10 min at setting 35, the resulting supernatant is transferred to a clean tube, and the liquid is evaporated using a Speed Vac apparatus. The residue in each tube is first dissolved in 40 uL 1 :1 acetonitrile:water, 2.5 mL of acetonitrile is added, and the centrifuge-transfer-evaporate step just described is repeated.
  • the dry residue resulting from the above-described experiments and sample preparation protocol may be analyzed for terfenadine metabolites using the HPLC method described below and may also be used to quantitate the inhibitory chemicals that were added to the reaction (see Protocol B).
  • Linear gradients are used for elution and are formed by mixing mobile phase A composed of water with mobile phase B composed of 0.025% (v/v) formic acid in acetonitrile (instrument: Hewlett Packard).
  • the elution time, in minutes, the percentage of mobile phase B present in the mixed mobile phase, and the flow rate (mL/min) are as follows: 0, 10, 0.10; 2, 10, 0.10; 3.5, 10, 0.20; 4, 10, 0.25; 5, 10, 0.25; 30, 55, 0.25; 32, 98, 0.25; 33, 98, 0.40; 39.8, 98, 0.40; 40, 98, 0.25; 45, 10, 0.25; 45.25, 10, 0.20; 50, 10, 0.20; 50.25, 10, 0.10.
  • the chromatographic column has dimensions of 150 mm length x 2.1 mm internal diameter, is packed with a proprietary material (Prism, Keystone Scientific.
  • Figure 2' shows that bergamottin and imperatorin are essentially ineffective first-pass inhibitors, while invention citrus-derived extracts C and D are effective inhibitors of the first- pass effect.
  • Figure 3' shows that the phototoxic citrus-derived constituents xanthotoxin and bergapten are far less effective than invention citrus-derived substance E at first-pass inhibition.
  • Figure 4' shows that lime extracts are as effective as grapefruit extracts.
  • Protocol A' Pretreatment of citrus oils prior to chromatography.
  • Cold-pressed citrus oil Food Chemicals Codex grade
  • Food Chemicals Codex grade Cold-pressed citrus oil (Food Chemicals Codex grade) is transferred to a suitable container, and all volatile materials are removed under reduced pressure (10 ⁇ 2 -10 "3 torr, 3-4 days).
  • the nonvolatile product yield is generally only 5-10%) of the original volume.
  • the citrus nonvolatiles are mixed with acetonitrile in a ratio of 2: 1 (w/w), the mixture is centrifuged (International Equipment Company, Model K-2, 5 min at setting 35), and the upper acetonitrile-containing phase is removed.
  • the extraction with acetonitrile is repeated once, the lower phase is discarded, the first and second acetonitrile phases are pooled, and acetonitrile is removed using Speed Vac concentrators (Savant Instruments; 12 h at 10 "2 - 10 3 torr, without added heat).
  • the nonvolatile material is mixed with ethanolic base (1 : 1 ethanol:water ⁇ v/v; each U.S. P. grade ⁇ containing 12.5 g potassium hydroxide/L) in a ratio of 1 :4 (w/v), the mixture is centrifuged for 5 min at setting 35, and the upper ethanolic phase is removed and discarded.
  • the nonvolatile material is washed an additional nine times with ethanolic base and, then, once with 1 : 1 ethanohwater (v/v).
  • the residue that remains is extracted twice with sufficient volumes of acetonitrile such that all colored material is removed.
  • the acetonitrile solution is washed six times with two volumes of hexane, with each hexane extract (upper layer) being removed and discarded, and the resulting acetonitrile solution is filtered through a 0.2 micron teflon membrane and evaporated to dryness using a Speed Vac concentrator.
  • the final product of the above process should appear as a viscous, deep red oil, but seasonal variations in the starting material (citrus oils) apparently can change the quality and appearance of the product of the above process. Hence, if a copious orange crystalline material contaminates the deep red oil, then the number of additional washes with ethanolic base should be increased from nine to nineteen.
  • Protocol B' Chromatography methods for processed citrus oils
  • the chromatographic column has dimensions of 250 mm length x 4.6 mm internal diameter, is packed with C18 bonded to 4 micron silica particles (9% carbon load; ODS-L80 YMC, Inc.), is protected with a 23 mm length x 4 mm internal diameter column containing the same material and with a 0.5 micron filter, and is maintained at 40 +/- 0.2 C.
  • the flow rate is maintained at 1.0 mL/min during the 22 min run cycle.
  • the column eluate from each 25 uL injection of oil obtained by Protocol A' is monitored at 400 +/- 200 nm and at 310 +/- 2 nm and is fractionated using a robotic collector (Gilson).
  • Linear gradients are used for elution and are formed by mixing mobile phase A composed of water with mobile phase B composed of acetonitrile (instrument: Hewlett Packard).
  • the elution time, in minutes, as well as the percentage of acetonitrile present in the mixed mobile phase are as follows: 0, 10; 5, 10; 30, 80; 40, 80; 41, 95; 50, 95; 53, 10; 60, 10.
  • the chromatographic column has dimensions of 150 mm length x 2.0 mm internal diameter, is packed with C18 bonded to 4 micron silica particles (14%o carbon load; ODS- M80.YMC, Inc.), is protected with a 2 mm internal diameter guard column packed with a proprietary material (Prism, Keystone Scientific, Inc.) and with a PTFE filter, and is maintained at 35 +/- 0.2 C. The flow rate is maintained at 0.20 mL/min during the 60 min run cycle.
  • the column eluate from each 10 uL injection is monitored for absorbance at 400 +/- -2 nm and at 310 +/- 2 nm and for fluorescence with excitation at 229 nm, emission at 450 nm, and bandpass filtration at 370 nm.
  • Protocol C Chemical degradation of citrus-derived dimer inhibitors of human cytochrome P450-mediated biotransformation
  • the elution time, in minutes, as well as the percentage of acetonitrile present in the mixed mobile phase are as follows: 0, 55; 5, 55; 10, 90; 1 1, 98; 17, 98; 18, 55; 22, 55.
  • the chromatographic column has dimensions of 250 mm length x 4.6 mm internal diameter, is packed with C18 bonded to 4 micron silica particles (9% carbon load; ODS-L80, YMC, Inc.), is protected with a 23 mm length x 4 mm internal diameter column containing the same material and with a 0.5 micron filter, and is maintained at 40 +/- 0.2 C.
  • the flow rate is maintained at 1.0 mL/min during the 22 min run cycle.
  • the column eluate from each 25 uL injection is monitored at 400 +/- 200 nm and at 310 +/- 2 nm and is fractionated using a robotic collector (Gilson).
  • Protocol D' Assessment of human cytochrome P450-mediated biotransformation
  • reaction buffer is composed of 0.10 M sodium phosphate, 1.0 mM ethylenediaminetetraacetic acid, and 5.0 mM magnesium chloride, pH 7.4 (all reagents: Fisher Scientific).
  • Inhibitory chemicals used are ketoconazole (Research Diagnostics, Inc.), ritonavir (NorvirTM, Abbott Laboratories), and fractions or degradation products resulting from Protocols B' and C above.
  • reaction mixture is completed by the addition of 10 uL of 500 uM saquinavir (InviraseTM, Roche Laboratories) dissolved in 1 : 1 ethanohwater and by gentle mixing, the samples are incubated for 15 min at 37 C, and the reaction is stopped by placing the tube on ice and adding 2.5 mL of acetonitrile.
  • 500 uM saquinavir InviraseTM, Roche Laboratories
  • the samples prepared above are readied for HPLC assessment using the protocol that follows. Each tube is vortex mixed and centrifuged for 10 min at setting 35, the resulting supernatant is transferred to a clean tube, and the liquid is evaporated using a Speed Vac apparatus. The residue in each tube is first dissolved in 40 uL 1 : 1 acetonitrile:water, 2.5 mL of acetonitrile is added, and the centrifuge-transfer-evaporate step just described is repeated.
  • the dry residue resulting from the above-described experiments and sample preparation protocol may be analyzed for saquinavir and saquinavir metabolites using the HPLC method described below and may also be used to quantitate the inhibitory chemicals that were added to the reaction (see Protocol B').
  • Linear gradients are used for elution and are formed by mixing mobile phase A composed of water with mobile phase B composed of acetonitrile (instrument: Hewlett Packard).
  • the elution time, in minutes, and the percentage of mobile phase B present in the mixed mobile phase are as follows: 0, 10; 5, 10; 30, 80; 31, 95; 40, 95; 43, 10; 48, 10.
  • the flow rate is 0.2 mL/min throughout the run.
  • the chromatographic column has dimensions of 150 mm length x 2.1 mm internal diameter, is packed with a proprietary material (Prism, Keystone Scientific, Inc.), is protected with a 2 mm internal diameter column containing the same material and with a PTFE filter, and is maintained at 35 +/- 0.2 C.
  • the dry sample residue is mixed with 50 uL of 1 : 1 acetonitrile: water just prior to each 48 min run cycle.
  • the column eluate from each 10 uL injection is monitored for absorbance at 239 +/- 2 nm.
  • Protocol A Pretreatment of citrus oils prior to chromatography.
  • citrus oils are not directly suitable for long-term preparative high pressure liquid chromatography because of the substantial presence of materials that show poor solubility in the preferred mobile phase systems.
  • sample preparation protocol that follows is used prior to chromatography.
  • Cold-pressed citrus oil Food Chemicals Codex grade
  • the nonvolatile product yield is generally only 5-10%) of the original volume.
  • the citrus nonvolatiles are mixed with acetonitrile in a ratio of 2: 1 (w/w), mixture is centrifuged (International Equipment Company, Model K-2, 5 min at setting 35), and the upper acetonitrile-containing phase is removed.
  • the extraction with acetonitrile is repeated once, the lower phase is discarded, the first and second acetonitrile phases are pooled, and acetonitrile is removed using Speed Vac concentrators (Savant Instruments; 12 h at 10 "2 -10 "3 torr, without added heat).
  • the nonvolatile material is mixed with ethanolic base (1 : 1 ethanol:water ⁇ v/v; each U.S. P. grade ⁇ containing 12.5 g potassium hydroxide/L) in a ratio of 1 :4 (w/v), the mixture is centrifuged for 5 min at setting 35, and the upper ethanolic phase is removed and discarded.
  • the nonvolatile material is washed an additional nine times with ethanolic base and, then, once with 1 : 1 ethanohwater (v/v).
  • the residue that remains is extracted twice with sufficient volumes of acetonitrile such that all colored material is removed.
  • the acetonitrile solution is washed six times with two volumes of hexane, with each hexane extract (upper layer) being removed and discarded, and the resulting acetonitrile solution is filtered through a 0.2 micron teflon membrane and evaporated to dryness using a Speed Vac concentrator.
  • HPLC conditions are preferred for preparative fractionation of the washed nonvolatile portion of citrus oil (product of Protocol A).
  • Linear gradients are used for elution and are formed by mixing mobile phase A composed of water with mobile phase B composed of acetonitrile (instrument: Hewlett Packard).
  • the elution time, in minutes, as well as the percentage of acetonitrile present in the mixed mobile phase are as follows: 0, 75; 5, 75; 10, 90; 1 1, 98; 17, 98; 18, 75; 22, 75.
  • the chromatographic column has dimensions of 250 mm length x 4.6 mm internal diameter, is packed with C18 bonded to 4 micron silica particles (9%> carbon load: YMC, Inc.), is protected with a 23 mm length x 4 mm internal diameter column containing the same material and with a 0.5 micron filter, and is maintained at 40 +/- 0.2 C.
  • the flow rate is maintained at 1.0 mL/min during the 22 min run cycle.
  • the column eluate from each 25 uL injection is monitored at 400 +/- 200 nm and at 310 +1-2 nm and is fractionated using a robotic collector (Gilson).
  • Protocol C Purification of Inhibitors (of human cytochrome P450-mediated biotransformation) 01, 02. 03, 04, 05, and 06 using a chiral HPLC column
  • Protocol D Chemical degradation of citrus-derived Inhibitors 01, 02, 03, 04, 05, and 06
  • the elution time, in minutes, as well as the percentage of acetonitrile present in the mixed mobile phase are as follows: 0, 55; 5, 55; 10, 90; 1 1, 98; 17, 98; 18, 55; 22, 55.
  • the chromatographic column has dimensions of 250 mm length x 4.6 mm internal diameter, is packed with C18 bonded to 4 micron silica particles (9%> carbon load: YMC, Inc.), is protected with a 23 mm length x 4 mm internal diameter column containing the same material and with a 0.5 micron filter, and is maintained at 40 +/- 0.2 C.
  • the flow rate is maintained at 1.0 mL/min during the 22 min run cycle.
  • the column eluate from each 25 uL injection is monitored at 400 +/- 200 nm and at 310 +/- 2 nm and is fractionated using a robotic collector (Gilson).
  • dimers it is preferred that the furan ring position of the furocoumarin rings A and B be completely free of substitution and attachment to, e.g., S or L. While the dimers are preferably pure they may be mostly pure as, e.g., isolated on HPLC.
  • One preferred S group is FI.
  • a method for processing citrus and selectively removing phototoxic furocoumarins from a first-pass effective citrus-derived substance comprising the step of extracting said citrus-derived substance with a mixture of at least one C 2 -C 4 alcohol and water, said first-pass effective citrus-derived substance maintaining anti-first-pass activity after said extraction.
  • a first-pass effective citrus-derived substance which has been extracted with a mixture of at least one C 2 -C 4 alcohol and water so as to reduce the amount of phototoxic furocoumarins therein.
  • a method for inhibiting the first-pass-effect of a material taken orally by a patient which is subject to the first-pass effect comprising the step of co-administering to said patient the first-pass effective citrus-derived substance of embodiment 4.
  • CAS Registry Number The following compounds, referred to here by CAS Registry Number, are not included as part of the invention compounds, inhibitors, trimers or dimers per se but are useful herein, if they fall within formula I above or the inhibitors in Figures 1-3 or dimers or trimers or compounds, or have bioenhancer properties, in invention methods for increasing bioavailability, in invention pharmaceutical compositions, etc. They are not included in the inventive compounds, inhibitors, trimers or dimers because they are a part of the prior art. Their use in increasing bioavailability is not disclosed in the art, however.
  • inventive dimer-excluded compounds include those coumarin-furocoumarin and furocoumarin-furocoumarin dimers found in:
  • the dimers described in Yoo. D. et al. Bull. Korean Chem. Soc. 1996, 17(6), 550-553, incorporated herein by reference may be used herein.
  • the invention dimers and trimers exclude these dimers.
  • the dimers of this reference are included in invention methods, compositions, mixtures with drugs, etc. in this preferred alternate embodiment.
  • the invention is directed to a method, composition, drug mixture, etc. using a compound comprising at least one furocoumarin ring in addition to at least one further coumarin or furcoumarin ring.
  • concentration of the drug is increased by the invention dimer or trimer as compared to the concentration when the dimer or trimer is not administered.
  • invention compositions comprising invention inhibitor, dimer and/or trimer (e.g., alone, mixed with drug(s), and/or diluent(s) and/or carrier(s) etc.) preferably comprise enough inhibitor(s), dimer(s) and/or trimer(s) such that they inhibit saquinavir biotransformation according to Protocol D' above better than pure ketoconazole on an equal molar or weight concentration basis.
  • invention compositions preferably provide a Y-axis value in Protocol D' (see Figure 5') of less than 0.5, preferably 0.45, 0.4, 0.35, 0.3, 0.25, 0.22, 0.2, 0.18, 0.15, 0.12, 0.1, 0.08, 0.05 or 0.03 or less when .01-.25 mg, including .02, .04, .06, .08, .1, .12, .14, .16, .18, .2, .22 and .24, and all ranges between all values, of composition are diluted or dissolved to one liter.

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Abstract

Cette invention se rapporte à une substance sure, dérivée d'un agrume et dotée d'une efficacité d'inhibition de l'effet de premier passage, ainsi qu'à des composés, inhibiteurs, trimères et dimères qui inhibent cet effet de premier passage. L'invention se rapporte également à des formulations contenant cette substance dérivée d'agrume, des dimères et des trimères ainsi qu'à des procédés visant à inhiber l'effet de premier passage.
PCT/US1997/023033 1996-12-06 1997-12-05 Composes et extrait d'agrume diriges contre l'effet de premier passage Ceased WO1998024316A1 (fr)

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AU53823/98A AU5382398A (en) 1996-12-06 1997-12-05 Anti-first-pass effect compounds and citrus extract

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US76408196A 1996-12-06 1996-12-06
US08/764,081 1996-12-06
US4387897P 1997-04-10 1997-04-10
US60/043,878 1997-04-10

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HISHMAT et al., "Synthesis of Furodiketochroman and Bisfurocoumarin Derivatives and Their Biological Activity", (1988), Vol. 110, Abstract No. 134920. *
LIFE SCIENCE, 1996, Vol. 59, Number 13, EDWARDS et al., "Naringin and Naringenin are not the Primary CYP3A Inhibitors in Grapefruit Juice", pp. 1025-1030. *
MEDLINE, 81264759, July 1981, RITSCHEL et al., "Pilot Study on Bioavailability of Coumarin and 7-Hydroxycoumarin Upon Peroral Administration of Coumarin in a Sustained-Release Dosage Form". *
PHARMAZIE, 1994, Volume 49, SIGUSCH et al., "Influence of Grapefruit Juice on the Pharmacokinetics of a Slow Release Nifedipine Formation", pages 522-524. *
THE LANCET, 15 April 1991, Vol. 345, YEE et al., "Effect of Grapefruit Juice on Blood Cyclosporin Concentration", pp. 955-956. *

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