U.S. patent application number 10/647444 was filed with the patent office on 2004-10-28 for anti-first-pass effect compounds.
This patent application is currently assigned to BIOAVAILABILITY SYSTEMS, LLC. Invention is credited to Harris, James W..
Application Number | 20040214847 10/647444 |
Document ID | / |
Family ID | 46203267 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214847 |
Kind Code |
A1 |
Harris, James W. |
October 28, 2004 |
Anti-first-pass effect compounds
Abstract
Safe, effective first-pass inhibiting compounds and
citrus-derived substances are provided. Formulations containing the
compounds are also provided as are methods for inhibiting the first
pass effect.
Inventors: |
Harris, James W.; (Cocoa
Beach, FL) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
BIOAVAILABILITY SYSTEMS,
LLC
Cocoa Beach
FL
|
Family ID: |
46203267 |
Appl. No.: |
10/647444 |
Filed: |
August 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10647444 |
Aug 26, 2003 |
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09916500 |
Jul 30, 2001 |
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6660766 |
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09916500 |
Jul 30, 2001 |
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09644860 |
Aug 24, 2000 |
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6309687 |
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09644860 |
Aug 24, 2000 |
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09255874 |
Feb 23, 1999 |
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6054477 |
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09255874 |
Feb 23, 1999 |
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08997259 |
Dec 23, 1997 |
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6063809 |
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60056382 |
Aug 26, 1997 |
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Current U.S.
Class: |
514/278 ;
546/18 |
Current CPC
Class: |
C07D 493/20 20130101;
A23L 2/39 20130101; C07D 493/10 20130101; A61K 36/752 20130101;
C07D 519/00 20130101; A61K 31/352 20130101; A23L 33/105 20160801;
A61K 31/37 20130101 |
Class at
Publication: |
514/278 ;
546/018 |
International
Class: |
A61K 031/4747; C07D
491/12 |
Claims
What is claimed is:
1. A substantially pure compound selected from the group consisting
of compounds of 181920
2. The substantially pure compound of claim 1, wherein said
compound is a compound of Formula V.
3. The substantially pure compound of claim 1, wherein said
compound is a compound of Formula VI.
4. The substantially pure compound of claim 1, wherein said
compound is a compound of Formula VII.
5. The substantially pure compound of claim 1, wherein said
compound is a compound of Formula VIII.
6. The substantially pure compound of claim 1, wherein said
compound is a compound of Formula IX.
7. The substantially pure compound of claim 1, wherein said
compound is a compound of Formula X.
8. A substantially pure compound of claim 1, having the Formula XI
21
9. A substantially pure compound of claim 1, having the Formula XII
22
10. A substantially pure compound of claim 1, having the Formula
XIII 23
11. A substantially pure compound of claim 1, having the Formula
XIV 24
12. A substantially pure compound of claim 1, having the Formula XV
25
13. A substantially pure compound of claim 1, having the Formula
XVI 26
14. A first-pass effective composition comprising at least one
compound of claim 1 and a pharmaceutically acceptable carrier.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to anti-first-pass effect
compounds, compositions, and methods for their use, preparation,
synthesis, and formulation. Preferably, the invention compounds and
compositions 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.
In addition to any other function they have, the invention
compounds and compositions function as inhibitors of the first-pass
effect of orally-administered drugs. Beneficiaries of this
invention are animals, preferably mammals, particularly humans, who
require drugs, etc. subject to the first-pass effect.
[0003] 2. Discussion of the Background
[0004] 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. For example, 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. Garteiz et al.,
Arzneim.-Forsch., 1982; 32:1185-1190). As a further example,
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.
[0005] 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, Nov. 18, 1989, incorporated herein by reference.
[0006] Although the agent(s), enzyme type(s), biological processes,
etc. responsible for the first-pass effect have not been fully
identified, research has focused on agents capable of inhibiting
the cytochrome P450 system. Inhibition of the P450 system is a
model for in vitro determination of in vivo bioavailability
enhancement. See, e.g., U.S. Pat. Nos. 5,478,723 and 5,567,592,
both incorporated herein by reference, for a more full description
of the P450 system. As reported by A. Keogh et al. (N. Eng. J.
Med., Vol. 333, No.10, p. 628, 1995) and S. Butman et al. (J. Heart
Lung Transpl., Vol. 10, No. 3, p.351, 1991), the dose of
cyclosporin required by heart transplant patients could be reduced
by approximately 85% when cyclosporin was co-administered with
ketoconazole. In economic terms, both references estimated the cost
savings to be equal to approximately $5,000 per year per patient.
Other drugs which are subject to the first-pass effect and whose
bioavailability is increased by inhibitors commonly given to humans
include midazolam (K. Olkkola et al, Clin. Pharmacol. Ther., 1993,
53:298-305), terfenadine (Seldane.RTM.) (P. Honig et al., JAMA,
Vol. 269, No. 12, 1513, 1993) and triazolam (Varhe, A. et al, Clin.
Pharmocol. Ther., 1994, 56:601-7).
[0007] In addition to ketoconazole, the drugs fluconazole,
ritonavir, itraconazole, miconazole, erythromycin and
troleandomycin have been identified as inhibitors of the first-pass
effect, in addition to any pharmacological effect they possess.
These compounds, however, 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. In fact, the emergence
of antifungal drug resistance in immunocompromised patients is
already known (T. J. Walsh: "Emergence of Antifingal Drug
Resistance in Immunocompromised Patients" Seminar, National
Institutes of Health, Feb. 7, 1996; Georgopapadakou, N. H. et al,
Antimicrobial Agents and Chemotherapy, February 1996, p.
279-291).
[0008] Dietary supplements, medicines, compounds, extracts, etc.
that are based on materials isolated from nature are increasingly
being studied and made available to consumers. This trend is
largely due to the fact that obtaining patent protection for these
materials has become routine (see, for example, U.S. Pat. Nos.
4,708,948, 5,409,938, 5,314,899,5,591,770 and 5,654,432, all
incorporated herein by reference). Not surprisingly, this trend is
now spreading to first-pass effective agents.
[0009] In 1991, Bailey et al. reported (Bailey, D. G., et al, The
Lancet, Vol. 337, Feb. 2, 1991, p. 268, incorporated herein by
reference) that grapefruit juice increased the bioavailability of
felodipine, and indicated that the inhibition of cytochrome P450
enzymes by bioflavonoids could explain their findings. This
identification of bioflavonoids as the active ingredient in
grapefruit juice was immediately challenged by R. Chayen et al.
(The Lancet, Vol. 337, Apr. 6, 1991, p.854) who suggested that
sesquiterpenoid compounds rather than flavonoids were the active
ingredients in grapefruit juice responsible for inhibition of the
first-pass effect. Although Bailey and Edgar were granted a patent
(U.S. Pat. No. 5,229,116, incorporated herein by reference)
directed to a method of increasing the bioavailability of a
pharmaceutical agent by co-administration of a flavonoid such as
naringin, their own recent work has openly brought into question
the accuracy of their initial identification of flavonoids as
active ingredient. See, for example, Bailey et al., Clin.
Pharmacokinet. 26 (2): 91-98, 1994, particularly pages 95 and 96
thereof. See also Edwards, D. J. et al, Life Sciences, Vol. 59, No.
13, pp. 1025-1030, 1996.
[0010] The reported effects of grapefruit juice as an effective
inhibitor of the first-pass effect has lead to numerous research
articles regarding the inhibition of the first-pass effect by
grapefruit juice on, e.g., nifedipine, nitrendipine, nisoldipine,
cyclosporin A, midazolam, triazolam, coumarin, and caffeine. As
these results have become better known, the so-called "grapefruit
juice effect" has become the subject of newspaper articles,
newsletters and medical texts intended for the general public. See,
for example, "The Medical Letter", Vol. 37 (issue 955) Aug. 18,
1995, The Peoples Pharmacy, Chapter 4 (St. Martin's Press) 1996, p.
41, the Feb. 19, 1991 newspaper article regarding felodopine and
grapefruit juice in the New York Times (section C, page 3, column
1) and a recent article in the Washington Post (Section A, p. 11,
8/30/96).
[0011] A review of the published studies that demonstrate the
grapefruit juice effect also shows that the magnitude of the effect
varies widely, and it is the present inventors' suspicion that this
variation is traceable to the source of the juice. In fact, the
production of commercial citrus juice involves a complicated series
of factors that increase the variability of the final product's
composition. These factors include the squeezing technique, the
concentration technique, the origin of the fruit, the ripeness of
the fruit at harvest, the admixture of fruits differing in origin
and ripeness, the admixture of juice and fruit tailings, etc.
Because the active agents in the grapefruit juice that inhibit the
first-pass effect were unknown or misidentified, scientists and
consumers could not choose a grapefruit juice preparation and rely
upon its utility to inhibit the first-pass effect.
[0012] Moreover, 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 furocoumarins 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.
[0013] Careful evaluation of the literature shows that psoralen and
certain low carbon number ether-substituted furocoumarins that are
given to humans in large doses do inhibit cytochrome P450. See, for
example, D. Bickers et al., J. Investigative Dermatology,
79:201-205, 1982, M. Tinel et al., Biochemical Pharmacology, Vol.
36, No. 6, 951-955, 1987, H. Fouin-Fortunet et al., J. Pharm.
Experimental Therapeutics, Vol. 36, No. 1, 237-247, 1986, and D.
Mays et al, Clin. Pharmacol. Ther., 42:621-626, 1987. Thus, and
because the known successful inhibitors of the first-pass effect
generally inhibit cytochrome P450, a tempting conclusion,
particularly in view of the recent disclaimers by Bailey, and
others, is that these low molecular weight furocoumarins present in
citrus are the active first-pass inhibitors in grapefruit juice. In
fact, and as will be described more fully below with regard to the
present invention, the present inventor has found that this is not
the case. Because the present inventor has discovered specific
compounds that inhibit the first-pass effect it is now possible to
produce a reliable, safe composition that both inhibits the
first-pass effect and, if desired, that is citrus-based or of
citrus origin and which contains no or reduced amounts of low
molecular weight phototoxic furocoumarins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows inhibitor results for various inhibitors.
[0015] FIG. 2 shows inhibitor results for various inhibitors.
OBJECTS OF THE PRESENT INVENTION
[0016] It is one object of this invention to provide chemical
compounds and compositions that inhibit the first-pass effect and
which are in a form, concentration, purity, etc. other than that
which is naturally or commercially occurring.
[0017] Another object of the present invention is to provide a
reliable, safe citrus-based or citrus-origin product that comprises
one or more invention compounds in non-naturally and
non-commercially occurring amounts and inhibits the first-pass
effect and which, optionally, is free of or contains a reduced
amount (as compared to a naturally or commercially occurring
amount) of phototoxic and, optionally, non-first-pass inhibiting
low molecular weight furocoumarins, which is useful as a food or
dietary supplement, a pharmaceutical, a drug, etc.
[0018] Another object of the present invention is to provide a
composition comprising one or more invention compounds that is
effective against the first-pass effect.
[0019] Another object of the present invention is to provide a
composition that contains one or more of the invention compounds
and no or reduced amounts as compared to naturally or commercially
occurring amounts of phototoxic low molecular weight
furocoumarins.
[0020] Another object of the present invention is to provide a
composition comprising at least one invention compound and
providing consistent and reliable first-pass inhibiting
activity.
[0021] Another object of the present invention is to provide the
above-described compounds and compositions as a component of
products and mixtures that provide active ingredients, therapeutic
agents, drugs, etc. or other substances that are subject to the
first-pass effect in humans.
[0022] Another object of the present invention is to provide
first-pass effect inhibiting compounds, also called bioenhancers
and inhibitors herein, in non-natural and non-commercially
occurring forms.
[0023] 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.
[0024] Another object of the present invention is a method for
inhibiting the first-pass effect in human patients, animals, etc.
taking drugs having a first-pass effect.
[0025] Another object of the present invention is a method for
preparing the above-described compositions, compounds, mixtures,
etc.
[0026] Another object of the present invention is a method for
preparing a citrus-based or citrus-origin composition containing no
or reduced amounts as compared to naturally and commercially
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 (in this application, "non-first-pass
inhibiting" includes first-pass activity provided by 2000 nM
bergamottin or imperatorin according to Protocol C or C'
herein).
[0027] Another object of the present invention is to provide and
use first-pass effective compounds and compositions containing a
first pass effective amount (in aggregate or individually) of at
least one invention compound 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 and/or in a form
comprising a higher concentration or purity of invention compounds
than found both in nature and commercially. As used herein
"commercially" means products produced and sold locally, nationally
and internationally, especially by the citrus-processing industry.
These forms, as their names specify, are different from the
invention compounds, as they naturally occur in, for example,
commercial citrus and in citrus products such as juice, cold
pressed oils, juice concentrates, oils, etc.
[0028] Another object of the invention is to provide a method of
inhibiting the first-pass effect by administration of at least one
invention compound, composition etc. to humans.
[0029] These and other objects will become apparent to those of
ordinary skill in this art upon a full appreciation of the
invention as described below with regard to preferred
embodiments.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] The present inventor has discovered chemical compounds which
inhibit the first-pass effect of orally administered drugs in
humans. The present inventor has also discovered that phototoxic
low molecular weight furocoumarins and certain ether-substituted
furocoumarins that are naturally present in citrus extracts,
juices, byproducts, etc. may be removed therefrom or reduced in
concentration without destroying the first-pass effect inhibiting
compounds therein. The present inventor has also discovered a
method for preparing citrus-based compositions using only FDA or
USP acceptable reagents. The present invention has been completed
on the basis of these findings and will be described in more detail
below.
[0031] The invention chemical compounds which inhibit the
first-pass effect in animals, including humans, are, in one
preferred embodiment, compounds according to the following Formulae
I-IV: 1
[0032] In each of the above structures, R is, independently, H or
an optionally substituted C.sub.1-C.sub.15 alkyl group,
[0033] L is an optionally substituted C.sub.1-C.sub.15 linear or
branched, saturated, monounsaturated or polyunsaturated alkyl group
optionally interrupted by one or plural nonadjacent sulfur or
oxygen atoms and optionally terminated at one or both ends by
oxygen,
[0034] HAr is an optionally substituted C.sub.6-C.sub.24 aromatic
group or heteroaromatic group optionally containing one or plural
ring atoms selected from the group consisting of N, O, S, and
P,
[0035] and E is --OH, --COOH, --COOR (where R is defined above) or
an optionally substituted C.sub.1-C.sub.8 linear or branched,
saturated, monounsaturated or polyunsaturated alkyl group
optionally interrupted by one or plural nonadjacent oxygen or
sulfur atoms, or E is a C.sub.3-C.sub.8 optionally substituted
cyclic saturated, monounsaturated or polyunsaturated alkyl group
optionally interrupted by one or plural nonadjacent oxygen or
sulfur atoms, or E is optionally substituted HAr. Preferably, the
compounds of Formulae I-IV as well as those described below do not
contain a peroxide (O--O) group. Disulfide groups (S--S) are not
preferred, but may be present. Preferably E is an epoxide or
dihydroxy radical such as --CH(OH).sub.2. E may also be an
acid-opened epoxide group.
[0036] The compounds of the invention as described above are
unlimited with regard to stereochemistry, E-Z isomerism and all
possibilities are included. Racemic mixtures are included as are
each and every enantiomer and diasteriomer. Preferred
stereochemistry is shown later.
[0037] The groups R, L, HAr, and E may optionally be substituted
with a C.sub.1-C.sub.6 linear, branched or cyclic alkyl group,
--OH, a halogen atom, a C.sub.1-C.sub.5 alkoxy group, a
C.sub.1-C.sub.5 alkyl carbonyloxy group, a C.sub.1-C.sub.5
alkoxycarbonyl group, etc. Such substituents also may be optionally
substituted directly on the ring structures of Formulae I-IV
regardless of whether such substituents appear on R, L, HAr or
E.
[0038] A second preferred embodiment of the present invention
chemical compounds which inhibit the first-pass effect are depicted
by Formulae V-X: 234
[0039] As noted above for Formulae I-IV, Formulae V-X are unlimited
with regard to stereochemistry, E-Z isomerism, etc.
[0040] The most preferred compounds according to the present
invention, which inhibit the first-pass effect, are those according
to the second embodiment above and having the following
stereochemistry (formulae XI-XVI): 567
[0041] The compositions of the present invention contain at least
one invention first-pass effective chemical compound preferably in
a first-pass effective amount. Citrus-based compositions of the
invention further contain a 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,
preferably, any citrus fruit naturally containing an invention
first-pass effect inhibiting compound or mixture of such 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.
[0042] First-pass effective compounds, substances and compositions
described herein are materials that prevent or retard the
degradation of orally administered drugs in the body. Preferably,
the first-pass effective materials of the invention, including
substances, compositions, mixtures, invention compounds, etc.
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. See U.S. Pat. No. 5,567,592 incorporated herein by
reference. A several-fold, including 5, 10, 15, 20-fold, etc.
increase in bioavailability (i.e., several hundreds or thousands of
percent AUC increase) is not unusual with the present invention.
The first-pass effectiveness of invention compounds, composition,
mixtures, materials, etc. may also be measured by, and preferably
meet the criteria of, the methods and characterizations described
in WO 97/15269, U.S. Pat. No. 5,665,386 and PCT/US96/09607, all
incorporated herein by reference.
[0043] 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.
[0044] The citrus-derived substance present in invention
compositions may make up the entire citrus-based composition or may
be only a part thereof. Thus, if the citrus-based substance is
prepared such that it contains one or more compounds according to
Formulae I-XVI in a first-pass effective amount no further compound
need be added. Food grade or pharmaceutically acceptable diluents,
excipients, carriers, etc., may be added, if desired.
[0045] 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. Preferably, these furocoumarins are
completely removed, meaning that they are removed to an extent such
that their presence is undetectable by liquid and, preferably, gas
chromatography.
[0046] The invention method for removing phototoxic low molecular
weight furocoumarins from invention citrus-derived components
preferably comprises optional removal of volatile components
(components removed after 12-48 h at a pressure of
10.sup.-2-10.sup.-3 Torr) and extraction with mixtures of at least
one C.sub.1-C.sub.10 alcohol (preferably ethanol) and water,
optionally in the presence of base. In certain situations it is
preferable not to remove volatile components such as
naturally-occurring terpenes but rather to use these volatiles
essentially as solvent in further processing. The extraction
mixture of alcohol and water may be discarded and what is left is
useful herein. C.sub.2-C.sub.5 alcohols are also preferred as are
C.sub.2 and C.sub.3 and C.sub.4 alcohols. The alcohol (ethanol) may
either be 100% alcohol or 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.
[0047] 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 either be 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 20-80 or 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, 10/90, 15/85, 20/80,
25/75, 30/70, 35/65, 40/60, 45/55, 40/60, 35/65, 30/70, etc.
alcohol/water. The extractions may be accomplished by any method
known in the art including liquid-liquid extraction, liquid-solid
extraction, continuous extraction etc. When the raw material used
to prepare the invention citrus-derived extract is, for example, an
oil, the alcohol (ethanol)/water 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.
[0048] As noted above, base is preferably used in removing
phototoxic furocoumarins and may be added to the water or alcohol
or both. Preferred bases are the alkali and alkaline earth
hydroxides and oxides, most preferably sodium hydroxide and
potassium hydroxide. The base is generally present in amounts from
0.01-80 grams per liter of alcohol/water mixture.
[0049] Preferably, 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, marmin, 6',7'-dihydroxybergamottin, and
imperatorin.
[0050] The invention citrus-derived substance, invention
compositions, invention mixtures, invention pharmaceutical
compositions, etc. preferably contain a first-pass effective amount
of at least one first-pass effective compound of Formulae I-XVI
above. In the alternative, several compounds of Formulae I-XVI may
be present, each in non-first-pass effective amounts where the sum
of the concentrations of said compounds provides first-pass
effectiveness.
[0051] In addition to the description above, one or more of the
hydrogen atoms depicted in these formulae (i.e., Formulae I-XVI)
may be replaced by one or any combination of two or more of
hydroxy, halogen, linear or branched C.sub.1-C.sub.40 hydrocarbon,
C.sub.1-C.sub.40 linear or branched ether (--OR where R is linear
or branched hydrocarbon), C.sub.1-C.sub.40 alkylhydroxy (--ROH
where R is linear or branched hydrocarbon and OH is bonded to a
primary, secondary or tertiary carbon), etc. As used herein
"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. All salts, particularly pharmaceutically acceptable salts,
and stereoisomers, physical forms, etc. are also included. The
compounds described in formulae I-XVI 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. Now that they have
been identified they can also be isolated from a citrus-derived
substance as shown herein.
[0052] Preferred methods for making the invention compounds of
Formulae I-XVI include the following schemes: 89
[0053] Such reactions are within the skill of the ordinary artisan.
See, for example, Chemistry Letters, 2019-2022, 1990, Can. J.
Chem., 63: 2673-2678, 1985, Australian Journal of Chemistry, 42:
1235-1248, 1989, East German patent DD 275687 and Soviet Union
patent SU 1397449, all incorporated herein by reference. Although
each of the reaction schema shown above yield either Formulae I+II
or Formulae III+IV, each method can give Formulae I-IV if the
appropriate reactants are used. The present invention compounds,
citrus-derived substance, mixtures, pharmaceutical compositions,
etc. preferably contain at least one compound according to Formulae
I-XVI above. Mixtures may be used.
[0054] The use of present invention citrus-derived substance,
compositions, mixtures, inhibitors, compounds, etc. are not limited
and may preferably be administered in amounts of 2 nanograms-2 g
and more per patient per day to increase the bioavailability of
drugs taken orally by a patient. Compositions of the invention may
contain, preferably, more of the invention compounds 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.
Dosage forms include oral administration forms, topical
administration forms, injection forms. The invention compounds,
citrus-derived substance, compositions, mixtures, etc. 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.
[0055] The citrus-derived substance, mixtures, compositions and
compounds 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 that increases the
oral bioavailability 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 in such a situation. A
"first-pass effective" invention citrus-derived substance, mixture,
composition or compound is a material that inhibits the observed
first-pass effect of at least one drug in an animal, preferably a
human, 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, etc. subject to the first-pass effect. "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 compounds,
citrus-derived substance, mixture, composition, etc. 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.) as well as binders, salt forms,
excipients, etc. which are useful are found in, e.g., U.S. Pat.
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 compounds are preferably provided in an amount
that provides consistent, reliable potency from batch to batch
regardless of the form in which it is provided.
[0056] The word "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 Merck Index, Twelfth Ed., Merck Research Laboratories,
Whitehouse Station, NJ, 1996, "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 pro-active, 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. Preferably, a
hydrophobic drug is defined as a drug more soluble in octanol than
in water. See U.S. Pat. No. 5,567,592, incorporated herein by
reference. The invention can be used with humans and animals such
as mammals.
[0057] The present invention compounds and citrus-derived
substances may be co-formulated with drugs, preferably drugs that
are subject to the first-pass effect. Preferably the drug has an
oral bioavailability of 92% or less, more preferably 50% or less.
Examples include, in addition to those incorporated by reference
above, saquinavir, indinavir, L-deprenyl, tacrolimus, cyclosporin A
(Sandimmune.RTM.), cyclosporin A (Neoral.RTM.), nelfinavir,
VX478/141W94, felodipine, nifedipine and sumatriptan. Such
co-formulations include the invention citrus-derived substance
and/or one or more compounds 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 compounds 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.
[0058] Other products useful for co-formulation herein are any and
all drug, medical food, or other products that are subject to the
first-pass effect. Examples of drugs are listed in the Merck Index.
Twelfth Ed., Merck Research Laboratories, Whitehouse Station, N.J.,
1996, incorporated herein by reference. Determining whether a
substance is subject to the first-pass effect is within the skill
of the average artisan in this field.
[0059] It is preferred that invention materials be protected from
stomach acid by, e.g., a coating. Such coatings are well known in
the art, and include enteric coatings, etc. See the Kirk-Othmer
Encyclopedia of Chemical Technology, 3rd Ed. Vol. 17, p. 281 ff,
incorporated herein by reference. Other useful pharmaceutical forms
may also be used, such as time-release forms (coatings), hard-and
soft-shell gelatin capsules, etc.
EXAMPLES
[0060] Synthesis of a Spiro Ortho Ester (Formula XVII): 10
[0061] Benzyl 6,7-epoxygeranyl ether was placed in an evacuated
chamber (0.1 torr) overnight in order to remove extraneous water
from the sample; 190 mg (730 .mu.mol) was weighed. Three
equivalents of 7-methoxycoumarin (0.386 g) was dissolved in 3 mL
CH.sub.2Cl.sub.2, and this liquid was transferred to a closed glass
container. Helium was used to purge the system during the reaction
period, the container was maintained at 5-6.degree. C., and the
reaction solution was magnetically stirred. Forty-three .mu.L of a
1 M solution of SnCl.sub.4 in CH.sub.2Cl.sub.2 and 16 .mu.mol of
BF.sub.3.Et.sub.2O were added; 5 minutes passed, then the epoxide,
dissolved in 4 mL CH.sub.2Cl.sub.2, was added in 4 equal portions;
each portion was followed by 4 .mu.mol BF.sub.3.Et.sub.2O. The
reaction was held at 5-6.degree. C. and stirred for 3.5 hours, and
the reaction apparatus was then disassembled. The reaction mixture
was treated immediately as follows: the mixture contains a total of
75 .mu.mol of BF.sub.3 and 5 nCl.sub.4, so 3 equivalents of
pyridine (225 .mu.mol) were added (reaction mixture held at
5-6.degree. C., with stirring) to destroy the Lewis acid catalysts.
After 30 minutes of stirring, the solvent was removed, and the
residue was dissolved in 3.16 mL of 95% ethanol; 0.6 mL of 50% KOH
in water (w/v) was added, and the solution was mixed vigorously.
Water (2.24 mL) was added, and this solution was then placed in a
Speed Vac apparatus overnight to remove the ethanol. Approximately
50% of the original volume remained. The aqueous solution was
extracted twice with 3 mL CH.sub.2Cl.sub.2, and the pooled
CH.sub.2Cl.sub.2 was extracted twice with 10 mL 5% KOH in water
(w/v) and twice with 10 mL 5% NaCl in water (w/v). The
CH.sub.2Cl.sub.2 was removed, and the residue was dissolved in
acetonitrile.
[0062] This acetonitrile solution may be used directly for HPLC
purification of the spiro ortho ester product via the preferred
conditions that follow. Linear gradients were used for elution and
were 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 were as follows: 0, 55; 5, 55;
10, 90; 11, 98; 17, 98; 18, 55; 22, 55. The chromatographic column
had dimensions of 250 mm length.times.4.6 mm internal diameter, was
packed with C18 bonded to 4 micron silica particles (9% carbon
load; YMC, Inc.), was protected with a 23 mm length.times.4 mm
internal diameter column containing the same material and with a
0.5 micron filter, and was maintained at 40.+-.0.2.degree. C. The
flow rate was maintained at 1.0 mL/min during the 22 min run cycle.
The column eluate from each 10 .mu.L injection was monitored at
259.+-.2 and was fractionated using a robotic collector (Gilson).
The retention time of Formula XVII in this system was 13.4 minutes.
.sup.1H N.M.R. .delta. (400 MHZ, CD.sub.3OD) 1.15, s and 1.26, s
and 1.39, s, 4'-Me (diasteriomers); 1.64, s, 8'-Me; 1.6-1.9, m,
H6',6'; 2.0-2.3, m, H7'; 3.70, s, 7-OMe; 3.83, m and 4.20, t, H5'
(diasteriomers); 4.01, d, H10'; 4.45, s, H12'; 5.39, t, H9'; 5.45,
d, H3; 6.40, s, H8; 6.49, d, H6; 6.71, d, H4; 7.06, d, H5;
7.20-7.35, m, phenyl. Mass spectrum m/z (electrospray): MS, 437
(MH.sup.+); MS/MS, 261, 177, 153 (fragments of MH.sup.+).
[0063] Bulk Pretreatment of Cold-Pressed Grapefruit Oil for
Subsequent Fractionation By Reversed-Phase HPLC
[0064] 1. Prepare 1:1 ethanol:water (v/v) containing 12.5 g KOH/L
using denatured reagent alcohol.
[0065] 2. Mix 1.0 L of cold-pressed grapefruit oil and 330 mL of
the basic ethanolic solution in a 2 L separatory funnel for 2.0
min.
[0066] 3. Wait 5.0 min, then remove the bottom ethanolic phase from
the funnel.
[0067] 4. Repeat steps 2 & 3 four times using fresh 330-mL
portions of the basic ethanolic solution.
[0068] 5. Repeat steps 2 & 3 once using a 330-mL portion of:
1:1 ethanol:water that is prepared without KOH. When KOH is absent,
a several-hours wait is required (preferably overnight) in order
for the phases to clearly separate. Assessment of the bottom
ethanolic phase with pH paper should show that the solution pH is
near neutral (.congruent.6, in most cases).
[0069] 6. Place the oil phase (yield should be greater than 0.9 L)
in a vacuum chamber and place the system under vacuum (0.5 torr or
better) for 3-4 days. The process is complete when swirling the
viscous liquid while under vacuum no longer initiates boiling.
[0070] 7. Wash the nonvolatile material with portions of
acetonitrile, to a total of 200 mL, and separate the
acetonitrile-soluble and insoluble materials by centrifugation (5
min at speed 50, IEC model K2).
[0071] 8. Remove the acetonitrile from the acetonitrile-soluble
phase (step 7) using a Speed Vac apparatus, and weigh the residue
(22-25 g would be expected).
[0072] 9. Add acetonitrile to the residue such that each 5-mL
portion contains 1.5 grams of residue, and divide the solution into
5-mL portions.
[0073] 10. Add 15-mL of iso-octane to each 5-mL portion, cap,
vortex mix, and centrifuge the mixture (2 min at speed 35). Discard
the top iso-octane phase.
[0074] 11. Repeat step 10 nine times; acetonitrile should be added
occasionally to insure that the bottom phase volume approximates 5
mL.
[0075] 12. Remove the acetonitrile from the bottom phase (step 11)
using a Speed Vac apparatus, and weigh the residue (approximately
20% of the original weight {step 8} would be expected).
[0076] 13. Dissolve the residue (step 12) in acetonitrile such that
a 0.25-0.30 g/mL solution is established, filter the solution
through a 0.2 .mu.m Teflon cartridge, and store the solution at
-20.degree. C.
[0077] Bulk Pretreatment of Cold-Pressed Grapefruit Oil for Further
use as Dietary Food Supplement, Drug, ConFormulation Ingredient,
etc.
[0078] 1. Prepare a 70:30 water:ethanol solution (v/v) that
contains 5% potassium hydroxide (w/v) using USP-grade ethanol,
NF/FCC-grade KOH, and purified water.
[0079] 2. Mix the ethanolic solution prepared in step 1 with an
equivalent volume (or slight excess) of whole, untreated
cold-pressed grapefruit oil (Food Chemicals Codex grade), and
transfer the mixture to a heat-and pressure-resistant food-grade
container.
[0080] 3. The sealed container is maintained at 95-100.degree. C.
for 1 hour. The container is cooled, the ethanolic phase (lower
phase of two-phase system) is removed, and a fresh portion of
ethanolic solution (equivalent to the volume used in step 2) is
added.
[0081] 4. Repeat the boiling cycle (step 3) until the desired
degree of sample purity is achieved. Ten cycles will remove >99%
of polar coumarins and furocoumarins, will remove >90% of
prominent nonpolar coumarins and furocoumarins (i.e.,
epoxyaurapten, epoxybergarnottin), and will not appreciably
decrease the content of the inhibitory spiro ortho esters.
[0082] 5. Wash the oil with purified water until the discarded wash
water pH becomes neutral.
[0083] 6. Place the oil phase under vacuum (0.1-0.3 torr) until
volatile materials are no longer removed from the sample (as
assessed by, for example, inspection of an empty in-line trap
maintained at -60 to -90.degree. C.). In general, approximately 95%
of the volume of the sample will be removed in this step.
[0084] 7. Mix the product of step 6 with an equivalent volume of
USP ethanol, and centrifuge the mixture. Repeat until the bottom
phase is substantially free of spiro ortho ester inhibitors. This
method removes ethanol-insoluble materials from the oil novolatile
preparation.
[0085] 8. Optionally, but preferably, place the pooled ethanol
extracts from step 7 under vacuum until the ethanol has been
substantially removed (e.g., 99%) or reduced (e.g. 10%).
[0086] Because adulteration of raw materials is known in the food,
flavor, and fragrance industries, citrus-derived components of the
invention including cold-pressed citrus oils should preferably be
assessed before they are used further in the production of, e.g.,
compositions of dietary supplements containing a first-pass
effective amount of one or a mixture of compounds of Formulae
I-XVI. One strategy consists of sample preparation (Protocol A;
Protocol A'), followed by chromatography (Protocol B; Protocol B';
Protocol B"), and ending with comparisons to historical standards.
Such assessment can provide consistent batches.
[0087] The following protocols are useful in preparing various
embodiments of the invention.
[0088] Protocol A: Preparation of Citrus Oils for Further
Chromatographic Processing or for Administration to Humans by
Removal of Toxic, Low Molecular Weight Furocoumarins.
[0089] A volume of cold-pressed citrus oil (Food Chemicals Codex
grade) was transferred to a container, and all volatile materials
were removed. Although several methods exist for removing volatiles
(e.g., distillation, distillation under reduced pressure,
evaporation under ambient conditions), the preferred method uses
Speed Vac concentrators (Savant Instruments; process requires 12-24
h and pressures of 10.sup.-2-10.sup.-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.
[0090] Low molecular weight, phototoxic furocoumarins were removed
from the nonvolatile preparation by liquid-liquid extraction: 16
times the volume of viscous liquid of 1:1 ethanol:water (v/v; each
U.S.P. grade) were added to the nonvolatile preparation, the
container capped, the solution mixed vigorously, the container
centrifuged (International Equipment Company, Model K-2, 5 min at
setting 35), and the top ethanolic phase discarded. The extraction
was repeated twice. Extraneous water and ethanol may be removed
from the preparation if desired by use, e.g., of a Speed Vac
apparatus. The product of this process may be used for human
administration in, e.g., filled capsules.
[0091] Protocol A': Pretreatment of Citrus Oils Prior to
Chromatography.
[0092] Most 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. Hence the sample preparation
protocol that follows is used prior to chromatography.
[0093] Cold-pressed citrus oil (Food Chemicals Codex grade) is
transferred to a suitable container, and all volatile materials are
removed under reduced pressure (10.sup.-2-10.sup.-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.sup.-2-10.sup.-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
ethanol:water (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 or iso-octane, with each hexane extract
(upper layer) being removed and discarded, and the resulting
acetonitrile solution is filtered through a 0.2 micron Teflon.RTM.
membrane and evaporated to dryness using a Speed Vac
concentrator.
[0094] 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.
[0095] Protocol B: Chromatography Methods for Processed Citrus
Oils
[0096] 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. Hence the 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.RTM.
membrane. The filtered solution is stored in a closed container at
-20.degree. C. 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.
[0097] If preparative fractionation of the washed nonvolatile
portion of citrus oil is desired, then the HPLC conditions given
below are preferred. 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; 11, 98; 17, 98; 18, 75; 22, 75. The chromatographic column has
dimensions of 250 mm length.times.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.times.4
mm internal diameter column containing the same material and with a
0.5 micron filter, and is maintained at 40+/-0.2.degree. 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).
[0098] If qualitative or quantitative assessments of citrus oils,
fractions thereof, or reference standards are desired, then the
HPLC conditions given below are preferred. 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.times.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.degree. 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 nmn, emission at 450 nm, and bandpass filtration
at 370 nm.
[0099] Protocol B': Chromatography Methods for Processed Citrus
Oils
[0100] If preparative fractionation of the washed nonvolatile
portion of citrus oil (product of Protocol A') is desired, then the
HPLC conditions given below are preferred. 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; 11, 98; 17, 98; 18, 75; 22, 75. The
chromatographic column has dimensions of 250 mm length.times.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.times.4 mm internal diameter column containing the
same material and with a 0.5 micron filter, and is maintained at
40+/-0.2.degree. 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 acetonitrile solution obtained by Protocol A' is
monitored at 400+/-200 nm and at 310+/-2 nm and is fractionated
using a robotic collector (Gilson).
[0101] If qualitative or quantitative assessments of citrus oils,
fractions thereof, or reference standards are desired, then the
HPLC conditions given below are preferred. 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.times.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 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+/-200 nm and at 310+/-2 nm and for fluorescence with excitation
at 229 run, emission at 450 nm, and bandpass filtration at 370
nm.
[0102] Protocol B": Purification of Invention Compounds Using a
Chiral HPLC Column
[0103] Pooled residues that result from fractionation of the
11-12.5 min region (Protocol B or B') and solvent removal (Speed
Vac, no heat added) are subjected to chiral liquid chromatography.
Isocratic elution is employed (mobile phase consists of 3.4 L
iso-octane, 0.6 L 95% ethanol (remainder is water), and 0.2 L
isopropanol) to elute Inhibitors XI-XVI from the column
(250.times.4.6 mm, Keystone Scientific, Inc., Chiral from each 25
uL injection (residue is dissolved in mobile phase) is monitored at
400+/-200 nm and at 310+/-2 nm and is fractionated using a robotic
collector (Gilson).
[0104] Protocol C: Assessment of Human Cytochrome P450-Mediated
Biotransformation
[0105] The process of preparing incubation mixtures begins by
mixing 10 uL of ethanol or an ethanolic solution containing an
inhibitor with 100 uL of 100 mg/mL bovine serum albumin (Sigma)
dissolved in reaction buffer at room temperature. 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, A', B, B', or B" above.
When possible, 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.degree. 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.degree. 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.
[0106] 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.
[0107] 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 Protocols B and 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.times.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.degree. C. The dry
sample residue is mixed with 60 uL of 1:1 acetonitrile:water
followed by 40 uL water just prior to each 50.25 min run cycle. The
column eluate from each 10 uL injection is monitored for
fluorescence with excitation at 228 nm, emission at 291 nm, and
bandpass filtration at 280 nm. Under these conditions, the
retention times of terfenadine alcohol metabolite, terfenadine
carboxylic acid metabolite, and the internal standard are 16.2 min,
17.4 min, and 22.2 min, respectively.
[0108] Protocol C': Assessment of Human Cytochrome P450-Mediated
Biotransformation
[0109] The process of preparing incubation mixtures begins by
mixing 10 uL of ethanol (control reactions) or an ethanolic
solution containing an inhibitor with 100 uL of 100 mg/mL bovine
serum albumin (Sigma) dissolved in reaction buffer at room
temperature. 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 (Norvir.TM., Abbott Laboratories), inhibitory
chemicals described in Protocol C, and fractions resulting from
Protocol B" above. Final inhibitor concentrations were expressed in
molarity by calculation from the weighed material or by use of
Beer's law. 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.degree. C. in a Dubnoff-type water bath, the reaction
mixture is completed by the addition of 10 uL of 500 uM saquinavir
(Invirase.TM., Roche Laboratories) dissolved in 1:1 ethanol:water
and by gentle mixing, the samples are incubated for 15 min at
37.degree. C., and the reaction is stopped by placing the tube on
ice and adding 2.5 mL of acetonitrile.
[0110] 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.
[0111] 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 Protocols B and 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.times.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.degree. C. In order to
minimize the degradation of analytes, 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. Under these conditions, the
retention times of saquinavir principal metabolite A, saquinavir
principal metabolite B, and saquinavir are 24.2 min, 26.0 min, and
30.0 min, respectively.
[0112] Demonstration of Effectiveness
[0113] In order to demonstrate the first-pass effectiveness of the
present invention, experiments with invention compounds and
invention citrus-derived substances were conducted according to
Protocol C' above where generation of saquinavir metabolites were
measured in the presence of various concentrations of inhibitor.
Citrus-derived substances according to the present invention were
prepared according to Protocols A', B', and B" above and were
compared to known inhibitor Ketoconazole. FIG. 1 shows results for
invention compounds of Formulae XI and XIII and also shows that
bergamottin and imperatorin are essentially ineffective first-pass
inhibitors. FIG. 2 shows how invention compounds compare to known
inhibitors Ritonavir and Ketoconazole.
[0114] In the invention compounds it is preferred that the furan
ring position of the furocoumarin rings be completely free of
substitution.
[0115] With regard to purification and processing methods, the
following embodiments are preferred:
[0116] A. 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.sub.2-C.sub.4 alcohol,
water, and optionally base, said first-pass effective citrus
derived substance maintaining anti-first-pass activity after said
extraction.
[0117] B. The method of embodiment A, wherein said citrus-derived
substance is a cold-pressed citrus oil.
[0118] C. The method of embodiment A, wherein said mixture of
ethanol and water is a 30/70 volume/volume mixture of ethanol and
water optionally containing 1-10% potassium hydroxide (W/V).
[0119] Other preferred embodiments include:
[0120] D. A first-pass effective citrus-derived substance which has
been extracted with a mixture of at least one C.sub.2-C.sub.4
alcohol and water so as to reduce the amount of phototoxic
furocoumarins therein.
[0121] E. The citrus-derived substance of embodiment D, wherein
said substance is a cold-pressed citrus oil.
[0122] F. The citrus-derived substance of embodiment D, which has
been extracted with a 30/70 volume/volume mixture of ethanol and
water optionally containing 1-10% potassium hydroxide (W/V).
[0123] G. 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
embodiments A and D.
[0124] Invention compositions preferably comprise invention
inhibitor material (compound, etc.) (e.g., alone, mixed with
drug(s), and/or diluent(s) and/or carrier(s) etc.) such that they
inhibit saquinavir biotransformation according to Protocol C' above
better than pure ketoconazole on an equal molar (preferred) or
weight concentration basis. Alternatively, invention compounds,
compositions, mixtures, formulations, etc. (materials) preferably
provide a Y-axis value in Protocol C' (see FIG. 1) 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 0.01-0.25 mg, including
0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22 and
0.24, and all ranges between all values, of invention material is
diluted or dissolved to one liter.
[0125] A commercial form of citrus containing the invention
compounds is cold-pressed grapefruit oil, which has a total
concentration of compounds of Formulae XI-XVI in the range of
perhaps up to 0.15-0.25 mg/ml. The six compounds are distributed
as: XI+XII+XIII+XIV equals approximately 50%, with XV and XVI the
remainder. For XI-XIV the distribution is about 3:2:2:1,
respectively and about 2:1 for XV:XVI. Compositions according to
the invention in one embodiment thus preferably contain higher
concentrations of invention compounds (i.e., total concentration of
all invention compounds therein) than those which occur in nature
and commercial forms of citrus. These concentrations are referred
to as "concentrated amounts". Examples of preferred concentrations
include greater than 0.25 mg/ml, 0.3, 0.8, 1, 2, 5, 8, 32, 128, 200
mg/ml, etc. With regard to invention compounds, the compounds in
one embodiment of the invention are in a form distinct from that
found in nature or commercially due to purity. The term
"substantially pure" and "substantially pure form" refers to a
purity greater than that found commercially and in nature for the
invention compounds. These concentrations and forms are easily
derminable by those of ordinary skill now that the present inventor
has identified the active compounds responsible for the "grapefruit
effect". Other language, phrases, etc. useful to describe the
embodiments of the present invention and distinguish them
patentably and otherwise from naturally or commercially occurring
forms are found in the following patents assigned to the U.S.
government and to others, all incorporated herein by reference:
U.S. Pat. Nos. 4,708,948, 5,409,938, 5,455,251, 4,977,244,
5,462,956, 5,314, 899, 5,104,977, 5,484,889, 5,591,770, 5,599,839,
5,672,607, 5,674,900, 5,648,354, 5,691,386, 5,681,829 and
5,654,432. Another description of how the present invention may be
used, in what amounts, and how administered appears in U.S. Pat.
No. 5,665,386. WO 97/15269 and WO 96/40192, all incorporated herein
by reference.
[0126] The following patent applications, provisional or otherwise,
are incorporated herein by reference: 60/056,382, 60/048,183,
60/043,878, 08/764,081 and 08/673,800 (Attorney Docket Numbers
7552-001-99, 7552-004-99, 7552-005-99, 7552-006-99 and
7552-007-99).
[0127] Other compounds useful herein are described by the following
formulae where R, L, E and HAr are as described above. As with the
above compounds, these compounds include all stereoisomers, E-Z
isomers, etc. Where naturally or commercially occurring, these
compounds are preferably in the forms described above regarding
purity, concentration, etc. These compounds may be optionally
substituted as compounds of Formulae I-XVI are. 111213
[0128] In the invention compounds a preferred group of sustituents,
optional and otherwise, comprise the following: hydrogen,
C.sub.1-C.sub.4 alkyl, --S(C.sub.1-C.sub.4 alkyl),
--O(C.sub.1-C.sub.4 alkyl), --NH.sub.2, --NH(C.sub.1-C.sub.4
alkyl)-N(C.sub.1-C.sub.2 alkyl) (C.sub.1-C.sub.4 alkyl), hydroxy,
--O(C.sub.1-C.sub.2 alkyl), fluoro, C.sub.1-C.sub.6 alkyl, chloro,
bromo, iodo, C.sub.1-C.sub.4 alkoxy, --CF.sub.3,
--C(.dbd.O)O--(C.sub.1-C.sub.4) alkyl, --OC(.dbd.O)(C.sub.1-C.sub.4
alkyl), --OC(.dbd.O)N(C.sub.1-C.sub.4 alkyl) (C.sub.1-C.sub.2
alkyl), --NHCO(C.sub.1-C.sub.4 alkyl), --COOH,
--COO(C.sub.1-C.sub.4 alkyl), --CONH(C.sub.1-C.sub.4 alkyl),
--CON(C.sub.1-C.sub.4 alkyl) (C.sub.1-C.sub.2 alkyl),
--S(C.sub.1-C.sub.4 alkyl), --CN, --NO.sub.2, --SO(C.sub.1-C.sub.4
alkyl), --SO.sub.2(C.sub.1-C.sub.4 alkyl),
--SO.sub.2NH(C.sub.1-C.sub.4 alkyl) and --SO.sub.2N(C.sub.1-C.sub.4
alkyl) (C.sub.1-C.sub.2 alkyl).
[0129] Another group of preferred substituents, optional and
otherwise, comprise: C.sub.1-C.sub.12 alkyl, aryl, (C.sub.1-C.sub.4
alkylene) aryl, phenyl, naphthyl, thienyl, benzothienyl, pyridyl,
quinolyl, pyrazinyl, pyrimidinyl, imidazolyl, furanyl,
benzofuranyl, benzothiazolyl, isothiazolyl, pyrazolyl, pyrrolyl,
indolyl, pyrrolopyridyl, oxazolyl and benzoxazolyl, C.sub.3-C.sub.8
cycloalkyl or (C.sub.1-C.sub.6 alkylene) (C.sub.3-C.sub.8
cycloalkyl), C.sub.1-C.sub.4 alkyl, benzyl, C.sub.1-C.sub.4
alkanoyl, C.sub.1-C.sub.6 alkoxy, --OC(.dbd.O) (C.sub.1-C.sub.6
alkyl), --OC(.dbd.O)N(C.sub.1-C.sub.4 alkyl)(C.sub.1-C.sub.2
alkyl), --S(C.sub.1-C.sub.6 alkyl), amino, --NH(C.sub.1-C.sub.2
alkyl), --N(C.sub.1-C.sub.2 alkyl) (C.sub.1-C.sub.4 alkyl),
--N(C.sub.1-C.sub.4 alkyl)-CO--(C.sub.1-C.sub.4 alkyl),
--NHCO(C.sub.1-C.sub.4 alkyl), --COOH, --COO(C.sub.1-C.sub.4
alkyl), --CONH(C.sub.1-C.sub.4 alkyl), --CON(C.sub.1-C.sub.4 alkyl)
(C.sub.1-C.sub.2 alkyl), --SH, --SO(C.sub.1-C.sub.4 alkyl),
--SO.sub.2(C.sub.1-C.sub.4 alkyl), --SO.sub.2NH(C.sub.1-C.sub.4
alkyl) and --SO.sub.2N(C.sub.1-C.sub.4 alkyl) (C.sub.1-C.sub.2
alkyl)
[0130] A third group of preferred substituents, optional and
otherwise, comprise: --S(C.sub.1-C.sub.4 alkyl) or
--SO.sub.2(C.sub.1-C.sub.4 alkyl) (C.sub.1-C.sub.6 alkyl),
--N(C.sub.1-C.sub.4 alkyl) (C.sub.1-C.sub.2 alkyl),
--S(C.sub.1-C.sub.4 alkyl), --SO(C.sub.1-C.sub.4 alkyl),
--CO(C.sub.1-C.sub.4 alkyl), --C(.dbd.O)H,
--C(.dbd.O)O(C.sub.1-C.sub.4 alkyl), C.sub.1-C.sub.3 alkoxy,
dimethylamino, methylamino, ethylamino, --NHC(.dbd.O)CH.sub.3,
C.sub.1-C.sub.3 thioalkyl, --COOH, --C(.dbd.O)O(C.sub.1-C.sub.4
alkyl), --C(.dbd.O)O(C.sub.1-C.sub.4 alkyl), --NO.sub.2, phenyl,
naphthyl, thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl,
furanyl, benzofuranyl, benzothiazolyl, benzisothiazolyl,
benzisoxazolyl, benzimidazolyl, indolyl, benzoxazolyl or
C.sub.3-C.sub.8 cycloalkyl, chloro, C.sub.1-C.sub.6 alkyl,
--O(C.sub.1-C.sub.6 alkyl) bromo, iodo, formyl, --CN, --CF.sub.3,
--NO.sub.2, --NH.sub.2, --NH(C.sub.1-C.sub.4 alkyl),
--N(C.sub.1-C.sub.2 alkyl) (C.sub.1-C.sub.6 alkyl),
--C(.dbd.O)O(C.sub.1-C.sub.4 alkyl), --C(.dbd.O) (C.sub.1-C.sub.4
alkyl), --COOH, --SO.sub.2NH(C.sub.1-C.sub.4 alkyl),
--SO.sub.2N(C.sub.1-C.sub.2 alkyl) (C.sub.1-C.sub.4 alkyl),
--SO.sub.2NH.sub.2, --NHSO.sub.2(C.sub.1-C.sub.4 alkyl),
--S(C.sub.1-C.sub.6 alkyl) and --SO.sub.2(C.sub.1-C.sub.6 alkyl),
fluoro, hydroxy, amino, methylamino, dimethylamino, acetyl,
hydrogen, C.sub.1-C.sub.4 alkyl, halo (e.g., chloro, fluoro, iodo
or bromo), hydroxy, --O(C.sub.1-C.sub.4 alkyl), --C(.dbd.O)
(C.sub.1-C.sub.4 alkyl), --C(.dbd.O)O(C.sub.1-C.sub.4 alkyl),
--OCF.sub.3, --CF.sub.3, --CH.sub.2OH or
--CH.sub.2O(C.sub.1-C.sub.2 alkyl) hydroxy, methoxy and fluoro.
[0131] Within these three groups of preferred substituents are also
specific examples of HAr (i.e., C.sub.6-C.sub.24 aromatic groups or
heteroaromatic groups).
[0132] In the present invention prodrugs and active metabolites of
the invention compounds, compositions, etc. are included. Such
prodrugs are compounds which give rise to an invention compound
upon administration to a mammal such as a human. Active metabolites
are compounds formed upon administration of an invention compound,
composition, etc. to a mammal, preferably a human, which are
first-pass effective. Some examples of invention prodrugs and
metabolites include: 14151617
[0133] Invention compounds, metabolites, prodrugs, etc. may
preferably be substituted with deuterium and/or fluorine to
increase residence time in the patient/animal/etc.
[0134] Pharmaceutical carriers, diluents, excipients, etc. are
known to those of skill in this art. Examples are provided in
several above-noted patents and publications.
* * * * *