Note: Descriptions are shown in the official language in which they were submitted.
CA 02593140 2007-10-18
INHIBITORS AND ENHANCERS OF URIDINE DIPHOSPHATE-
GLUCURONOSYLTRANSFERASE 2B (UGT2B)
BACKGROUND OF THE INVENTION
1. Field of the invention
This invention aims at enhancing drug bio-availability by providing an
effective UGT2B inhibitor as well as a UGT2B enhancer for increasing the
detoxification ability of individuals.
2. Description of the prior art
The drug metabolism process in human body, especially the metabolism of
high fat-soluble drugs, includes two biotransformation steps: phase I reaction
that
catalyzes fat-soluble molecules to polarized molecules, and phase II reaction
that
produces highly polarized molecule through conjugation, such that the drugs
can
be metabolized efficiently and excreted to urine or feces.
The most common and important conjugation is glucuronidation by uridine
diphosphate ( UDP ) -glucuronosyltransferases (refers to as UGTs , EC 2.4.1.17
hereafter).
The UGTs is one of the major enzymes in phase II reaction in human. It is
now evident that UGTs have more than 110 isoenzymes. UGTs can catalyze the
conjugation of UDP-glucuronic acid (UDPGA) and the endogenous fat-soluble
compounds' chemical bonds, such as hydroxyl, sulfonyl, carboxylic acid, amine,
or amide, to facilitate the 0-glucuronidation, N-glucuronidation, or S-
glucuronidation (King et al., 2000, Cuff. Drug Metab., 1(2): 143-61), and thus
enhances the polarity of the fat-soluble molecules.
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CA 02593140 2007-10-18
According to a review article published by Radominska-Pandya et al (Drug
Metab Rev. 31(4):817-99, 1999), most human UGTs belong to the UGT1A and
UGT2B families. The UGT1A family consists of UGT1A1, UGT1A2P, UGT1A3-
10, UGTIA1 1P and UGT1Al2p, while UGT2B family consists of UGT2B4,
UGT2B7, UGT2B10, UGT2B15 and UGT2B17.
In addition, UGTs posses extensive substrate specificity. The UGT1A and
UGT2B metabolize different compounds. The UGT1A family mainly metabolizes
phenolic compounds such as estrone, 2-hydroxyestrone, 4-nitrophenol, 1-
naphthol,
etc. with the involvement of bilirubin. The UGT2B family metabolizes steroid
compounds such as androsterone, linoleic acid, etc. with the involvement of
bile
acids.
It was reported that UGTs can either enhance the bio-activity of some
compounds or, under certain circumstances, transform some compound into toxic
substances, such as morphine, steroids, bile acids and mid retinoids. (see
Vore et
al. (1983a) Life Sciences 32:2989-2993; Vore et al. (1983b) Drug Metabolism
Reviews 14:1005-1019 ; Abbott and Palmour (1988) Life Sciences 43:1685-169).
It was also reported that UGTs have involved in the activation of polycyclic
aromatic hydrocarbons (PAH) and heterocyclic aromatic amines. (Munzel et al.
(1996) Archives of Biochemistry and Biophysics, 355: 205-210; Bock et al.
(1998)
Advances in Enzyme Regulation, 38: 207-222).
UGTs can be found in several tissues including liver, kidney, bile duct,
esophagus, stomach, intestine, rectum, ileum, jejunum, spleen, mammary gland,
skin, lung, and brain. However, the distribution of various UGTs in human body
differs by type. For instance, UGT2B7 exists mainly in esophagus, liver,
intestine,
colon, kidney, and spleen; while UGT1A1 can be found in liver, bile duct,
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CA 02593140 2007-10-18
stomach and colon ( Tukey et al. (2000) Annu. Rev. Pharmacol. Toxicol., 40:
581-616. Review ) .
Studies by Burchell and Coughtrie (Burchell B and Coughtrie MW (1997)
Environmental Health Perspectives 105: 739-747) found differences among
individuals in their abilities to metabolize medicines, due to the genetic
polymorphisms in UGT genes. Therefore, the information regarding the
regulatory function of UGTs in individual's drug metabolism process is
essential
in evaluating a drug's potential pharmaceutical efficacy and its interaction
with
other drugs.
The UGTs is also an important detoxification system in human body. In
addition to the endogenous fat-soluble compounds, the exogenous fat-soluble
compounds can also become water-soluble through glucuronidation, and thus
enhances the excretion of the exogenous fat-soluble compounds and maintains
human body's normal detoxification function.
Therefore, the glucuronidation will be hampered by defected UGTs
activities in individuals who suffered from liver diseases. Consequently, the
liver's lower clearance rate in metabolizing drug will increase the toxic
reaction
and the rate of carcinogenesis in an individual with liver diseases.
According to literatures, butylated hydroxyanisole (BHA) (Buetler et al.
(1995) Toxicology & Applied Pharmacology 135(1): 45-57) and pregnenolone-16
a -carbonitrile (PCN) (Viollon-Abadie et al., 1999, Toxicology & Applied
Pharmacology., 155(1):1-12) may activate UGT2B.
Before circulating through the entire body, most drugs that are absorbable
to gastroenterological tract will enter the liver through portal circulation.
This is
the so-called "first-pass effect". It had been confirmed that the ubiquitous
UGTs
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CA 02593140 2007-10-18
in the intestine and the liver is one of the major enzymes that are necessary
to the
"first-pass effect" of the drug absorbance process. Such "first-pass effect"
will
stabilize a drug's bio-availability.
Owing to this phenomenon, the pharmacological scientists are aggressively
looking for safe, effective, and reversible UGT inhibitors to apply to drugs
with
low bio-availability due to their fast metabolism, for the purpose of
increasing
their efficacy. Such a need is especially evident in oral medicines.
Studies in UGT inhibitors and their interactions with drugs have been
conducted in recent year. Reported UGT inhibitors include silymarin
(Venkataramanan et al. 2000, Drug Metabolism and Disposition 28: 1270-1273),
quinoline (Dong et al., 1999, Drug Metabolism & Disposition 27:1423-1428),
oltipraz (Vargas et al., 1998, Drug Metabolism & Disposition 26:91-97),
tacrolimus (Zucker et al., 1999, Therapeutic Drug Monitoring 21:35-43), octyl
gallate, apigenin, imipramine, clozapine, acetaminophen, and emodin
(Radominska-Pandya et al., 1999, as mentioned earlier).
It was also reported that diazepam and flunitrazepam (FM2) can strongly
inhibit the activity of UGT2B (Grancharov et al., 2001, harmacol
Ther.,89(2):171-
86).
Since the aforementioned UGT inhibitors are active drug ingredients by
themselves and will induce prominent physical responses, they are not good
candidates as drug absorption enhancers.
It was well recognized among those who are familiar with the techniques
that a good UGT inhibitor for enhancing the bio-availability of drugs should
at
least posses the following three characteristics: (1) No or minimum
pharmacological effect, except inhibiting UGT; (2) The inhibition should be
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CA 02593140 2007-10-18
reversible. In other words, UGTs should be able to restore its normal
functions,
after the inhibitors were excreted or metabolized; and (3) The efficacy of the
inhibitor should be able to prominently lower the activity of UGTs in the
intestine
and the liver with a minimum dose.
It was known in recent years that grapefruit juice and certain components of
other natural products, such as narigin, naringenin, hesperidine and other
flavonoids, can inhibit some pharmacological activities.
US 6,121,23 depicts that by using essential oil, one can enhance the bio-
availability of an oral medicine in the intestine of a mammal. The method
involves co-administration of a therapeutic dose of the pharmaceutical
compound
and an essential oil or a component of essential oil where 10% inhibition was
demonstrated with the presence of no more than 0.01 wt. of essential oil or a
component of essential oil. In this US patent, it was also demonstrated that
essential oil enhances the bio-availability of the drug through its inhibition
of
cytochrome P450.
Another study indicated that flavonoids compounds prepared from the liver
of Long-Evants rat, such as naringenin, hesperetin, kaempferol, quercetin,
rutin,
flavone, a-naphthoflavone, and 13-naphthoflavone can inhibit the metabolism of
estrone and estradiol in microsomes (Zhu et al. 1998, J Steroid Biochem Mol
Bio1,64(3-4) : 207-15).
According to Chinese herbal medicine literature, due to its relatively milder
toxicity than synthetic compounds, Chinese herbal enhancers (CHEs) were widely
used in about 30%-75% of Chinese herbal medicine prescriptions. According to
Japan's Food and Drug Administration, among 210 official Chinese herbal
compound prescriptions, Glycyrrhizae radix is the most frequently used CHE (in
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150 or 71.4% prescriptions), followed by Zingiberis (in 42.9% prescriptions)
and
Zizyphi fructus (in 31.9% prescriptions). In the Japanese National Formulary
(2nd
Edition), the most frequently used CHEs are Glycyrrhizae radix (71.4%),
Zingiberis (42.9%), Holen (poria) (35.2%), Paeoniae radix (32.9%), Zizyphi
fructus (31.9%) and Cinnamami cortex (29.5%).
Other than these frequently used CHEs, studies regarding other CHEs are
rare. For the purpose of developing new UGT inhibitors, further investigations
on
some other CHEs are worthwhile. For instance, flavonoids-containing CHEs like
Scutellariae radix (contains baicalin, wogonin, baicalein, skullcap-flavon I
and
wogoin glucuronide), Artemisiae cpillaris herba (contains capillarisin,
cirsilineol,
cirsimaritin, genkwanin, rhamrcocitrin); and terpenoids-containing CHEs like
Alismatis rhizome (contains alisol monoacetate and triterpenoids), Moutan
radicis
cortex (contains paeoniflorin, oxypaeoniflorin and its benzoyl derivatives),
Aconiti tuber (contains aconitine, mesaconitine, jesaconitine and atisine),
Tragacantha (contains tragoside I and tragoside II), Persical semen (contains
24-
methylenecycloartanol), Cimicifugae rhizome (contains cimigenol, cimigenol
xyloside and its 12-hydroxyl derivatives and dahurinol).
Possible mechanisms of CHEs as enhancers of drug absorption include: (1)
catalysts: new active ingredients may be derived from the cooking or the
preparation process of Chinese herbal medicine; (2) carrier: carrying the
drug's
active ingredients going through barriers to reach its targets; (3) enzyme
inhibitor:
take UGT inhibitor as an example, if a drug's active ingredients cannot be
absorbed by oral administration due to UGT metabolism, it may become orally
absorbable by combining with a CHEs that lowers or restricts the "first-pass
effect".
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This application's inventor found that glycyrrhizin in Glycyrrhizae radix
and oleanolic acid and P-myrcene in Zizyphi fructus can enhance the partition
coefficient in drugs like acyclovir, buprenorphine, or buprenorphine and hence
increase these drug's transdermal permeation more than 1,000 percent in either
in
vitro or clinical trials. Reference: Dr. Oliver Yoa-Pu Hu's acyclovir Patent
(Taiwan Patent No.084682, US Patent No.6,162,459, Japan Patent No.2681881);
buprenorphine Patent (Taiwan Patent No.137835, US Patent No.6,004,969); and
piroxi cam Patent (Taiwan Patent No.133855).
New opiods drugs such as buprenorphine, nalbuphine and butorphanol were
developed recently. They are classified as narcotic agonist-antagonist
analgesics,
due to their dual agonistic and antagonistic effects on opiods-receptors
(Schmidt,
W.K. et al, Drug Alcohol Depend. 14, 339, 1985). In addition to having high
affinity to opiods-receptors, these dual-effect drugs can also be used as an
antagonist to compensate for the drawbacks of narcotic analgesics, such as to
lower their addictive effect and to drastically minimize their respiratory
inhibition.
According to Schmidt et al (1985), nalbuphine possesses both the affinity to
Kappa receptor (0P2) and the antagonist effect to Mu receptor (0P3). There is
no
obvious addiction or synergistic effect with only a slight respiratory
inhibition,
after a six-month continuous usage of nalbuphine. Therefore, in clinical
trials,
nalbuphine is safer than the traditional narcotic analgesics, and has
exhibited an
excellent therapeutic effect.
The drawback of this drug is its poor absorbability when delivered orally.
In Goodman and Gillman's study, the bio-availability of nalbuphine is 11 4 %,
while the bio-availability of nalbuphine in animal is 2.7 0.4%, a shorter
half-life.
In current clinical pharmacology, the drug can only be administered through
IV,
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CA 02593140 2007-10-18
not orally. The nalbuphine's pharmacokinetic studies indicate its half life
through
the excretion of liver is 5 hours, and about 7% of the drug are excreted to
the urine
in its original form (Birgit et al. 1996, Drug metabolism and Disposition,
25(1):1-
4; Birgit et al. 1998, Drug Metabolism and Disposition, 26(1):73-77; and
Richard
et al. 1990, Clin. Pharmacol Ther., 47:12-19).
Published literature has proved that nalbuphine is mainly metabolized
through UGT2B7 (Radominska-Pandya et al., 1999).
US 6,004,969 depicts a transdermal delivery of buprenorphine. The
method includes the delivery of a drug to patients that contains the following
pharmaceutical ingredients: 1) about 0.8% of buprenorphine or its HC1 salt; 2)
about 10-20% of one or a combination of the following drug enhancers: 2-
pinene,
trans-cinnamic acid, 13-myrcene or 13-myrcene; and 3) about 79.2-89.2% of one
or
a combination of the following inert ingredients: stearyl alcohol, sodium
carboxymethyl-cellulose, glycerol, cetyl alcohol, 1,3-propylene glycol, and
water.
Studies regarding the use of Chinese herbal medicine in diseases treatment
and prevention have become prominent in recent years. However, the issues
about what are the herbal medicines that can inhibit UGT and how to apply them
to the UGT-related therapeutic usages are still need to be addressed.
Besides, there are a lot of studies indicate that many pharmaceutical
excipients might have effect on the activity of cytochrome P450. It is thought
generally that most of the excipients are used to increase the volume, the
solubility or stability and so on of drugs. However, such point of view might
need
to be changed.
Mountfield et al. (Mountfield et al.,2000) indicates at 2000 that some of the
excipients influence CYP3A4 enzyme activity in microsomal enzyme experiment
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in vitro. Some other excipients are also reported in other researches, for
example,
PEG400 (Johnson et al., 2003), CremophorTm RH40(Wabdel et al., 2003) both
have effect on the enzyme activity of CYP3A4.
Bravo et al. (Bravo et al., 2003) have studied the effect of surfactant on
activity of enzyme. The medicine studied is colchicine that is metabolized by
P-gp
and CYP3A4 in vivo. The control group is colchicine dissolved in 0.9% NaC1
solution, and the experimental group is colchicine dissolved in 5% solutol
HS15-
NaC1 solution. The result shows that the concentration of experimental group
is
above two times of controlled group. The clearing rate of experimental group
is
obviously two folds lower than controlled group. And in-vitro hepatocyte
experiment shows that 0.003% solutol HS15 inhibits clearing rate of
colchicine. It
is worth noted that this kind of surfactants are thought to destroy cell
membrane in
other studies, affect metabolism of cell (Silva et al., 2004), change the
usage of
co-factor in catalysis reaction of enzymes or interaction between substrate
and
enzyme. However, there is no evidence showing that solutol HS15 at such
concentration has any impact on the intact of the cell under light microscope.
Therefore, the detailed mechanism needs to be investigated in the future.
Generally speaking, the research and development of new medicine could
be divided into several stages: New Chemical Entity (NCE), pre-clinical
toxicity
testing, pharmacological testing and finally, the clinical testing.
Traditional
chemical selection is to modify the structure on those who has pharmacological
activity or extract the biological chemicals from plants. Unfortunately, most
of
these chemicals are fat-soluble compound, thus, different excipients are added
to
improve solubility and stability of these chemicals. For pharmaceutical
industry, it
is inevitable to use excipient. Hence, this invention not only studies the
effect of
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using CHEs as UGT inhibitor and enhancer, but also the effect of excipients on
enzyme activity of UGT in order to provide more UGT inhibitors and enhancers
that are safe and effective.
In sum, the development of a safe, effective and reversible UGT inhibitor
will enable the oral administration of high "first-pass effect" drugs, and
will
minimize the side effect and the dosage of highly variable drugs. In addition,
the
toxicity of the carcinogenic compounds caused by UGT activities can also be
reduced.
Developing a safe, efficient and reversible UGT enhancer is also a desirable
goal of the pharmaceutical industry. It will help patients with low clearance
rate in
drug metabolism due to reduced liver functions to metabolize the drugs, and
enhance the detoxification function of the liver.
SUMMARY OF THE INVENTION
First of all, this invention provides a UGT2B inhibitor that can increase the
bio-availability of drugs. This inhibitor is a compound in a free base or a
pharmaceutically acceptable salt form that is selected from the group
consisting of:
capillarisin, isorhamnetin, 13-naphthoflavone, a-naphthoflavone, hesperetin,
terpineol,
(+)-limonene, P-myrcene, swertiamarin, eriodictyol, cineole, apigenin,
baicalin,
ursolic acid, isovitexin, lauryl alcohol, puerarin, trans-cinnamaldehyde, 3-
phenylpropyl acetate, isoliquritigenin, paeoniflorin, gallic acid, genistein,
glycyrrhizin,
protocatechuic acid, ethyl myristate, umbelliferone, terpineol, (+)-limonene,
13-
myrcene, cineole, apigenin, ursolic acid, gallic acid, glycyrrhizin,
protocatechuic
acid, PEG (Polyethylene glycol) 400, PEG 2000, PEG 4000, TweenTm 20, TweenTm
CA 02593140 2012-11-02
60, TweenTm 80, BRIJ 58, BRIJ 76, Pluronic F68, Pluronic F127, and a
combination thereof.
This invention also provides a pharmaceutical composition that includes the
aforementioned UGT2B inhibitor as its active ingredient, and a
pharmaceutically
acceptable inert ingredient. The pharmaceutical composition in this invention
can
decrease the enzymatic activity of UGT2B, and thus increase the bio-
availability
of analgesics such as morphine.
Secondly, this invention provides an UGT2B enhancer that can improve the
detoxification function of the liver. This enhancer is a compound with a free
base
or a pharmaceutically acceptable salt comprises of selected materials from the
following: nordihydroguaiaretic acid, wogonin, trans-cinnamic acid, baicalein,
quercetin, daidzein, oleanolic acid, homoorientin, hesperetin, narigin,
neohesperidin, (+)-epicatechin, hesperidin, liquiritin, eriodictyol,
formononetin,
quercitrin, genkwanin, kaempferol, isoquercitrin, (+)-catechin, naringenin,
daidzin,
(-)-epicatechin, uteolin-7-glucoside, egosterol, rutin, luteolin, ethyl
myristate,
apigenin, 3-phenylpropyl acetate, umbelliferone, glycyrrhizin, protocatechuic
acid,
poncirin, isovitexin, 6-gingerol, cineole, genistein, and trans-
cinnamaldehyde.
This invention also provides a pharmaceutical composition that includes the
aforementioned UGT2B enhancer as its active ingredient, and a pharmaceutically
acceptable inert ingredient. The pharmaceutical composition in this invention
can
increase the enzymatic activity of UGT2B, and thus increase the clearance rate
of
a drug.
According to an aspect, the invention provides for a pharmaceutical
composition comprising: an opioid; an Uridine diphosphate ( UDP ) -
glucuronosyltransferases 2B (UGT2B) inhibitor, the inhibitor being a free base
or
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a pharmaceutically acceptable salt of capillarisin, isorhamnetin, fl-
naphthoflavone,
a-naphthoflavone, hesperetin, swertiamarin, eriodictyol, baicalin, isovitexin,
puerarin, isoliquritigenin, paeoniflorin, genistein, ethyl myristate,
umbelliferone,
polyethylene glycol (PEG) 400, PEG 2000, PEG 4000, Tween0 20, Tweent 60,
Tween0 80, BRIJ 58, BRIJ 76, Pluronic0 F68, Pluronic0 F127, or a mixture
thereof; and a pharmaceutically acceptable inert ingredient.
According to another aspect, the invention provides for a pharmaceutical
composition comprising a mixture intended for oral administration, the mixture
comprising: an opioid being (-)-morphine, naloxone, nalorphine, oxymorphone,
hydromorphone, dihydromorphine, codeine, naltrexone, naltrindole, nalbuphine,
buprenorphine, or a mixture thereof; an Uridine diphosphate ( UDP ) -
glucuronosyltransferases 2B (UGT2B) inhibitor, the inhibitor being a free base
or
a pharmaceutically acceptable salt of capillarisin, isorhamnetin, fl-
naphthoflavone,
a-naphthoflavone, hesperetin, swertiamarin, eriodictyol, baicalin, isovitexin,
lauryl alcohol, puerarin, trans-cinnamaldehyde, 3-phenylpropyl acetate,
isoliquritigenin, paeoniflorin, genistein, ethyl myristate, umbelliferone,
terpineol,
(+)-limonene, 13-myrcene, cineole, apigenin, ursolic acid, gallic acid,
glycyrrhizin,
protocatechuic acid, polyethylene glycol (PEG) 400, PEG 2000, PEG 4000,
Tween0 20, Tween0 60, Tween0 80, BRIJ 58, BRIJ 76, Pluronic0 F68,
PluronicCD F127, or a mixture thereof; and a pharmaceutically acceptable inert
ingredient.
These features and advantages of the present invention will be fully
understood and appreciated from the following detailed description of the
accompanying figures.
11 a
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BRIEF DESCRIPTION OF THE FIGURES
Figure 1 depicts the temporal effect of capillarisin on the concentration of
nalbuphine in blood, after the SD rats were orally treated with nalbuphine.
Figure 2 depicts the temporal effect of capillarisin on the concentration of
nalbuphine in blood, after the SD rats were intravenously injected with
nalbuphine.
Figure 3 compares the temporal effect of the concentration of nalbuphine in
blood of SD rats between the samples and the controls. The control animals
were
treated with nalbuphine orally as well as intravenously while the sample
animals
were treated with nalbuphine and capillarisin.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
In the first dimension, this invention provides a UGT2B inhibitor that is a
compound in a free base or a pharmaceutically acceptable salt form that is
selected from the group consisting of: capillarisin, isorhamnetin, $ -
naphthoflavone, a -naphthoflavone, hesperetinõ terpineol, (+)-limonene, 16 -
myrcene, swertiamarin, eriodictyol, cineole, apigenin, baicalin, ursolic acid,
isovitexin, lauryl alcohol, puerarin, trans-cinnamaldehyde, 3-phenylpropyl
acetate,
isoliquritigenin, paeoniflorin, gallic acid, genistein, glycyrrhizin,
protocatechuic
acid, ethyl myristate, umbelliferone, PEG (Polyethylene glycol) 400, PEG 2000,
PEG 4000, Tween 20, Tween 60, Tween 80, BRIJ 58, BRIJ 76, Pluronic F68,
Pluronic F127, and a combination thereof.
In a good example of this invention, the UGT2B inhibitor is a compound in
a free base or a pharmaceutically acceptable salt form that is selected from
the
group consisting of: capillarisin, isorhamnetin, 13' -naphthoflavone, a -
naphthoflavone, hesperetinõ terpineol, (+)-limonene, 13 -myrcene, swertiamarin
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and eriodictyol; or a combination of them. In a better example, the UGT2B
inhibitor contains capillarisin.
It was confirmed that UGT2B inhibitor of this invention can increase the
bio-availability of a drug. The applicability of UGT2B inhibitor in the
preparation of pharmaceutical compositions, especially the morphine
analgesics,
is also predicted in this invention.
Therefore, this invention provides a pharmaceutical composition which
contains:
(a) a pharmacologically active ingredient of aforementioned UGT2B inhibitor,
and
(b) a pharmaceutically acceptable inert ingredient.
In the second dimension, this invention provides an UGT2B enhancer that
is a compound in a free base or a pharmaceutically acceptable salt form that
is
selected from the group consisting of: nordihydroguaiaretic acid, wogonin,
trans-
cinnamic acid, baicalein, quercetin, daidzein, oleanolic acid, homoorientin,
hesperetin, narigin, neohesperidin, (+)-epicatechin, hesperidin, liquiritin,
eriodictyol, formononetin, quercitrin, genkwanin, kaempferol, isoquercitrin,
(+)-
catechin, naringenin, daidzin, (-)-epicatechin, uteolin-7-glucoside,
egosterol, rutin,
luteolin, ethyl myristate, apigenin, 3-phenylpropyl acetate, umbelliferone,
glycyrrhizin, protocatechuic acid, poncirin, isovitexin, 6-gingerol, cineole,
genistein, and trans-cinnamaldehyde.
In a good example of this invention, the UGT2B enhancer is a compound in
a free base or a pharmaceutically acceptable salt form that is selected from
the
group consisting of: nordihydroguaiaretic acid, wogonin, trans-cinnamic acid,
baicalein, quercetin, daidzein, oleanolic acid, homoorientin, hesperetin,
narigin,
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neohesperidin, (+)-epicatechin, hesperidin, liquiritin and eriodictyol. A
better
example of UGT2B enhancer contains nordihydroguaiaretic acid.
It was confirmed that the UGT2B enhancer of this invention can increase
the clearance rate of a drug. The applicability of the UGT2B enhancer in the
preparation of pharmaceutical compositions is also predicted in this
invention.
Therefore, this invention provides a pharmaceutical composition which
contains:
(a) a pharmacologically active ingredient of aforementioned UGT2B enhancer,
and
(b) a pharmaceutically acceptable inert ingredient.
In the pharmaceutical composition of this invention, the UGT2B enhancer
is used along with a pharmaceutically effective amount of liver disease drugs.
The liver diseases include, but not limited to, viral hepatitis, chronic
hepatitis,
alcoholic liver cirrhosis, compensated cirrhosis, and hepatic failure.
The UGT2B inhibitor or enhancer used in this invention is easily obtainable
to persons familiar with the technology. It can be chemically synthesized in
the
laboratory, purchased from chemical company, or purified from pertinent
natural
sources. The UGT2B inhibitor or enhancer used in the following experimental
examples are purchased from Sigma Chemical Co., Nacalai Tesque (Kyoto, Japan)
and Indofine Chemical Co., Inc. (Somerville, New Jersey).
In this invention, the term "pharmacologically active ingredient" refers to a
pharmaceutical composition that can either inhibit or enhance the UGT2B
activity
when a proper quantity was used in therapeutic purposes. The proper quantity
of
this active ingredient varies by type of disease, patient's weight, age,
health
condition, and the way the drug was delivered. It should be determined by
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technically qualified persons.
In this invention, the term "pharmaceutically acceptable" means that the
salt component of an UGT2B inhibitor or enhancer must be compatible with other
ingredients of the pharmaceutical composition, and will not hamper the
application of this composition onto an individual.
The pharmaceutical composition of this invention can be used exclusively
on an individual or in combination with morphine-like analgesic agents.
In a good example of this invention, the morphine-like analgesic agents
include: (-)-morphine, naloxone, nalorphine, oxymorphone, hydromorphone,
dihydromorphine, dihydromorphine, naltrexone, naltrindole, nalbuphine, and
buprenorphine. In a better example, the morphine-like analgesic is nalbuphine.
The pharmaceutical composition of this invention can be manufactured as
an intravenously- or orally-administered substance.
The pharmaceutical composition of this invention can be manufactured in a
form for intravenous administration with the addition of sterile water or non-
water
solution, dispersion, suspension or emulsion, and sterile powder that can be
reconstituted to sterile solution for injection. Examples of suitable water or
non-
water inert ingredients, diluents, solvents, or carriers include: water,
alcohol,
propylene glycol, polyethylene glycol, glycerol, or other similar compounds,
and
organic ester such as ethyl oleate.
It is better that the pharmaceutical composition of this invention be
manufactured in a form that can be administered orally. The oral forms include
solid substances (e.g., capsule, tablet, powder, and granule) and liquid
substance
(e.g., emulsion, solution, dispersion, suspension).
In addition, the pharmaceutical composition of this invention and other
CA 02593140 2007-10-18
drugs can be presented in different forms. For example, it can be delivered as
a
tablet, by injection, or as an oral syrup. Furthermore, it is noteworthy that
the
pharmaceutical composition of this invention can be administered with other
drugs at the same time or sequentially. For instance, as a tablet, it can be
concentrated in a single tablet or spread to several tablets and can be given
simultaneously or sequentially. All combinations, delivery methods and
sequences can be flexibly administered by persons who are familiar with these
techniques.
The dose and frequency in delivering the pharmaceutical composition of
this invention varies according to: the severity of the illness to be treated,
route of
delivery, and the patient's weight, age, physical condition and the response.
In
general, the dosage of the pharmaceutical composition of this invention is
estimated to fall in the range of 0.01 mg/Kg weight to 20 mg/Kg, as a single
dose
or several doses, and can be delivered through a non-gastroenterological or
oral
route.
A detailed description of better examples
The following experiments further describe the invention in detail. Please
be advised that these experiments are examples. They are not the limitations
of
this invention's applications.
Experiment 1. in vitro experiment of UGT2B inhibitor
Material and method:
1. The preparation of UGT2B inhibitor
In the following experiment, 27 different kinds of CHEs and 10 different
excipients were used as UGT2B inhibitor in this invention. These CHEs are pure
compounds available commercially, and were purchased from Sigma Chemical
16
CA 02593140 2007-10-18
Co., Nacalai Tesque (Kyoto, Japan) and Indofine Chemical Co., Inc.
(Somerville,
New Jersey). Their category, name, source and chemical formula are listed in
Table 1. These UGT2B inhibitors are dissolved in alcohol at the concentration
of
1, 10, 100pM for the following experiment.
Besides, these excipients are commercially available pure compounds, they
are PEG (Polyethylene glycol) 400, PEG 2000, PEG 4000, Tween 20, Tween 60,
Tween 80, BRIJ 58, BRIJ 76, Pluronic F68, Pluronic F127. These excipients
are dissolved in water and make up to 0.5%, 5%, 50%(wt%, w/v) for the
following experiment.(Note: BRIJ is a registered trademark of ICI Americas,
Inc.;
Pluronic is a registered trademark of BASF Corporation.)
Table 1. The category, name, source and chemical formula of UGT2B inhibitors
Source
Chemical formula and molecular
Category Name
(Scientific name) weight
OH
HO 0
Flavonoid Apigenin Chamomillae flos
OH 0
MW: 270.24
OH ¨
40
Isovitexin Swertiae herba HO 0
Glc0
OH 0
MW : 432.38
OH
HO 0
OMe
Isorhamnetin Sennae folium
OH
OH 0
MW : 316.27
HO io 0 0
Aurantii fructus
Umbelliferone
immaturus (7-Hydroxycoumarin)
MW :162.14
17
CA 02593140 2007-10-18
Source Chemical
formula and molecular
Category Name
(Scientific name) weight
=H
0 OMe
RutO 0 0
_ 1
Diosmin
OH 0
MW : 608.55
0 OMe
0
OH
Hesperetin Citri reticulatae HO 0
OH 0
MW : 302.28
Glucuronic acid-0 is 0 40
I
baicalin Scutellariae radix
HO
OH 0
MW : 446.35
=H
=H
0
0
CH2OH
HO 0
Puerarin Puerariae radix 40 I
. SOH
MW : 416.38
HO 0 0 0 0
I
Artemisiae
Capillarisin Me0 OH
capillaris herba OH 0
MW : 316.27
HO 0
SI I
Genistein Puerariae radix
OH
OH 0 410
MW : 270.24
0 0
a - 0 1
Naphthoflavone _
o
MW : 272.3
0
/3- 0 0 ,
_
Naphthoflavone
0 0
MW : 272.3
18
CA 02593140 2007-10-18
Source Chemical
formula and molecular
Category Name
(Scientific name) weight
0 OH
0
Cinnamami HO 0 OH
Eriodictyol
cortex
OH 0
MW : 288.25
0
,---.....
3-Phenylpropyl Cinnamami H3C 0
1101
Acetate cortex
MW :178.23
0
4,
H. ,C,
40 C=C, H
Trans- Cinnamami
Cinnamaldehyde cortex H
MW :132.16
cH2
Amome I
1
-,,, CH2 3-Myrcene cardamomi
I
fructus
113C-CH,
MW :136.23
CH3
essential oil
el
Cinae
Terpineol
flos(Santonica)
H3C CH3
OH
MW :154.25
CH3
Cardamomi el
(+)-Limonene
fructus
H3C CH,
MW :136.23
Ethyl Myristate Myristicae semen
Cinae ICH3
o
Cineole
flos(Santonica)
H3C CH3
MW :154.25
19
CA 02593140 2007-10-18
Source Chemical formula and molecular
Category Name
(Scientific name) weight
0
0 _Io_cHa
0
Tennins Paeoniflorin Paeoniae radix HOCH, 0 OH
HO HO
OH
MW: 480.47
0
OO
Swertiamarin Swertiae herba W=o-beta-D-glucose
OH
CH2
MW : 374.34
HO 40 OH
chalcon Isoliquritigenin Astragali radix
OH 0
MW 256.25
COOH
CO
OH awl*
Glycyrrhizae
Saponin Glycyrrhizin
0 WWI
radix
MW: 822.94
H,C
õleo COOH
Ursolic Acid Zizyphi fructus
HO
NC CH,
MW : 456.70
COOH
Protocatechuic Cinnamami
401
acid cortex OH
OH
MW :154.12
COOH
Gallic Acid
HO OH
OH
MW 170.12
H3C OH
Lauryl Alcohol
MW :186.33
CA 02593140 2007-10-18
2. The preparation of liver microsome
The Sprague-Dawley male rats, weighted 300-400 g are used as model
animals in these experiments. The microsome is prepared as follows:
i. Sacrifice a rat after 12-16 hours fasting, take its liver; wipe dry the
liver and
weigh it.
ii. Add 300% (by volume) of cold 0.3M sucrose soluation, and homogenize the
liver.
iii. Centrifuge the homogenized liver suspension at 9000 x g speed (KS-800,
Kubota, made in Japan) for 10 min at 4 C. Collect the supernatant.
iv. Centrifuge the supernatant at 105,000xg speed (L8-60M, Beckman, made in
USA) for 60 min at 4 C.
v. Remove the supernatant, add 0.3M sucrose (of the same volume) and
repeat
the homogenization. The homogenized liver suspension is the microsome.
Store the microsome at -70 C.
Thaw the microsome before use. Add 5p1/m1 BRIJO35 (Fisher et al. 2000,
Drug Metabolism & Disposition. 28(5):560-6) into microsome by a ratio of
microsome : BRIJS35 = 8 : 1 (v/v).
[noteBRIJO 35 SOLUTION 30% WN , BRIJ is a registered trademark of
ICI Americas, Inc.]
3. Measure the protein amount in microsome
The protein amount is determined as follows:
i. Take 0.1 ml microsome and dilute to 5m1 0.85 % NaC1 (50-fold dilution,
v/v).
Take 0.2 ml of the diluted microsome and place it in a capped test tube
(triple
experiments). Separately, replacing microsome with 0.2 ml NaCl as the
control.
21
CA 02593140 2007-10-18
ii. Add 2.2 ml Biuret regent (SIGMA, 690-A) into each test tube, mix well and
store at room temperature for 10 min.
iii. Add 0.1 ml folin regent (SIGMA, 690-A), mix immediately, and store at
room
temperature for 30 min. Measure the absorbance at 550 nm wave length
within the 30 min. Measure the concentration of protein in microsome based
on the standard curve generated by various bovine albumin concentration and
absorbance.
4. The measurement of the inhibition of UGT2B activity in vitro
i. (A)solution: 17p1 1M Tris-HC1 buffer, 17p1 50mM MgCl2, 40p1 microsome and
10p1 150mM UDPGA solution.
(B)solution: mix 20mM nalbuphine solution with the inhibitor to be tested at
the 1 : 1 ratio.
ii. Mix solution (A) and 17p1 solution (B) thoroughly, place in a 37 C water
bath
and shake at 125 rpm for 60 min.
iii. Add lml ACN to interrupt the reaction.
iv. Centrifuge at 130,000 xg speed at 4 C for 5 min, and
v. Take 150 Ill supernatant and analyze the concentration of nalbuphine in a
high
performance liquid chromatography (HPLC).
5. The measurement of nalbuphine concentration
(1) Condition of HPLC
The mobile phase consists of 15% sodium acetate (5 mM/L, pH 3) and 85%
ACN with a 1.0 ml/min flow rate. Set the spectrophotometer's (RF-551,
Shimadzu, Kyoto, made in Japan) excitation wave length at 210 nm and emission
wave length at 345 nm. Set the Ultra-violet detector's (SPD-10A, Shimadzu,
Kyoto, made in Japan) wave length to 210 nm.
22
CA 02593140 2007-10-18
(2) Prepare the standard solution
Prepare nalbuphine solution at 0.5, 1, 2.5, 5, 10, 15, 18, 20 mM
concentrations. Resolve the standard solution in water, but dilute the
solution with
alcohol if it contains inhibitor.
Use the same protocol as described in step 4 (The measurement of the
inhibition of UGT2B activity in vitro), except that the de-ionized water,
instead of
the "150mM UDPGA solution" be used in the (A) solution.
After HPLC analysis, obtain a corrected curve by plotting HPLC reading of
nalbuphine wave heights against its relative concentration. Analyze the
standard
deviation (SD), coefficient of variance (%CV), and %error to examine the
accuracy.
Results:
The result is displayed in Table 2. Capillarisin has the best inhibitory
effect
on the metabolism of nalbuphine in microsome. The inhibition rate could reach
111.077 ( 21.807)%. Other CHEs including isorhamnetin,I3-naphthoflavone,a-
naphthoflavone, hesperetin, terpineol, (+)-limonene, 13 -myrcene,
swertiamarin,
and eriodictyol also have at least 30%inhibition rate.
Table 2. The effect of the inhibitor on the metabolism of nalbuphine in liver
microsome
Name % Inhibition (Mean SD)
8.5 M 85 M 850 M
apigenin -26.668
13.062 53.998 15.763 -16.067 17.864
isovitexin 29.821 9.786 -5.528
9.096 -12.377 8.912
isorhamnetin 88.419 11.605 85.132
14.703 106.846 8.102
umbelliferone -14.707 5.810 4.596 8.236
-20.590 18.244
hesperetin 51.736 21.691 -59.096
18.879 -124.34 49.356
23
CA 02593140 2007-10-18
baicalin 36.298 9.403 46.253 22.923 44.262 2.879
puerarin 3.919 5.607 18.289 7.685
10.103 5.841
capillarisin 111.077 21.807
105.410 21.808 105.257 19.306
genistein 15.630 6.046 5.733 6.406 -
9.200 5.182
a- naphthoflavone 72.33 2.811 97.79 4.370
90.81 7.175
13.- naphthoflavone 95.82 5.461 86.02 16.487
99.72 15.877
eriodictyol -1.061 3.714 -17.133 2.297 14.114 2.878
3-phenylpropyl acetate -26.288 27.337 17.280 5.837
2.390 22.463
trans-cinnamaldehyde -6.973 3.782 -3.099 9.457 18.284 10.350
13 -myrcene 16.812 1.716 35.290 0.220
34.883 7.296
terpineol 40.558 6.511 62.367 2.582
58.164 4.241
(+)-limonene 18.284 0.793 45.284 7.844
44.238 2.284
ethyl myristate -27.845 14.692 -12.134 5.706
6.326 1.484
cineole -8.413 18.562 -
12.107 6.679 58.890 8.558
paeoniflorin 12.093 8.544 17.797 9.248 -
2.966 9.529
swertiamarin 28.239 2.248 27.930 4.129 -
2.499 6.899
isoliquritigenin -2.482 5.506 17.592 4.565
18.637 16.623
glycyrrhizin 9.926 6.659 -20.298 3.674 -5.653 6.620
ursolic acid 34.171 18.576 17.267 14.316 6.482 18.840
protocatechuic acid -20.210 5.957 -4.563 3.372
9.844 1.872
gallic acid 15.842 4.418 8.051 3.024
12.223 4.080
lauryl alcohol 14.422 3.370 19.496 3.464
6.232 6.752
Besides, figure 3 and 4 depict the result about the excipients in the
experiment above. PEG 4000 has the best inhibitory effect on the metabolism of
nalbuphine in microsome, the inhibitory rate reaches 108.222 ( 3.356) %; the
24
CA 02593140 2007-10-18
best inhibitory rate of the other excipients reach 60% at least.
Table 3 The effect of the excipient on the metabolism of nalbuphine in liver
microsome-1
Name % Inhibition (Mean SD)
Concentration of 0.0425% 0.425% 4.25%
excipients ( wt%,
w/v )
PEG 400 102.137 6.156 97.030
12.875 93.359 10.893
PEG 2000 32.615 5.192 86.635
7.948 97.011 4.588
PEG 4000 71.410 3.109 108.222 3.356 107.329
6.242
Tween 20 83.734 6.465 99.863 8.487 93.926
7.862
Tween 60 78.475 4.915 30.917 7.429 27.327
9.644
Tween 80 91.163 5.861 92.387 6.302 99.651
3.344
Table4 The effect of the excipient on the metabolism of nalbuphine in liver
microsome-2
Name % Inhibition (Mean SD)
Concentration of 0.02125% 0.2125% 2.125%
excipient ( wt%,
w/v )
BRIJ 58 78.117 6.634 41.592 5.606 -33.648
11.873
BRIJ 76 40.488 2.327 29.959 2.572 60.439
7.751
Pluronic F68 97.431 5.764 98.925 4.475 65.768
2.371
Pluronic F127 14.499 28.385 70.247 36.296 83.845
16.451
CA 02593140 2007-10-18
Experiment 2. in virto experiment of UGT2B enhancer
This experiment uses the same protocol described in Experiment 1, except
by testing the 40 CHEs listed in Table 5 as the UGT2B enhancer. Those CHEs are
commercially available pure compounds, acquired from Sigma Chemical Co.,
Nacalai Tesque (Kyoto, Japan) and Indole Chemical Co. Inc (Somerville, New
Jersey). Their categories, names, sources, and chemical compositions are
described in Table 5.
Table 5. Category, name, source, and chemical composition of the UGT2B
enhancers.
Source
Chemical formula and molecular
Category Name
(scientific name) weight
0 OH
Me0 o
Artemisiae
Flavonoid Genkwanin go I
cpillaris herba
OH 0
MW : 284.27
0 OH
0
1
Apigenin Chamomillae flos HO 0
OH 0
MW : 270.24
= H
0 OH
0 Luteolin Digitals folium HO 0I
OH 0
MW : 286.24
= H
0 OH
Glc0 0 0
Lute0lin_7 -
Glucoside Digitals folium I
OH 0
MW: 448.39
26
CA 02593140 2007-10-18
Source Chemical
formula and molecular
Category Name
(scientific name) weight
OH
HO 401 0
SOH
I
Homoorientin Swertiae herba
Glc0
OH 0
MW: 448.39
HO 0 I 0 0 OH
Isovitexin Swertiae herba
Go()
OH 0
MW : 432.38
OMe
XO 0
SOH
Neohesperidin
Aurantii fructus
0
immaturus OH 0
X: Neohesperidoside
MW : 610.57
HO 401 0
I
Formononetin Astragali radix
o el
OMe
MW : 268.27
0 OH
401
I
KaeMpfer01 Sennae folium HO 0
OH
OH 0
MW : 286.24
sH
0 OH
Hydrangeae HO o
Isoquercitrin
dukis folium 0 1
0-beta-D-glucose
OH 0
MW: 464.38
0 OH
6-Gingerol Zingiberis HO lel CH3
OMe
MW : 294.38
27
CA 02593140 2007-10-18
Source Chemical
formula and molecular
Category Name
(scientific name) weight
0
HO 0 ,,,
1.1101-1,
0
Liquiritin Glycyrrizae radix
0
KOHY
OH
OH
MW
OH
HO o
Aurantii fructus
naringenin
immaturus
OH 0
MW 272.26
HO 0 0
Aurantii fructus
Umbelliferone
immaturus (7-Hydroxycoumarin)
MW 162.14
00 OH
OH
Rutin Sophorae flos HO 0
ORut
OH 0
MW 664.58
OMe
HAcE7 0,3 ¨jor;
0
Aurantii fructus 3 H
"IIP OH
Hesperidin OH OH __
immaturus OH
OH 0
MW 610.57
=H
OMe
RutO 0
Diosmin I
OH 0
MW 608.55
OMe
OH
Hesperetin Citri reticulatae HO 0
OH 0
MW 302.28
28
CA 02593140 2007-10-18
Source Chemical
formula and molecular
Category Name
(scientific name) weight
OMe
0 010
I
Wogonin Scutellariae radix HO 0
OH 0
MW : 284.27
HO 0 01
Baicalein Scutellariae radix SI
HO
OH 0
MW: 270.24
HO 0 0
I
Daidzein Puerariae radix
OH
o
MW : 254.24
tH.0:1,1
0 0 0
I
Daidzin Puerariae radix OH
0 I.
OH
OH
MW: 416.38
0 OH
io
I
Quercitrin Viscum coloratum HO 0 OH
ORha
OH 0
MW: 448.38
I* OH
0
I
Quercetin Viscum coloratum HO 0 OH
OH
OH 0
MW: 302.2
OH
CH3 40
Nordihydroguaiar HO
-
5 OH
etic acid CH,
HO
MW : 302.36
HO 0 0
I
Genistein Puerariae radix
OH
OH 0 SI
MW : 270.24
29
CA 02593140 2007-10-18
Source Chemical formula and molecular
Category Name
(scientific name) weight
OMe
Neohespendose 0 ial 0 1.1
Aurantii fructus
Poncirin 1W
immaturus
OH 0
MW : 594.37
,OH
XO ao 0
Narigin Aurantii fructus
immaturus OH 0
X:Rhamnoglucoside
MW : 580.54
0 OH
0
OH
Cinnamami HO 0
Eriodictyol
cortex
OH 0
MW : 288.25
0
3-Phenylpropyl Cinnamami H3C4:3
Acetate cortex
MW :178.23
0
H. ,C,
10 C=C H
Trans- Cinnamami \H
Cinnamaldehyde cortex
essential oil
MW :132.16
Ethyl Myristate Myristicae semen
Cinae
Cineole a-13>
0
flos(Santonica)
C
H3c CH3
MW :154.25
0 OH
itTennins (+)-Epicatechin Gambir HO 0 OH
OH
OH
MW : 290.27
CA 02593140 2007-10-18
Source Chemical
formula and molecular
Category Name
(scientific name) weight
OH
HO 0
OH
(+)-Catechin Paeoniae radix
OH
OH
MW 290.27
OH
HO 0
OH
(-)-Epicatechin Gambir
OH
OH
MW 290.27
cH,
CH,
CH3
Sterol Ergosterol Holen(poria) CH, OM
HO
MW 396.65
H,0,s COOH
CH, goo
Saponin Glycyrrhizin Glycyrrhizae OHOH
radix 6T4
H,C CH,
0
OH
MW : 822.94
1-13c C H3
CH3 00 COOH
Triterpenoid Oleanolic Acid Zizyphi fructus
HO
H3C CH3
MW: 456.70
COOH
PrOtOCateChUiC Cinnamami
acid cortex OH
OH
MW 154.12
0
Trans-Cinnamic c=c'cOH
Acid ===
31
CA 02593140 2007-10-18
Source
Chemical formula and molecular
Category Name
(scientific name) weight
MW :148.16
Result:
The results are summarized in Table 6. The nordihydroguaiaretic acid
exhibited the best enhancement effect on the metabolism of nalbuphine in liver
microsome. It could reach a -188.09( 16.566)%rate of inhibition. The other
CHEs
including wogonin, trans-cinnamic acid, baicalein, quercetin, daidzein,
oleanolic
acid, homoorientin, hesperetin, narigin, neohesperidin, (+)-epicatechin,
hesperidin,
liquiritin, eriodictyol could have at least 30% enhancement rate.
Table 6. The effect of the enhancers on the metabolism of nalbuphine in liver
microsome
Enhancer % Inhibition (Mean SD)
8.5 1µ4 85 M 850 tiM
genkwanin -26.999 4.509 -77.684
20.682 -43.231 18.793
apigenin -26.668 13.062 53.998 15.763 -16.067
17.864
luteolin -25.508 26.594 -28.324 15.205 -
17.558 12.135
luteolin-7-glucoside -45.053 3.583 -36.109 9.203 -47.869
26.599
homoorientin -128.53
27.613 -127.21 28.005 -133.17 26.968
isovitexin 29.821 9.786 -5.528 9.096 -12.377
8.912
neohesperidin -66.586 9.614 -113.63 8.986 -113.46
16.721
formononetin -86.463 3.490 -31.471 4.775 -24.680
4.417
kaempferol -19.545 14.198 -76.028
27.291 -71.058 4.509
isoquercitrin -8.089 2.404 -19.119
21.887 -71.696 20.468
6-gingerol -14.156 4.469 -13.378 3.967 -12.341
1.579
liquiritin -9.382 1.803 -17.704 8.510 -29.167
2.737
32
CA 02593140 2007-10-18
6-gingerol -62.872 41.065 -48.35 13.179 -22.61 14.532
umbelliferone -14.707 5.810 4.596 8.236 -20.590 18.244
rutin -19.854 19.742 -
29.414 29.485 -33.458 13.373
hesperidin 2.163 2.725 -31.997 10.339 -42.02 4.245
hesperetin 51.736 21.691 -59.096 18.879 -124.34
49.356
wogonin -128.63 8.286 -153.23 6.491 -135.55 2.879
-145.76 8.474
baicalein -99.628 12.832 -125.44 8.620
(212.5 1.1,M)
-138.40 2.307
daidzein -127.64 20.806 -138.29 4.617
(4251.1M)
daidzin -29.524 21.990 -41.466 16.977 -61.04
21.066
quercitrin -81.27 15.027 -83.60 27.446 -55.57 12.151
quercetin -81.440 5.593 -142.98 18.532 -119.26
19.351
nordihydroguaiaretic
-142.15 41.001 -165.04 22.961 -188.09
16.566
acid
genistein 15.630 6.046 5.733 6.406 -9.200 5.182
poncirin -16.068 8.122 -8.448 8.261 -7.098
18.196
narigin -124.10 16.541 -80.70 4.927 -98.15 5.276
3-phenylpropyl acetate -26.288 27.337 17.280 5.837 2.390 22.463
trans-cinnamaldehyde -6.973 3.782 -3.099 9.457 18.284 10.350
ethyl myristate -27.845 14.692 -12.134 5.706 6.326 1.484
cineole -8.413 18.562 -12.107 6.679 58.890 8.558
(+)-epicatechin -32.553 1.578 -47.075 0.533 -8.118 1.256
(+)-catechin -68.387 7.344 -54.783 9.381 -65.261 47.038
(-)-epicatechin -28.68 17.634 -23.53 27.304 -48.35 39.354
33
CA 02593140 2007-10-18
ergosterol -12.501 28.884 -28.311 20.311 -
34.561 19.877
glycyrrhizin 9.926 6.659 -20.298 3.674 -5.653
6.620
oleanolic acid -87.200 24.408 -135.54 15.185 -
128.64 22.066
protocatechuic acid -20.210 5.957 -4.563 3.372
9.844 1.872
eriodictyol -1.061 3.714 -17.133 2.297 14.114
2.878
trans-cinnamic acid -153.03 24.865 -109.06 18.574 -
134.74 3.90
Experiment 3. The effect of UGT2B inhibitors on the concentration of
nalbuphine
taken orally
Material and methods:
1. Experimental animal
Healthy male Sprague-Dawley rat, weight 500-600g, acquired from
National Laboratory Animal Breeding and Research Center in Taiwan, are used.
After the acquisition, the animals are kept in a room with constant
temperature (at
25 1 C), humidity and day light (12 hours per day) for one week. Before the
experiment, the animals are fasted for 12-16 hours. The drugs are administered
orally.
2. Preparation of UGT2B inhibitor and nalbuphine solution
Standard solution of nalbuphine is dissolved in water, and all inhibitors are
dissolved in alcohol.
3. Methods:
i. Anesthetize the rat with 3-5mg/100g body weight of pentobarbital
intraperitoneally (I.P.). The rat will be anesthetized in about 20-30 min.
ii. Insert the PE-50 catheter tube into external jugular vein to sample the
blood.
iii. Orally administered 6 rats with UGT2B inhibitor--capillarisin (4mg/Kg
body
weight) and nalbuphine solution (100mg/Kg body weight). Use another 6 rats
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CA 02593140 2007-10-18
as the control. They were given only nalbuphine solution (100 mg/Kg body
weight). Take 0.3 mL blood sample at the 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6,
8,
12 and 24 hours after treated with these drugs, centrifuge at 10000 rpm, and
take 0.1 mL serum for an analysis of the concentration of nalbuphine.
4. Measurement of the concentration of nalbuphine
(1) Sample preparation
Place 0.1 mL serum in a 10 mL test tube and quickly transfer it to an ice
bath. Add 50 pL internal standard (buprenorphine 5 pg/mL) and 05 mL sodium
carbonate buffer (0.5 mole/L, pH=9.25) and mixed well. Extract the serum with
4
mL n-hexane and isoamyl alcohol mixture (9:1 (v/v)), and shake for 20 min.
Centrifuge at 1,080xg speed at 4 C for 15 min, then place it in a -80 C
freezer
until the water layer froze. Transfer the organic solvent layer into another
clean
test tube, lyophylized until dried. Add 100pL ACN to dissolve the dried
material,
auto-pipette 50pL and applied to HPLC for analyzing the concentration.
(2) Condition for HPLC analysis
Moving phase consists of 15%sodium acetate buffer (5 mM/L, pH=3.75)
and 85%ACN, with flow rate of 1.3 mL per min, an electrical chemical detector
(ECD (electrochemical detector), Coulochem II, ESA) is used for the detection
(E 1 =200mv, E2=400mv, E=500mv).
(3) Preparation of the standard solution
The Nalbuphine that was dissolved in ACN was diluted to the concentration
of 5, 10, 20, 50, 100, 200, 500, 1000, 2000, and 3000 ng/mL with serum.
Prepare the standard solution of various concentrations according to
protocols described in step (1) "sample preparation". After HPLC analysis,
obtain
a corrected curve by plotting HPLC reading of nalbuphine wave heights against
CA 02593140 2007-10-18
its relative concentration. Analyze the standard deviation (SD), coefficient
of
variance (%CV), and %error to examine the accuracy.
Result:
Table 7 shows the changes of nalbuphine in the blood from a
pharmacokinetic point of view. There are significantly different Tmax, AUC,
Cmax, CL/F, and V/F between the experimental and control sets. As shown in
Table 7, the Tmax is 25 5min and Cmax is 2582.3 906.6 ng/ml in SD rat after
being administered with both 100 mg/Kg nalbuphine and 4 mg/Kg capillarisin
orally. In comparison, the Tmax is 97 36 min and Cmax is 79.31 18 ng/ml in the
control group which was treated with 100 mg/Kg nalbuphine only.
The above results indicated that due to the inhibitive effect of capillarisin
on UGT2B, the adsorption of nalbuphine had a 30-fold increase from the
original
concentration that was orally administered; and the absolute bio-availability
increased to 108% from the original 5%.
On the other hand, no significant difference in MRT, k, t1/2 value was
observed between the sample and the control. It indicates the administration
of
capillarisin has no influence on the metabolism of nalbuphine.
Figure 1 shows the temporal effect of capillarisin on the blood nalbuphine
concentration after SD rats were given nalbuphine orally. According to Figure
1,
the concentration of serum nalbuphine in the experimental set was 32.68 times
higher than in the control set, at 0.25 hour after administration. The
difference in
the nalbuphine concentration diminishes by time.
Table 7 Pharmacokinetic analysis of orally administered nalbuphine in the
control
set (without capillarisin) and experimental set (with capillarisin)
PK Coefficient (unit) Control (n=6)
Experiment (n=6)
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CA 02593140 2007-10-18
Nalbuphine
Nalbuphine
+ capillarisin
(Mean SE)
(Mean SE)
Cmax * (ng/mL) 79 18 2582 906
Tmax (min) 97 36 25 5
AUC ' (0-t) (min*ng/mL) 21430 8823 218248
67598
AUC * (total) (min*ng/mL) 24356 8865 244071
69510
k (I /min) 0.0027 0.0007 0.0027
0.0006
t 1/2 (min) 332 62 310 59
MRT (mm) 496 90 316 103
CL/F ' (mL/min/kg) 5838 1049 826 364
V/F * (mL/kg) 2919123
863250 377412 170431
Note: * indicates < 0.05; ** indicates p 5_ 0.01
Experiment 4. Effects of UGT2B inhibitor on the concentration of intravenously
administered nalbuphine
Material and methods:
The experiment follows the same protocol as described in the "material and
methods" section of "Experiment 3", but an insertion of a catheter tube in the
carotid artery was added, in addition to those procedures described in
previous "3.
Methods" step. Such a design is for the purpose of delivering the drug via the
vein while obtaining blood samples from the artery. The insertion of a tube is
similar to the step ii in "Experiment 3", but a special attention is necessary
to
avoid blood loss by clamping the artery near the heart during the insertion of
the
tubing. Just insert about 2.5 cm into the artery will be enough.
Take 0.3 mL blood sample from the PE-50 catheter, at 15, 20, 30, 45, 60, 90,
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CA 02593140 2007-10-18
120, and 180 minutes after the drug was given to SD rats, and analyze the
concentration of nalbuphine in the blood in both the experimental and control
sets.
Result:
Table 8 depicts the change of nalbuphine in blood, from the
pharmacokinetic point of view. There are obvious differences in AUC, Cmax,
CL/F, and V/F between the control and the experimental animals. The highest
concentration in blood (Cmax) reaches 365 119 ng/ml, after SD rat was given
100 mg/Kg nalbuphine and 4mg capillarisin intravenously. While in the control
animals, the Cmax is a relatively lower 154 30 ng/ml with only 100 mg/Kg
nalbuphine was given.
The above result demonstrates that intravenous capillarisin administration
inhibits UCT2B. it increases the nalbuphine concentration to 32.68 times
higher
than in the control animals, and enhances absolute bio-availability by 2.7
0.4%.
In comparison, the oral administration of nalbuphine to SD rats with and
without
the addition of capillarisin will increase the absolute bio-availabilities to
127.85
36.41% and 12.759 4.64% respectively.
Figure 2 shows the temporal effect of capillarisin on the concentration of
nalbuphine in blood after SD rats were given nalbuphine intravenously.
The difference in the nalbuphine concentration between the control and
experimental animals increases gradually by time. After 180 minutes, the serum
nalbuphine concentration in the experimental animals was 2.37 times that of
the
control animals.
Table 8 Pharmacokinetic analysis of intravenous administration of nalbuphine
without capillarisin (control sets) and with capillarisin (experimental sets)
PK Coefficient (unit) Control (n=6)
Experiment (n=6)
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Nalbuphine
Nalbuphine
+ capillarisin
(Mean SE)
(Mean SE)
Cmax (ng/mL) 154 30 365 119
AUC (0-t) (min*ng/mL) 1731 295 5862 1188
AUC ' (total) (min*ng/mL) 1909 330 7135 1218
(1/min) 0.016 0.004 0.017 0.008
t 1/2 (min) 77 29 63 11
MRT (min) 59 12 89 17
CL/F * (mL/min/kg) 843 235 204 34
V/F * (mL/kg) 42194 6492 19379 6475
Note: * indicates < 0.05; ** indicates p 0.01
Experiment 5. Comparison of the effect of UGT2B inhibitor on the orally and
intravenously administered nalbuphine concentrations
Material and method:
This experiment uses the same "material and method" as described in
Experiments 3 and 4.
Deliver nalbuphine orally and intravenously to control animals, and
nalbuphine and capillarisin orally to experimental animals. Take 0.3 ml blood
samples from the PE-50 tubing to analyze the concentration of nalbuphine in
the
serum.
Result:
Figure 3 shows the changes of the nalbuphine concentration in SD rats at
different time, after nalbuphine was orally and intravenously given to the
control
group while nalbuphine and capillarisin were orally given to the experiment
group.
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CA 02593140 2007-10-18
The absorption of orally administered drugs is affected by three factors:
adsorption in the gastroenterological tract, first-pass effect, and other
metabolism;
while the intravenous route is affected mainly by metabolism other than the
first-
pass effect. Comparing the animals that were orally given inhibitor
(experiment
group) with those intravenously given drug without inhibitor (control group),
as
shown in Figure 3, the oral absorption is significantly improved with the
presence
of the inhibitor. Its absolute bio-availability increases from 5 % to 108 %.
In
addition, the AUC values are similar in both sets of animals, indicating the
addition of the inhibitor enhances the oral absorption of nalbuphine. [Note:
compare values in Table 5 and 6, the AUC value of orally administered
nalbuphine and capillarisinin in Table 5 was 244071 69510, while the AUC
value in the control set (intravenous administration) was 7135 1218. They
seem
to be inconsistent with the above conclusion. The discrepancy is caused by the
difference in quantity: when given the nalbuphine through oral route, the dose
was
100 mg / Kg, while the intravenous dose was 1 mg / Kg].
Many changes and modifications in the above described embodiment of the
invention can, of course, be carried out without departing from the scope
thereof.
Accordingly, to promote the progress in science and the useful arts, the
invention
is disclosed and is intended to be limited only by the scope of the appended
claims.
40
