U.S. patent application number 17/573143 was filed with the patent office on 2022-08-11 for metabolites of bictegravir.
The applicant listed for this patent is Gilead Sciences, Inc.. Invention is credited to Haolun Jin, Hyung-Jung Pyun, Bill J. Smith, Raju Subramanian, Jianhong Wang.
Application Number | 20220249499 17/573143 |
Document ID | / |
Family ID | 1000006272215 |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220249499 |
Kind Code |
A1 |
Jin; Haolun ; et
al. |
August 11, 2022 |
METABOLITES OF BICTEGRAVIR
Abstract
The present invention provides metabolites of the antiviral drug
bictegravir, including compositions and salts thereof, which are
useful in the prevention and/or treatment of HIV as well as
analytical methods related to the administration of
bictegravir.
Inventors: |
Jin; Haolun; (Foster City,
CA) ; Pyun; Hyung-Jung; (Fremont, CA) ; Smith;
Bill J.; (San Mateo, CA) ; Subramanian; Raju;
(San Mateo, CA) ; Wang; Jianhong; (Foster City,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gilead Sciences, Inc. |
Foster City |
CA |
US |
|
|
Family ID: |
1000006272215 |
Appl. No.: |
17/573143 |
Filed: |
January 11, 2022 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16251454 |
Jan 18, 2019 |
11253524 |
|
|
17573143 |
|
|
|
|
62619478 |
Jan 19, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/94 20130101;
A61K 45/06 20130101; C07D 498/18 20130101; A61K 31/553 20130101;
A61K 31/537 20130101; A61P 31/18 20180101; A61K 31/706 20130101;
C07H 17/00 20130101 |
International
Class: |
A61K 31/537 20060101
A61K031/537; A61K 31/553 20060101 A61K031/553; A61P 31/18 20060101
A61P031/18; A61K 31/706 20060101 A61K031/706; A61K 45/06 20060101
A61K045/06; C07D 498/18 20060101 C07D498/18; C07H 17/00 20060101
C07H017/00; G01N 33/94 20060101 G01N033/94 |
Claims
1. (canceled)
2. A composition comprising a compound selected from M15, M20, and
M23: ##STR00009## or a pharmaceutically acceptable salt thereof,
wherein the compound, or a pharmaceutically acceptable salt
thereof, is present in the composition in an amount greater than
about 25% by weight.
3. The composition of claim 2 wherein the compound, or a
pharmaceutically acceptable salt thereof, is present in the
composition in an amount greater than about 50% by weight.
4. The composition of claim 2 wherein the compound, or a
pharmaceutically acceptable salt thereof, is present in the
composition in an amount greater than about 75% by weight.
5. (canceled)
6. A pharmaceutical composition comprising a compound selected from
M15, M20, and M23: ##STR00010## or a pharmaceutically acceptable
salt thereof, and a least one pharmaceutically acceptable
carrier.
7. The pharmaceutical composition of claim 6 further comprising one
to three additional therapeutic agents.
8. The pharmaceutical composition of claim 7 wherein the one to
three additional therapeutic agents is an anti-HIV agent.
9. The pharmaceutical composition of claim 8 wherein each of the
one to three additional therapeutic agents is selected from the
group consisting of HIV protease inhibitors, HIV non-nucleoside
inhibitors of reverse transcriptase, HIV nucleoside inhibitors of
reverse transcriptase, HIV nucleotide inhibitors of reverse
transcriptase, and combinations thereof.
10. A method of preventing or treating an HIV infection in a human
comprising administering to the human a therapeutically effective
amount of a compound selected from M15, M20, and M23: ##STR00011##
or a pharmaceutically acceptable salt thereof.
11. The method of claim 10 further comprising administering to the
human a therapeutically effective amount of one to three additional
therapeutic agents.
12. The method of claim 11 wherein the one to three additional
therapeutic agents is an anti-HIV agent.
13. The method of claim 12 wherein the one to three additional
therapeutic agents is selected from the group consisting of HIV
protease inhibitors, HIV non-nucleoside inhibitors of reverse
transcriptase, HIV nucleoside inhibitors of reverse transcriptase,
HIV nucleotide inhibitors of reverse transcriptase, and other drugs
for treating HIV, and combinations thereof.
14. A method of detecting or confirming the administration of
bictegravir to a patient comprising identifying a compound selected
from M15, M20, and M23: ##STR00012## or a salt thereof, in a
biological sample obtained from the patient.
15. The method of claim 14 wherein the biological sample is derived
from plasma, urine, or feces.
16. A method of measuring the rate of metabolism of bictegravir in
a patient comprising measuring the amount of a compound selected
from M15, M20, and M23: ##STR00013## or a salt thereof, in the
patient at one or more time points after administration of
bictegravir.
17. The method of claim 16 wherein the amount of compound is
measured from a blood sample.
18. The method of claim 16 wherein the amount of compound is
measured from plasma.
19. The method of claim 16 wherein the amount of compound is
measured from a urine sample.
20. The method of claim 16 wherein the amount of compound is
measured from a feces sample.
21. A method of determining the prophylactic or therapeutic
response of a patient to bictegravir in the treatment of HIV
infection comprising measuring the amount of a compound selected
from M15, M20, and M23: ##STR00014## or a salt thereof, in the
patient at one or more time points after administration of
bictegravir.
22. A method of optimizing the dose of bictegravir for a patient in
need of treatment with bictegravir comprising measuring the amount
of a compound selected from M15, M20, and M23: ##STR00015## or a
salt thereof, in the patient at one or more time points after
administration of bictegravir.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 16/251,454, filed Jan. 18, 2019, which claims
the benefit of priority of U.S. Provisional Patent Application Ser.
No. 62/619,478, filed Jan. 19, 2018. The contents of each
application are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention provides metabolites of the antiviral
drug bictegravir, including compositions and salts thereof, which
are useful in the prevention and/or treatment of HIV as well as
analytical methods related to the administration of
bictegravir.
BACKGROUND OF THE INVENTION
[0003] The HIV/AIDS pandemic has claimed the lives of millions of
people, and millions more are currently infected. Antiretroviral
therapy has turned HIV infection into a chronic, manageable
disease; however, no cure yet exists for HIV. Patients must remain
on therapy for their whole lives making drug resistance an ongoing
issue. Additionally, as patients age, concomitant treatment for
other diseases and conditions becomes more common, increasing the
potential for drug-drug interactions with HIV antiviral treatment.
Accordingly, continued development of new antiviral drugs and
combination therapies are a priority in the field of HIV
therapeutics.
[0004] Integrase strand transfer inhibitors (INSTIs) are a class of
antiretroviral drugs that act by inhibiting the essential HIV
protein integrase from inserting the viral DNA genome into the host
cell's chromatin. An example INSTI is bictegravir (BIC) which is
currently being tested in human clinical trials in combination with
emtricitabine (FTC), and tenofovir alafenamide (TAF). Bictegravir
has the molecular structure shown below and is described in WO
2014/100212.
##STR00001##
[0005] In view of widespread HIV infection and the challenges in
overcoming drug resistances and drug-drug interactions, there is a
continuing need for new and improved antiviral agents. The
metabolites of bictegravir, as well as their compositions and
methods of use described herein, are directed toward fulfilling
this need.
SUMMARY OF THE INVENTION
[0006] The present invention provides a compound selected from:
##STR00002##
or a pharmaceutically acceptable salt thereof, which is
substantially isolated.
[0007] The present invention further provides compositions
comprising a compound of the invention, or pharmaceutically
acceptable salt thereof, and at least one pharmaceutically
acceptable carrier.
[0008] The present invention further provides preparations
comprising a compound of the invention, or a pharmaceutically
acceptable salt thereof.
[0009] The present invention further provides methods of preventing
or treating an HIV infection in a human by administering to the
human a therapeutically effective amount of a compound of the
invention, or a pharmaceutically acceptable salt thereof.
[0010] The present invention further provides methods of detecting
or confirming the administration of bictegravir to a patient,
comprising identifying a compound of the invention, or a salt
thereof, in a biological sample obtained from the patient.
[0011] The present invention further provides methods of measuring
the rate of metabolism of bictegravir in a patient comprising
measuring the amount of a compound of the invention, or a salt
thereof, in the patient at one or more time points after
administration of bictegravir.
[0012] The present invention further provides methods of
determining the prophylactic or therapeutic response of a patient
to bictegravir in the treatment of HIV infection, comprising
measuring the amount of a compound of the invention, or a salt
thereof, in the patient at one or more time points after
administration of bictegravir.
[0013] The present invention further provides methods of optimizing
the dose of bictegravir for a patient in need of treatment with
bictegravir, comprising measuring the amount of a compound of the
invention, or a salt thereof, in the patient at one or more time
points after administration of bictegravir.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the proposed structures of metabolites and
biotransformation pathways of bictegravir in human plasma, urine,
and feces following a single 100 .mu.Ci/100 mg oral dose of
[.sup.14C]bictegravir to healthy male adult human subjects.
[0015] FIG. 2 shows an extracted ion chromatogram of M15 from
analysis of a standard solution M15.
[0016] FIG. 3 shows a radiochromatogram and extracted ion
chromatogram of metabolite M15 from analysis of an 8-hour pooled
plasma sample obtained after a single oral dose of
[.sup.14C]bictegravir to male human subjects (100 mg, 100
.mu.Ci).
[0017] FIG. 4 shows extracted ion chromatograms from individual and
co-injection of a standard solution of M15 with an 8-hour pooled
plasma sample obtained after a single oral dose of
[.sup.14C]bictegravir to male human subjects (100 mg, 100
.mu.Ci).
[0018] FIG. 5A shows MS precursor and MS/MS product ion mass
spectra (m/z 626) of M15 from analysis of a standard solution of
M15.
[0019] FIG. 5B shows the proposed structure and proposed
fragmentation pattern of M15.
[0020] FIG. 6 shows MS precursor and MS/MS product ion mass spectra
(m/z 626) of metabolite M15 from analysis of an 8-hour pooled
plasma sample obtained after a single oral dose of
[.sup.14C]bictegravir to male human subjects (100 mg, 100
.mu.Ci).
[0021] FIG. 7 shows an extracted ion chromatogram from analysis of
a standard solution of M20.
[0022] FIG. 8 shows an extracted ion chromatogram and
radiochromatogram from analysis of an 8-hour pooled plasma sample
obtained after administration of a single oral dose of
[.sup.14C]bictegravir to male human subjects (100 mg, 100
.mu.Ci).
[0023] FIG. 9 shows extracted ion chromatograms from individual
injections and a co-injection of a standard solution of M20 with an
8-hour pooled plasma sample obtained after administration of a
single oral dose of [.sup.14C]bictegravir to male human subjects
(100 mg, 100 .mu.Ci).
[0024] FIG. 10A shows product ion (m/z 546) mass spectrum of M20
from analysis of a standard solution of M20.
[0025] FIG. 10B shows the proposed structure and proposed
fragmentation pattern of M20.
[0026] FIG. 11 shows product ion (m/z 546) mass spectrum of M20
from analysis of an 8-hour pooled plasma sample obtained after
administration of a single oral dose of [.sup.14C]bictegravir to
male human subjects (100 mg, 100 .mu.Ci).
[0027] FIG. 12 shows proposed structures of BIC metabolites M465a,
M465b, M465c, M305, M625, M641, and M611 identified in vitro.
DETAILED DESCRIPTION
[0028] The present invention is directed to metabolites of
bictegravir and uses thereof. In some embodiments, the metabolite
is bictegravir which has undergone (1) glucuronidation, (2)
dehydrogenation, (3) hydroxylation, (4) hydroxylation with a loss
of fluoride, (5) sulfation or glucuronic acid conjugation of the
hydroxy-bictegravir, (6) sulfation or glucuronic acid or cysteine
conjugation of desfluoro-hydroxy-bictegravir, or (7) a combination
thereof. In some embodiments, the metabolite is selected from M15,
M58, M51, M52, M21, M23, M54, M55, M22, M53, M20, M35, M12, M59,
M45, M56, M16, M57, M9, and M37 (See FIG. 1). In some embodiments,
the metabolite is selected from M465a, M465b, M465c, M305, M625,
M641, and M611 (see FIG. 12).
[0029] In some embodiments, the metabolite is a compound selected
from M15, M20, and M23:
##STR00003##
[0030] In some embodiments, the metabolite is a compound selected
from M15 and M20. In some embodiments, the metabolite is M15. In
some embodiments, the metabolite is M20. In some embodiments, the
metabolite is M23.
[0031] The present invention further includes salts of the
metabolites of the invention, such as pharmaceutically acceptable
salts. A salt generally refers to a derivative of a disclosed
compound wherein the parent compound is modified by converting an
existing acid or base moiety to its salt form. A pharmaceutically
acceptable salt is one that, within the scope of sound medical
judgment, is suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio. Examples of pharmaceutically
acceptable salts include, but are not limited to, mineral or
organic acid salts of basic residues such as amines; alkali or
organic salts of acidic residues such as carboxylic acids; and the
like. The pharmaceutically acceptable salts of the present
invention include the conventional non-toxic salts of the parent
compound formed, for example, from non-toxic inorganic or organic
acids.
[0032] The pharmaceutically acceptable salts of the present
invention can be synthesized from the parent compound which
contains a basic or acidic moiety by conventional chemical
methods.
[0033] Generally, such salts can be prepared by reacting the free
acid or base forms of these compounds with a stoichiometric amount
of the appropriate base or acid. Lists of suitable salts are found
in Remington's Pharmaceutical Sciences, 17.sup.th ed., Mack
Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of
Pharmaceutical Science, 66, 2 (1977), each of which is incorporated
herein by reference in its entirety. In one particular embodiment,
the pharmaceutically acceptable salt is a sodium salt.
[0034] In some embodiments, the metabolite compounds, or salts
thereof, are substantially isolated. By "substantially isolated" is
meant that the metabolite compound, or salt thereof, is at least
partially or substantially separated from the environment in which
it was formed or detected. Partial separation can include, for
example, a composition enriched in the compound of the invention.
Substantial separation can include compositions containing at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 95%, at least about 97%, or
at least about 99% by weight of the metabolite, or salt thereof. In
some embodiments, M15, M20, and M23 are substantially isolated.
[0035] A metabolite of the invention, or its salt, can be present
in a composition where the composition includes at least one
compound other than the metabolite. In some embodiments, the
composition includes more than one metabolite of the invention. In
some embodiments, the composition comprises one or more metabolites
of the invention, or salts thereof, and bictegravir, or a salt
thereof. Compositions can be mixtures containing a metabolite of
the invention, or salt thereof, and one or more solvents,
substrates, carriers, etc. In some embodiments, the composition
comprises a metabolite of the invention, or salt thereof, in an
amount greater than about 25% by weight. In some embodiments, the
composition comprises a metabolite of the invention, or salt
thereof, in an amount greater than about 50% by weight. In some
embodiments, the composition comprises a metabolite of the
invention, or salt thereof, in an amount greater than about 75% by
weight. In some embodiments, the composition comprises a metabolite
of the invention, or salt thereof, in an amount greater than about
80% by weight. In some embodiments, the composition comprises a
metabolite of the invention, or salt thereof, in an amount greater
than about 85% by weight. In some embodiments, the composition
comprises a metabolite of the invention, or salt thereof, in an
amount greater than about 90% by weight. In some embodiments, the
composition comprises a metabolite of the invention, or salt
thereof, in an amount greater than about 95% by weight.
[0036] A preparation of a metabolite of the invention, or salt
thereof, can be prepared by chemical synthesis or by isolation of
the metabolite from a biological sample. Preparations can have a
purity of greater than about 50%, greater than about 60%, greater
than about 70%, greater than about 80%, greater than about 90%, or
greater than about 95% purity. Purity can be measured by any of
conventional means, such as by chromatographic methods or
spectroscopic methods like NMR, MS, LC-MS, etc.
[0037] The metabolites of the invention are asymmetric (e.g.,
having one or more stereocenters). All stereoisomers, such as
enantiomers and diastereomers, are intended unless otherwise
indicated. Methods on how to prepare optically active forms from
optically active starting materials are known in the art, such as
by resolution of racemic mixtures or by stereoselective
synthesis.
[0038] Metabolites of the invention also include all isotopes of
atoms occurring in the metabolites. Isotopes include those atoms
having the same atomic number but different mass numbers. For
example, isotopes of hydrogen include tritium and deuterium. In
some embodiments, the metabolite includes at least one
deuterium.
[0039] The term, "compound" or "metabolite," as used herein is
meant to include all stereoisomers, geometric iosomers, tautomers,
and isotopes of the structures depicted.
[0040] The term, "metabolite" as used herein is meant to include
any and all metabolic derivatives of bictegravir, including
derivatives that have undergone one or more transformative
processes selected from (1) glucuronidation, (2) dehydrogenation,
(3) hydroxylation, (4) hydroxylation with a loss of fluoride, (5)
sulfation or glucuronic acid conjugation of the
hydroxy-bictegravir, and (6) sulfation or glucuronic acid or
cysteine conjugation of desfluoro-hydroxy-bictegravir. In some
embodiments, the metabolites of the invention have undergone more
than one transformative process, including metabolic transformation
of a derivative of bictegravir that has already undergone one or
more metabolic transformations.
[0041] The compound bictegravir can also be considered a prodrug of
the metabolites of the invention (e.g., a prodrug of metabolites
M15, M20, M23, and the like) because bictegravir metabolically
transforms upon administration to provide the metabolites of the
invention. Accordingly, bictegravir can be administered to a human
as a means of providing a metabolite of the invention to the human,
for example, for preventing or treating an HIV infection in the
human.
[0042] The present invention further includes a pharmaceutical
composition comprising a metabolite of the invention, or
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" is meant to refer to any
adjuvant, carrier, excipient, glidant, sweetening agent, diluent,
preservative, dye/colorant, flavor enhancer, surfactant, wetting
agent, dispersing agent, suspending agent, stabilizer, isotonic
agent, solvent, or emulsifier which has been approved by the United
States Food and Drug Administration as being acceptable for use in
humans or domestic animals.
Methods
[0043] The present invention further relates to a method of
preventing or treating an HIV infection (e.g., HIV-1 and/or HIV-2)
in a human by administering to the human a therapeutically
effective amount of a metabolite of the invention, or a
pharmaceutically acceptable salt thereof. The human may have or be
at risk of having the infection.
[0044] The term "treatment" or "treating" as used herein is
intended to mean the administration of a metabolite, composition
thereof, or preparation thereof, according to the present invention
to alleviate or eliminate symptoms of HIV infection and/or to
reduce viral load in a patient.
[0045] The term "prevention" or "preventing" refers to the
administration of a metabolite, composition thereof, or preparation
thereof, according to the present invention post-exposure of the
human to the virus but before the appearance of symptoms of the
disease, and/or prior to the detection of the virus in the blood.
The terms also refer to prevention of the appearance of symptoms of
the disease and/or to prevent the virus from reaching detectible
levels in the blood. The term includes both pre-exposure
prophylaxis, as well as post-exposure prophylaxis. The term also
refers to prevention of perinatal transmission of HIV from mother
to baby, by administration to the mother before giving birth and to
the child within the first days of life.
[0046] The terms "effective amount" or "therapeutically effective
amount" refer to an amount of a metabolite according to the
invention, which when administered to a patient in need thereof, is
sufficient to effect treatment for disease-states, conditions, or
disorders for which the compounds have utility. Such an amount
would be sufficient to elicit the biological or medical response of
a tissue system, or patient that is sought by a researcher or
clinician. The amount of a metabolite according to the invention
which constitutes a therapeutically effective amount will vary
depending on such factors as the compound and its biological
activity, the composition used for administration, the time of
administration, the route of administration, the rate of excretion
of the compound, the duration of the treatment, the type of
disease-state or disorder being treated and its severity, drugs
used in combination with or coincidentally with the compounds of
the invention, and the age, body weight, general health, sex and
diet of the patient. Such a therapeutically effective amount can be
determined routinely by one of ordinary skill in the art having
regard to their own knowledge, the state of the art, and this
disclosure.
[0047] Administration of the metabolites of the invention, or their
pharmaceutically acceptable salts, can be carried out via any of
the accepted modes of administration of agents for serving similar
utilities. The pharmaceutical compositions of the invention can be
prepared by combining a metabolite of the invention, or a
pharmaceutically acceptable salt thereof, with an appropriate
pharmaceutically acceptable carrier and, in specific embodiments,
are formulated into preparations in solid, semi solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants, gels,
microspheres, and aerosols. Exemplary routes of administering such
pharmaceutical compositions include, without limitation, oral,
topical, transdermal, inhalation, parenteral, sublingual, buccal,
rectal, vaginal, and intranasal. In a particular embodiment,
pharmaceutical compositions of the invention are tablets. In
another embodiment, pharmaceutical compositions of the invention
are injection (intramuscular (IM) or intraperitoneal (IP)).
Pharmaceutical compositions of the invention are formulated so as
to allow the active ingredients contained therein to be
bioavailable upon administration of the composition to a patient.
Compositions that will be administered to a subject or patient take
the form of one or more dosage units, where for example, a tablet
may be a single dosage unit, and a container of a compound of the
invention in aerosol form may hold a plurality of dosage units.
Actual methods of preparing such dosage forms are known, or will be
apparent, to those skilled in this art; for example, see Remington:
The Science and Practice of Pharmacy, 20th Edition (Philadelphia
College of Pharmacy and Science, 2000). The composition to be
administered will, in any event, contain a therapeutically
effective amount of a compound of the invention, or a
pharmaceutically acceptable salt thereof, for treatment of a
disease or condition of interest in accordance with the teachings
described herein.
[0048] The present invention further relates to a method of
detecting or confirming the administration of bictegravir to a
patient comprising identifying a metabolite of the invention, or
salt thereof, in a biological sample obtained from the patient. In
some embodiments, the biological sample is derived from plasma,
urine, or feces.
[0049] The present invention further relates to a method of
measuring the rate of metabolism of bictegravir in a patient
comprising measuring the amount of metabolite, or salt thereof, in
the patient at one or more time points after administration of
bictegravir.
[0050] The present invention further relates to a method of
determining the prophylactic or therapeutic response of a patient
to bictegravir in the treatment of HIV infection comprising
measuring the amount of a metabolite of the invention, or salt
thereof, in the patient at one or more time points after
administration of bictegravir.
[0051] The present invention further relates to a method of
optimizing the dose of bictegravir for a patient in need of
treatment with bictegravir comprising measuring the amount of a
metabolite of the invention, or salt thereof, in the patient at one
or more time points after administration of bictegravir. The amount
of metabolite may be indicative of the rate at which the patient
metabolizes bictegravir. Patients who metabolize bictegravir more
quickly or more effectively than other patients may form higher
amounts of metabolite and potentially require higher doses of
bictegravir, or additional doses, compared with patients who
metabolize bictegravir more slowly. Patients who metabolize
bictegravir less quickly or less effectively than other patients
may form lower amounts of metabolite and potentially require lower
doses of bictegravir, or fewer doses, compared with patients who
metabolize bictegravir more quickly. Accordingly, the method of
optimizing the dose of bictegravir may further include determining
whether the measured amounts of metabolite are higher or lower than
average, and adjusting the dosage of bictegravir accordingly.
[0052] Measuring the amount of metabolite, or salt thereof, in a
patient can be carried out by obtaining a biological sample from
the patient and measuring the amount of metabolite, or salt
thereof, in the sample. In some embodiments, the sample is blood.
In other embodiments, the sample is plasma. In other embodiments,
the sample is urine. In other embodiments, the sample is feces.
[0053] The term "patient" is meant to refer to a human or other
mammals such as laboratory animals and household pets (e.g., cats,
dogs, swine, cattle, sheep, goats, horses, rabbits), and
non-domestic animals such as non-human primates, mammalian
wildlife, and the like, that are in need of therapeutic or
preventative treatment for a viral infection, such as HIV
infection.
Combination Therapies
[0054] One or more additional pharmaceutical agents can be used in
combination with the compounds, salts, and compositions of the
present invention for preventing or treating an HIV infection
(e.g., in a human patient). In some embodiments, the composition of
the invention further comprises one or more additional therapeutic
agents. In some embodiments, the composition of the invention
further comprises one to three additional therapeutic agents (e.g.,
one to three anti-HIV agents). In some embodiments, the one or more
one additional therapeutic agents is an anti-HIV agent.
[0055] In the above embodiments, the additional therapeutic agent
may be an anti-HIV agent. For example, in some embodiments, the
additional therapeutic agent is selected from the group consisting
of HIV protease inhibitors, HIV non-nucleoside inhibitors of
reverse transcriptase, HIV nucleoside inhibitors of reverse
transcriptase, HIV nucleotide inhibitors of reverse transcriptase,
HIV integrase inhibitors, HIV non-catalytic site (or allosteric)
integrase inhibitors, entry inhibitors (e.g., CCR5 inhibitors, gp41
inhibitors (i.e., fusion inhibitors) and CD4 attachment
inhibitors), CXCR4 inhibitors, gp120 inhibitors, G6PD and
NADH-oxidase inhibitors, compounds that target the HIV capsid
("capsid inhibitors"; e.g., capsid polymerization inhibitors or
capsid disrupting compounds such as those disclosed in WO
2013/006738 (Gilead Sciences), US 2013/0165489 (University of
Pennsylvania), and WO 2013/006792 (Pharma Resources),
pharmacokinetic enhancers, and other drugs for treating HIV, and
combinations thereof. In some embodiments, the anti-HIV agent is an
HIV protease inhibitor, an HIV non-nucleoside inhibitor of reverse
transcriptase, an HIV nucleoside inhibitor of reverse
transcriptase, an HIV nucleotide inhibitors of reverse
transcriptase, a pharmacokinetic enhancer, or combination thereof.
In some embodiments, the anti-HIV agent is an HIV nucleoside
inhibitor of reverse transcriptase, an HIV nucleotide inhibitors of
reverse, or combination thereof.
[0056] In further embodiments, the additional therapeutic agent is
selected from one or more of:
[0057] (1) HIV protease inhibitors selected from the group
consisting of amprenavir, atazanavir, fosamprenavir, indinavir,
lopinavir, ritonavir, nelfinavir, saquinavir, tipranavir,
brecanavir, darunavir, TMC-126, TMC-114, mozenavir (DMP-450),
JE-2147 (AG1776), L-756423, RO0334649, KNI-272, DPC-681, DPC-684,
GW640385X, DG17, PPL-100, DG35, and AG 1859;
[0058] (2) HIV non-nucleoside or non-nucleotide inhibitors of
reverse transcriptase selected from the group consisting of
capravirine, emivirine, delaviridine, efavirenz, nevirapine, (+)
calanolide A, etravirine, GW5634, DPC-083, DPC-961, DPC-963,
MIV-150, TMC-120, rilpivirene, BILR 355 BS, VRX 840773, lersivirine
(UK-453061), RDEA806, KM023 and MK-1439;
[0059] (3) HIV nucleoside inhibitors of reverse transcriptase
selected from the group consisting of zidovudine, emtricitabine,
didanosine, stavudine, zalcitabine, lamivudine, abacavir,
amdoxovir, elvucitabine, alovudine, MIV-210, .+-.-FTC, D-d4FC,
emtricitabine, phosphazide, fozivudine tidoxil, apricitibine
(AVX754), KP-1461, GS-9131 (Gilead Sciences) and fosalvudine
tidoxil (formerly HDP 99.0003);
[0060] (4) HIV nucleotide inhibitors of reverse transcriptase
selected from the group consisting of tenofovir, tenofovir
disoproxil fumarate, tenofovir alafenamide (Gilead Sciences),
GS-7340 (Gilead Sciences), GS-9148 (Gilead Sciences), adefovir,
adefovir dipivoxil, CMX-001 (Chimerix) or CMX-157 (Chimerix);
[0061] (5) HIV integrase inhibitors selected from the group
consisting of raltegravir, elvitegravir, dolutegravir,
cabotegravir, curcumin, derivatives of curcumin, chicoric acid,
derivatives of chicoric acid, 3,5-dicaffeoylquinic acid,
derivatives of 3,5-dicaffeoylquinic acid, aurintricarboxylic acid,
derivatives of aurintricarboxylic acid, caffeic acid phenethyl
ester, derivatives of caffeic acid phenethyl ester, tyrphostin,
derivatives of tyrphostin, quercetin, derivatives of quercetin,
S-1360, AR-177, L-870812, and L-870810, BMS-538158, GSK364735C,
BMS-707035, MK-2048, BA 011, and GSK-744;
[0062] (6) HIV non-catalytic site, or allosteric, integrase
inhibitors (NCINI) including, but not limited to, BI-224436,
CX0516, CX05045, CX14442, compounds disclosed in WO 2009/062285
(Boehringer Ingelheim), WO 2010/130034 (Boehringer Ingelheim), WO
2013/159064 (Gilead Sciences), WO 2012/145728 (Gilead Sciences), WO
2012/003497 (Gilead Sciences), WO 2012/003498 (Gilead Sciences)
each of which is incorporated by references in its entirety
herein;
[0063] (7) gp41 inhibitors selected from the group consisting of
enfuvirtide, sifuvirtide, albuvirtide, FB006M, and TRI-1144;
[0064] (8) the CXCR4 inhibitor AMD-070;
[0065] (9) the entry inhibitor SP01A;
[0066] (10) the gp120 inhibitor BMS-488043;
[0067] (11) the G6PD and NADH-oxidase inhibitor immunitin;
[0068] (12) CCR5 inhibitors selected from the group consisting of
aplaviroc, vicriviroc, maraviroc, cenicriviroc, PRO-140, INCB15050,
PF-232798 (Pfizer), and CCR5mAb004;
[0069] (13) CD4 attachment inhibitors selected from the group
consisting of ibalizumab (TMB-355) and BMS-068 (BMS-663068);
[0070] (14) pharmacokinetic enhancers selected from the group
consisting of ritonavir, cobicistat and SPI-452; and
[0071] (15) other drugs for treating HIV selected from the group
consisting of BAS-100, SPI-452, REP 9, SP-01A, TNX-355, DES6,
ODN-93, ODN-112, VGV-1, PA-457 (bevirimat), HRG214, VGX-410,
KD-247, AMZ 0026, CYT 99007A-221 HIV, DEBIO-025, BAY 50-4798,
MDX010 (ipilimumab), PBS 119, ALG 889, and PA-1050040 (PA-040), and
combinations thereof.
[0072] In certain embodiments, a metabolite disclosed herein, or a
pharmaceutically acceptable salt thereof, is combined with two,
three, four or more additional therapeutic agents. The two, three
four or more additional therapeutic agents can be different
therapeutic agents selected from the same class of therapeutic
agents, or they can be selected from different classes of
therapeutic agents. In a specific embodiment, a metabolite
disclosed herein, or a pharmaceutically acceptable salt thereof, is
combined with an HIV nucleotide or nucleoside inhibitor of reverse
transcriptase and an HIV non-nucleoside inhibitor of reverse
transcriptase. In another specific embodiment, a metabolite
disclosed herein, or a pharmaceutically acceptable salt thereof, is
combined with an HIV nucleotide or nucleoside inhibitor of reverse
transcriptase, and an HIV protease inhibiting compound. In a
further embodiment, a metabolite disclosed herein, or a
pharmaceutically acceptable salt thereof, is combined with an HIV
nucleotide or nucleoside inhibitor of reverse transcriptase, an HIV
non-nucleoside inhibitor of reverse transcriptase, and an HIV
protease inhibiting compound. In an additional embodiment, a
metabolite disclosed herein, or a pharmaceutically acceptable salt
thereof, is combined with an HIV nucleotide or nucleoside inhibitor
of reverse transcriptase, an HIV non-nucleoside inhibitor of
reverse transcriptase, and a pharmacokinetic enhancer.
[0073] In certain embodiments, when a metabolite disclosed herein
is combined with one or more additional therapeutic agents as
described above, the components of the composition are administered
as a simultaneous or sequential regimen. When administered
sequentially, the combination may be administered in two or more
administrations.
[0074] In certain embodiments, a metabolite disclosed herein is
combined with one or more additional therapeutic agents in a
unitary dosage form for simultaneous administration to a patient,
for example as a solid dosage form for oral administration (e.g., a
fixed dose combination tablet).
[0075] In certain embodiments, a metabolite disclosed herein is
administered with one or more additional therapeutic agents.
Co-administration of a metabolite disclosed herein, or a
pharmaceutically acceptable salt thereof, with one or more
additional therapeutic agents generally refers to simultaneous or
sequential administration of a compound disclosed herein and one or
more additional therapeutic agents, such that therapeutically
effective amounts of the metabolite and one or more additional
therapeutic agents are both present in the body of the patient.
[0076] Co-administration includes administration of unit dosages of
the metabolites disclosed herein before or after administration of
unit dosages of one or more additional therapeutic agents, for
example, administration of the metabolites disclosed herein within
seconds, minutes, or hours of the administration of one or more
additional therapeutic agents. For example, in some embodiments, a
unit dose of a metabolite disclosed herein is administered first,
followed within seconds or minutes by administration of a unit dose
of one or more additional therapeutic agents. Alternatively, in
other embodiments, a unit dose of one or more additional
therapeutic agents is administered first, followed by
administration of a unit dose of a metabolite disclosed herein
within seconds or minutes. In some embodiments, a unit dose of a
metabolite disclosed herein is administered first, followed, after
a period of hours (e.g., 1-12 hours), by administration of a unit
dose of one or more additional therapeutic agents. In other
embodiments, a unit dose of one or more additional therapeutic
agents is administered first, followed, after a period of hours
(e.g., 1-12 hours), by administration of a unit dose of a
metabolite disclosed herein.
Pharmaceutical Formulations and Dosage Forms
[0077] The pharmaceutical compositions disclosed herein can be
prepared by methodologies well known in the pharmaceutical art. For
example, in certain embodiments, a pharmaceutical composition
intended to be administered by injection can prepared by combining
a metabolite of the invention with sterile, distilled water so as
to form a solution. In some embodiments, a surfactant is added to
facilitate the formation of a homogeneous solution or suspension.
Surfactants are compounds that non-covalently interact with the
compound of the invention so as to facilitate dissolution or
homogeneous suspension of the compound in the aqueous delivery
system.
[0078] The metabolites of the invention, or their pharmaceutically
acceptable salts, can be administered in a therapeutically
effective amount, which will vary depending upon a variety of
factors including the activity of the specific compound employed;
the metabolic stability and length of action of the compound; the
age, body weight, general health, sex, and diet of the patient; the
mode and time of administration; the rate of excretion; the drug
combination; the severity of the particular disorder or condition;
and the subject undergoing therapy.
[0079] The invention will be described in greater detail by way of
specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of noncritical parameters which can be changed or modified
to yield essentially the same results.
EXAMPLES
Example 1: Results of a Phase 1 Study to Evaluate the
Pharmacokinetics, Metabolism, and Excretion of Bictegravir in
Healthy Subjects
Study Design
[0080] The objectives of this study were: (1) to determine the mass
balance of bictegravir following administration of a single, oral
dose of radiolabeled carbon-14 ([.sup.14C])bictegravir; (2) to
evaluate the pharmacokinetics (PK) of bictegravir and its
metabolite(s), where possible; (3) to determine the metabolite
profile of bictegravir in humans following administration of a
single, oral dose of radiolabeled [.sup.14C]bictegravir; and (4) to
assess the safety and tolerability of bictegravir.
[0081] This was a Phase 1, open-label, single center, mass-balance
study to evaluate the PK, metabolism, and excretion of bictegravir
following administration of a single, oral dose of radiolabeled
[.sup.14C]bictegravir in healthy subjects. A total of eight patents
were enrolled. Subjects were healthy male nonsmokers, 18 to 45
years of age, inclusive, with a body mass index (BMI) from 19 to 30
kg/m2, inclusive, normal 12-lead electrocardiogram (ECG), normal
renal function, no significant medical history, and in good general
health, as determined by the investigator at the screening
evaluation performed no more than 28 days prior to the scheduled
first dose.
[0082] Treatment involved a single, oral dose followed by a 6- to
21-day sample collection period, the exact duration of which was
based on recovery of radiolabeled drug. The dose was 100 mg
bictegravir (99 mg of nonradiolabeled bictegravir [as the sodium
salt form] plus approximately 100 .mu.Ci [1 mg] radiolabeled
[.sup.14C]bictegravir) administered orally as an approximately
40-mL ethanolic solution (4:6 [v/v] water:ethanol, pH adjusted with
HCl). Following administration, the dosing container was rinsed
twice, each rinse with approximately 50 mL of cranberry juice, and
administered to the subject. The entire study drug solution and
cranberry juice rinse (total of approximately 140 mL) was taken
within a 10 minute window.
[0083] Individual data and summary statistics for the percentage
and cumulative percentage of total [.sup.14C]-radioactive dose
recovered in urine, stool, and both samples were provided per
sampling time. Individual, mean (standard deviation [SD]), and
median (first quartile [Q1], third quartile [Q3]) cumulative
percentage of total [.sup.14C]-radioactive dose recovered versus
time profiles in urine, stool, and both samples were presented in
time linear and
semi-log scales.
[0084] Individual subject concentration data and summary statistics
of plasma, whole blood, urine, and stool samples per sampling time
were presented for total [.sup.14C]-radioactivity. The whole
blood-to-plasma ratio of total [.sup.14C]-radioactivity
concentration was determined for each subject and tabulated with
descriptive statistics. In addition, individual subject
concentration data and summary statistics of plasma and urine
samples per sampling time were presented for bictegravir.
[.sup.14C]Bictegravir Metabolite Profiling in Plasma, Urine, and
Feces
[0085] A total of 20 metabolites of bictegravir were identified in
the metabolite profiling results by high performance liquid
chromatography (HPLC)-MS/TopCount method. These metabolites were
generated through several biotransformation pathways, including
direct glucuronidation (M15 and M58), dehydrogenation (M51 and
M52), hydroxylation (M21, M23, M54, and M55), hydroxylation with a
loss of fluoride (M22 and M53), sulfation or glucuronic acid
conjugation of
the hydroxy-bictegravir (M20, M35, M12, M59, and M45), sulfation or
glucuronic acid or cysteine conjugation of
desfluoro-hydroxy-bictegravir (M56, M16, M57, M9, and M37) (FIG.
1).
[0086] Plasma: Metabolite profiling and quantitation in plasma was
performed with samples pooled for individual subjects between 0 to
144 hours postdose. Bictegravir and 13 metabolites were identified
in human plasma. [.sup.14C]Bictegravir was the major circulatory
radioactive component and M20 (hydroxy-bictegravir-sulfate) and M15
(bictegravir-glucuronide) were the major metabolites in plasma,
accounting for 67.9%, 20.1%, and 8.6%, respectively, of the plasma
AUC0-72 h (area under the concentration time curve) of total
radioactivity. The AUC0-72 h ratios of minor metabolites M21
(hydroxy-bictegravir), M52 (dehydrogenation product), and M23/M51
(hydroxy-bictegravir/dehydrogenation product) relative to that of
the total radioactivity were 2.0%, 0.6%, and 0.2%, respectively.
All of the metabolites were BLQ (below limit of quantification) by
144 hours after dosing, indicating no long-lived metabolites.
[0087] Urine: Metabolite profiling and quantitation in urine was
performed with samples pooled for individual subjects within the
period of 0 to 96 hours postdose. Bictegravir and 20 metabolites
were identified in human urine. M15 (co-eluted with M58, both
bictegravir-glucuronides), was the major radioactive component in
the urine, accounting for 21.4% of the administered dose. Minor or
trace level metabolites (see FIG. 1) were below 2.2% of the dose.
Recovered unchanged bictegravir was low in urine (3.6% of the
dose), consistent with the LC/MS/MS results of bictegravir.
[0088] Feces: Metabolite profiling and quantitation in feces was
performed with the feces samples pooled for individual subjects.
The unchanged parent, M9 (desfluoro-hydroxy-bictegravir-cysteine
conjugate), M21/M22
(hydroxy-bictegravir/desfluoro-hydroxy-bictegravir co-eluted), and
M23 (hydroxy-bictegravir) accounted on average for 30.6%, 13.0%,
8.1%, and 3.6%, respectively, of the administered dose
(quantitation averaged from 8 subjects within 0 to 144 hours
postdose).
[0089] Identification of metabolites: Metabolites were identified
by LC-MS/TopCount method. First, product ion mass spectra of the
authentic bictegravir and [.sup.14C]bictegravir reference standards
were acquired on an LTQ ion trap mass spectrometer and an LTQ
Orbitrap high resolution mass spectrometer. Then their major
fragmentation patterns were proposed and the elemental compositions
of the corresponding fragment ions were confirmed. Second, the
retention times of the metabolites observed on
LC-Radio-chromatograms were compared to the corresponding retention
times on LC-MS chromatogram operating in a full scan positive
ionization mode and the molecular ions of the metabolites were
determined. Product ion mass spectra were then acquired for the
molecular ions of the potential metabolites. Accurate mass spectra
were also acquired on an LTQ Orbitrap high resolution mass
spectrometer to confirm the chemical formulas of the proposed
molecular ions and their product ions. The plausible fragmentation
pathways and the putative metabolite structures were proposed (FIG.
1).
[0090] Metabolite M15 and M58 eluted at .about.25.74 and 25.44 min
on LC-MS chromatogram and had molecular ions at m/z 626. Accurate
mass measurement of these ions provided a chemical formula of
C.sub.27H.sub.27F.sub.3N.sub.3O.sub.11.sup.+ with mass errors from
0.02 to 0.2 ppm suggesting an addition of C.sub.6H.sub.8O.sub.6
moiety to the parent molecular ion. CID of these molecular ions
resulted in similar fragmentation and showed major ions at m/z 450,
corresponding to a neutral loss of -176 Da from the molecular ions.
MS3 of the ion at m/z 450 showed spectra that matched the reference
standard of bictegravir, indicating that M15 and M58 were the
glucuronides of bictegravir.
[0091] Metabolites M54, M21, and M23 eluted at .about.31.40, 33.18,
and 33.97 min, respectively, on the LC-MS chromatogram and all had
molecular ions at m/z 466. Accurate mass measurement of these ions
provided a chemical formula of
C.sub.21H.sub.19F.sub.3N.sub.3O.sub.6.sup.+, with mass errors from
0.2 to 0.4 ppm, suggesting an addition of 0 atom (+16 Da), to the
parent molecular ion. CID of the molecular ions of M23 resulted in
major ions at m/z 448, 423, 307, and 289. CID of the molecular ions
of M21 and M54 resulted in major ions at m/z 448, 423, 323, 305,
and 289. Accurate mass measurement of these product ions confirmed
their chemical formulas. MS spectra suggested that M23, M21, and
M54 were the mono hydroxylation metabolites of bictegravir.
[0092] Metabolite M20 eluted at 29.92 min on LC-MS chromatogram and
had molecular ions at m/z 546. Accurate mass measurement of this
ion provided a chemical formula of
C.sub.21H.sub.19F.sub.3N.sub.9O.sub.9S.sup.+, with a mass error of
0.3 ppm, suggesting an addition of SO.sub.4 moiety to the parent
molecular ion. CID of this molecular ion resulted in a major ion at
m/z 466, corresponding to a neutral loss of SO.sub.3 moiety (-80
Da) from the parent molecular ion. MS3 spectra resulted in fragment
ions at m/z 448, 423, 307, and 289. Mass spectra of M20 suggested
that it was a sulfate conjugate of the hydroxylated product of
bictegravir.
[0093] Metabolite Quantitation: LC-Radio-chromatograms of the
pooled plasma, urine, and feces samples were obtained. Quantitation
of [.sup.14C]-bictegravir and its metabolites was based on
integration of their corresponding peaks on the radio-chromatograms
and the radioactivity concentration/radioactive dose recovered in
the corresponding sample. The concentrations of bictegravir and its
metabolites in the excreta were reported as percent of dose
administered. Concentrations measured in the plasma samples were
reported as ng equivalent bictegravir/mL.
[0094] Radioactivity Recovery: The average extraction recovery from
the fecal homogenates and plasma was 95.5% and 100.4%,
respectively. The average reconstitution recovery from the dried
feces and plasma extract residues was 99.9% and 100.3%,
respectively. The recovery from the urine centrifugation process
was 100.8%. The recovery from the urine concentration process was
100.6%. The radioactivity recovery from the HPLC column was
99.3%.
Separation and Quantitation of M21 and M22 Metabolites in Pooled
Feces
[0095] Liquid chromatography was used to separate M21
(hydroxy-bictegravir) and M22 (desfluoro-hydroxy-bictegravir) in
pooled feces samples because these two metabolites co-eluted during
the metabolite profiling process. M22 eluted as a single peak and
the radioactivity was quantified, however, M21 co-eluted with M23.
The percent of M21 was calculated by subtraction of M22 from
M21/M22 mixture.
[0096] M21 and M22 accounted on average for 4.8% and 3.3% of the
dose, respectively, through
144 hours postdose in the feces samples pooled per individual
subject. In the feces samples pooled per collection interval, M21
and M22 accounted for 2.9% and 4.1% of the dose, respectively,
through 144 hours after dose. These analyses indicated that the
levels of hydroxy-bictegravir (M21) and
desfluoro-hydroxy-bictegravir (M22) were similar, ranging from
approximately 3% to 4% of the dose.
Summary of Results
[0097] Pharmacokinetics Results: This mass balance study
demonstrated that recovery of bictegravir was primarily from feces
relative to urine. Metabolism is the major clearance pathway for
bictegravir in humans. A total of 20 metabolites of bictegravir
were identified by high performance liquid chromatography
(HPLC)-MS/TopCount method. Direct glucuronidation, hydroxylation,
defluorination, dehydrogenation, and Phase II conjugation of
oxidized metabolites were the major metabolic pathways for
bictegravir.
[0098] In human plasma, [.sup.14C]bictegravir was the major
circulatory radioactive component and M20 (sulfate of
hydroxy-bictegravir) and M15 (glucuronide of bictegravir) were the
major metabolites in plasma, accounting for approximately 67.9%,
20.1%, and 8.6%, respectively, of the plasma AUC0-72 h of total
radioactivity. In human urine, M15 (co-eluted with M58, both direct
glucuronides of bictegravir) was the major metabolite (21.4% of
dose). The radioactivity in feces samples pooled by time intervals
and for individual subjects was accounted for mainly by bictegravir
(31% to 34% of dose), the cysteine conjugate of
desfluoro-hydroxy-bictegravir (10% to 13% of dose),
hydroxy-bictegravir co-eluted with desfluoro-hydroxy-bictegravir
(7% to 8% of dose for the co eluted peak), and minor oxidation
products. The levels of M21 (hydroxyl-bictegravir) and M22
(desfluoro-hydroxy-bictegravir) were similar, each ranging on
average from approximately 3% to 4% of the dose in the M21/M22
mixture in feces from humans.
Example 2: Synthesis and Characterization of M15, M20, and M23
Preparation of M15
##STR00004##
[0100] Anomeric bromination of 1 with 33% HBr in acetic acid gave
bromide 2 in 70% crystallized yield. Treatment of phenol 3 with
bromide 2 in the presence of Ag.sub.2CO.sub.3 in acetonitrile at
60.degree. C. produced compound 4 in 75% yield after reverse phase
chromatography. Deprotection of 4 with triethylamine in
methanol/water gave clean conversion to glucuronic acid 5 (M15).
The reaction was stopped at 90% conversion since the amount of 3
present in the reaction started to increase. The mixture was
purified by reverse phase chromatography with 0.1% TFA in the
chromatography solvent. The free acid was isolated after
lyophilization, however it contained a significant amount of 3. It
was discovered that the free acid 5 was unstable in either neutral
or acidic conditions. The triethylamine salt of M15 (5) appeared to
be stable. Another batch was purified using the same method as
before but the TFA was neutralized with triethylamine before
lyophilization. The compound was stable to the conditions but
contained a large amount of triethylammonium trifluoroacetate. Not
all salt was removed. The material was purified by reverse phase
chromatography but without TFA in the solvent. After lyophilization
the triethylamine salt of M15 (5) was isolated in high purity and
24% overall yield.
##STR00005##
Step 1.
[0101] A solution of compound 6 (333 mg, 0.70 mmol),
4-(dimethylamino)pyridine (85 mg, 0.70 mmol), and triethylamine
(0.2 mL, 1.44 mmol) in tetrahydrofuran (10 mL) was stirred at room
temperature as a solution of 2,2,2-trichloroethyl sulfurochloridate
(218 mg, 0.88 mmol) in tetrahydrofuran (2.5 mL) was added over
.about.2 min. After 3.5 h, additional triethylamine (0.1 mL, 0.72
mmol) and 2,2,2-trichloroethyl sulfurochloridate (100 mg, 0.40
mmol) were added. After 1.5 h since addition, the reaction mixture
was diluted with ethyl acetate (50 mL) and washed with water
(.times.1), 10% citric acid (.times.1), water (.times.1), and brine
(.times.1). After the aqueous fractions were extracted with ethyl
acetate (.times.1), the organic fractions were combined, dried
(MgSO.sub.4), and concentrated. The residue was purified by
CombiFlash (40 g column) eluting 0-10% methanol in dichloromethane
to get 458 mg (95%) of compound 7. .sup.1H NMR (400 MHz,
Chloroform-d) .delta. 10.40 (t, J=5.8 Hz, 1H), 8.33 (s, 1H), 6.86
(td, J=9.4, 2.2 Hz, 1H), 5.37-5.30 (m, 2H), 4.95 (s, 2H), 4.67 (d,
J=5.9 Hz, 2H), 4.63 (s, 1H), 4.19 (dd, J=12.7, 3.9 Hz, 1H), 4.02
(s, 3H), 3.98 (dd, J=12.7, 9.2 Hz, 1H), 2.13-1.95 (m, 4H), 1.83
(qd, J=8.7, 7.6, 3.5 Hz, 1H), 1.57 (ddd, J=12.3, 4.1, 2.9 Hz, 1H).
.sup.19F NMR (376 MHz, Chloroform-d) .delta. -110.32 (dt, J=9.7,
5.3 Hz), -122.76 (dd, J=9.9, 5.6 Hz), -125.40 (d, J=4.7 Hz).
LCMS-ESI.sup.+ (m/z): [M+H]+ calculated for
C.sub.24H.sub.22Cl.sub.3F.sub.3N.sub.3O.sub.9S: 690.01; found:
690.27.
Step 2.
[0102] A solution of compound 7 (255 mg, 0.37 mmol) in acetonitrile
(3 mL) was stirred at room temperature as magnesium bromide (180
mg, 0.98 mmol) was added. The resulting suspension was stirred at
50.degree. C. bath. After 30 min, the reaction mixture was stirred
at 0.degree. C. as several drops of 0.1 N HCl was added until the
insoluble material was dissolved. After the resulting solution was
diluted with water (30 mL), the product was extracted with
dichloromethane (25 mL.times.3). The combined extracts were dried
(Na.sub.2SO.sub.4) and concentrated. The residue was purified by
CombiFlash (40 g column) eluting 0-15% methanol in dichloromethane
to get 178 mg (71%) of compound 8. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 12.46 (s, 1H), 10.44 (t, J=5.9 Hz, 1H), 8.42
(s, 1H), 7.63 (td, J=10.5, 9.9, 2.0 Hz, 1H), 5.43 (dd, J=9.5, 4.0
Hz, 1H), 5.36 (s, 2H), 5.09 (s, 1H), 4.72-4.48 (m, 4H), 4.01 (dd,
J=12.7, 9.5 Hz, 1H), 1.93 (s, 4H), 1.83 (d, J=12.1 Hz, 1H),
1.62-1.50 (m, 1H). .sup.19F NMR (376 MHz, DMSO-d.sub.6) .delta.
-110.83 (dd, J=9.8, 5.2 Hz), -124.08 (dd, J=10.9, 5.5 Hz), -126.44.
LCMS-ESI.sup.+ (m/z): [M+H].sup.+ calculated for
C.sub.23H.sub.20Cl.sub.3F.sub.3N.sub.3O.sub.9S: 675.99; found:
676.26.
Step 3.
[0103] A solution of compound 8 (50 mg, 0.074 mmol) in methanol
(2.5 mL) was stirred at room temperature as ammonium bicarbonate
(721 mg, 9.12 mmol) and zinc powder (217 mg, 3.32 mmol) were added.
The resulting suspension was stirred at room temperature for 18 h.
The reaction mixture was concentrated at room temperature and the
residue was dried in vacuum for 30 min. The residual solids was
triturated with 0.01 N ammonium bicarbonate (50 mL) with sonication
for .about.2 min, and the resulting slurry was left at room
temperature for 30 min before filtered through celite. After the
flask and the celite pad were washed with additional 0.01 N
ammonium bicarbonate (10 mL), the combined filtrate and washing
were loaded on a reverse phase CombiFlash column (15.5 g), which
was previously equilibrated with .about.200 mL of 0.01 M ammonium
bicarbonate in .about.50% aqueous acetonitrile, followed by 0.01 M
ammonium bicarbonate in 100% water. The column was eluted with
CombiFlash eluting 0-45% solvent B in solvent A (solvent A: 0.01 M
ammonium bicarbonate in 100% water; solvent B: 0.01 M ammonium
bicarbonate in 80% acetonitrile in water) and the product
containing fractions were combined and freeze dried to get 31 mg
(75%) of compound 9 (M20) as ammonium salt. LCMS-ESI.sup.- (m/z):
[M-H].sup.- calculated for
C.sub.21H.sub.22Cl.sub.3F.sub.3N.sub.3O.sub.9S: 690.01; found:
690.27.
Preparation of M23
##STR00006##
[0104] Step 1.
[0105] A suspension of 2,4,6-trifluoro-3-methoxybenzaldehyde (10,
2990 mg, 15.7 mmol), hydroxylamine hydrochloride (1352 mg, 19.5
mmol), and sodium acetate (1598 mg, 19.5 mmol) in ethanol (60 mL)
was stirred vigorously at room temperature for 2.5 h. The
suspension was diluted with water (60 mL) and stirred at ice bath
for 1 h. The resulting solid was filtered, washed with cold 50%
aqueous ethanol, and dried in vacuum overnight to obtain 2942 mg
(91%) of compound 11. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
11.86 (s, 1H), 8.08 (s, 1H), 7.38 (td, J=11.0, 2.2 Hz, 1H), 3.90
(s, 3H). .sup.19F NMR (376 MHz, DMSO-d.sub.6) .delta. -117.25 (dd,
J=10.9, 3.5 Hz), -125.34 (ddd, J=11.9, 8.6, 3.6 Hz), -128.35 (dd,
J=8.5, 2.2 Hz). LCMS-ESI.sup.+ (m/z): [M+H].sup.+ calculated for
C.sub.8H.sub.7F.sub.3NO.sub.2: 206.04; found: 206.00.
Step 2.
[0106] A solution of compound 11 (601 mg, 0.98 mmol) in acetic acid
(6 mL) was stirred at 65.degree. C. while zinc powder (1500 mg,
7.65 mmol) was added portionwise over 30 min. After addition, the
mixture was stirred at 65.degree. C. After 1.5 h, the reaction
mixture was filtered and the filtrate was concentrated to dryness.
The residue was dissolved in water and washed with diethyl ether
(.times.1). After the organic fraction was extracted with water
with 2 drops of acetic acid, the two aqueous fractions were
combined, diluted with saturated aqueous NaHCO.sub.3 until it
became neutral, and extracted with ethyl acetate (.about.25
mL.times.5). The extracts were combined, dried (MgSO.sub.4), and
concentrated to get 440 mg (79%) of the corresponding crude amine
12. LCMS-ESI.sup.+ (m/z): [M+H].sup.+ calculated for
C.sub.8H.sub.9F.sub.3NO: 192.06; found: 191.86.
Step 3.
[0107] A solution of the above amine 12 (440 mg, 2.30 mmol) in
dichloromethane (1.5 mL) was stirred at room temperature as 1 M
boron tribromide in dichloromethane (7 mL, 7 mmol) was added. After
2 h, additional 1 M boron tribromide in dichloromethane (1 mL, 1
mmol) was added to the solution. After 2 h since addition, the
reaction mixture was cooled at ice bath and methanol (15 mL) was
added slowly. The solution was concentrated and the residual oil
was dissolved with methanol (.about.15 mL) before concentration,
which was repeated 4 times. The resulting residue was dissolved in
methanol, and stirred in ice bath before addition of triethylamine
(1.5 mL, 10.76 mmol) followed by di-tert-butyl dicarbonate (593 mg,
2.72 mmol). The resulting mixture was stirred at 0.degree. C. for 2
h and then at room temperature overnight. After the resulting
solution was concentrated, the residue was dissolved in ethyl
acetate (.about.30 mL) and water (.about.30 mL), and acidified with
10% citric acid. Two fractions were separated and the aqueous
fraction was extracted with ethyl acetate (.times.1). After the
organic fractions were washed with brine (.times.1), the combined
organic fractions were dried (MgSO.sub.4) and concentrated. The
residue was purified by CombiFlash (40 g column) eluting 0-100% EA
in hexane to get 530 mg (83%) of compound 13. .sup.1H NMR (400 MHz,
Chloroform-d) .delta. 6.70 (ddd, J=10.3, 9.4, 2.3 Hz, 1H), 5.62
(br, 1H), 4.89 (s, 1H), 4.37 (d, J=5.8 Hz, 2H), 1.44 (s, 9H).
.sup.19F NMR (376 MHz, Chloroform-d) .delta. -125.17--126.42 (m,
1F), -132.92--134.28 (m, 1F), -137.39 (m, 1F). LCMS-ESI.sup.+
(m/z): [M-C.sub.4H.sub.8+H].sup.+ calculated for
C.sub.8H.sub.7F.sub.3NO.sub.3: 222.04; found: 221.95.
Step 4.
[0108] A solution of compound 13 (530 mg, 1.91 mmol) in
dichloromethane (4.8 mL) was stirred at 0.degree. C. as 4 M HCl in
dioxane (4.8 mL, 19.2 mmol) was added. After addition, the mixture
was stirred at room temperature. After 1.75 h, the reaction mixture
was concentrated and the residual white solid was co-evaporated
with toluene (.about.20 mL.times.2) before being dried in vacuo
overnight to obtain 399 mg (98%) of the crude amine hydrochloride
salt.
[0109] A suspension of the acid 14 (600 mg, 1.87 mmol), the above
amine HCl salt (399 mg, 1.87 mmol), and
2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU, 748 mg, 1.97 mmol) in dichloromethane
(12 mL) was stirred at room temperature as
N,N-diisopropylethylamine (1.65 mL, 9.47 mmol) was added. After 15
min, additional HATU (712 mg, 1.87 mmol), N,N-diisopropylethylamine
(1.65 mL, 9.47 mmol), and DMF (3 m) were added to the mixture.
After 15 min since the addition, the solution was concentrated to
remove the most of dichloromethane and the residue was diluted with
methanol (25 mL). After the resulting solution was stirred at room
temperature for 1 h, it was concentrated to remove most of the
methanol, and diluted with ethyl acetate (.about.70 mL) before
washing with aqueous ammonium chloride (.times.2), aqueous
NaHCO.sub.3(.times.2), and brine (.times.1). After the aqueous
fractions were extracted with ethyl acetate (.about.75 mL.times.1),
the organic fractions were combined, dried (MgSO.sub.4), and
concentrated. The residue was purified by CombiFlash (80 g column)
eluting with 0-11% methanol in dichloromethane to get 647 mg (72%)
of compound 6. .sup.1H NMR (400 MHz, Chloroform-d) .delta. 10.32
(t, J=5.8 Hz, 1H), 8.33 (s, 1H), 7.25 (s, 1H), 6.65 (ddd, J=10.7,
9.4, 2.2 Hz, 1H), 5.36 (dd, J=9.6, 3.7 Hz, 2H), 4.68-4.53 (m, 3H),
4.25 (dd, J=12.8, 3.8 Hz, 1H), 4.02 (dd, J=13.0, 9.9 Hz, 1H), 3.99
(s, 3H), 2.14-1.93 (m, 4H), 1.91-1.78 (m, 1H), 1.61-1.52 (m, 1H).
.sup.19F NMR (376 MHz, Chloroform-d) .delta. -125.85 (d, J=9.2 Hz),
-132.78 (t, J=10.6 Hz), -136.03 (d, J=10.7 Hz). LCMS-ESI.sup.+
(m/z): [M+H].sup.+ calculated for
C.sub.22H.sub.21F.sub.3N.sub.3O.sub.6: 480.14; found: 480.27.
Step 5.
[0110] To a solution of compound 6 (300 mg, 0.63 mmol) in
acetonitrile (.about.10 mL) was added magnesium bromide (299 mg,
1.63 mmol) at room temperature and the resulting mixture was
stirred at 50.degree. C. bath for 20 min. The reaction mixture was
concentrated and the residue was triturated with water (100 mL) and
dichloromethane (100 mL). The suspension was stirred while in an
ice bath and 1 N HCl was added to make the mixture strongly acidic
(pH, .about.2). After the insoluble product was filtered, the two
fractions were separated and the aqueous fraction was extracted
with dichloromethane (.about.100 mL.times.2). The combined organic
fractions were dried (MgSO.sub.4), concentrated, and purified by
CombiFlash (24 g column) eluting 0-20% methanol in dichloromethane.
The obtained product was combined with the previously obtained
solid product, dissolved in methanol, filtered to remove debris,
and concentrated to get amorphous solid. The amorphous solid was
crystalized in acetonitrile (.about.10 mL) and the crystal formed
was filtered, washed with cold acetonitrile, and dried in vacuum to
get 209 mg (72%) of compound 15 (M23). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 12.44 (s, 1H), 10.33 (t, J=5.7 Hz, 1H), 10.11
(s, 1H), 8.44 (s, 1H), 7.27-7.01 (m, 1H), 5.44 (dd, J=9.6, 4.1 Hz,
1H), 5.09 (d, J=3.8 Hz, 1H), 4.67 (dd, J=12.9, 4.1 Hz, 1H), 4.60
(s, 1H), 4.59-4.43 (m, 2H), 4.02 (dd, J=12.7, 9.6 Hz, 1H), 1.93 (d,
J=5.1 Hz, 4H), 1.84 (d, J=12.1 Hz, 1H), 1.57 (dt, J=12.2, 3.5 Hz,
1H). .sup.19F NMR (376 MHz, DMSO-d.sub.6) .delta. -127.13, -131.52
(t, J=11.2 Hz), -134.59 (d, J=11.4 Hz). LCMS-ESI.sup.+ (m/z):
[M+H].sup.+ calculated for C.sub.21H.sub.19F.sub.3N.sub.3O.sub.6:
466.12; found: 466.26.
Example 3: Confirmation of Chemical Structure of M15
General Methods
[0111] All times, centrifuge speeds, temperatures, and volumes are
approximate due to the normal accuracy constraints of laboratory
equipment. Unless otherwise noted, centrifugation was conducted as
outlined in the following table.
TABLE-US-00001 TABLE 1 Time Matrix Force (Minutes) Temperature
Plasma.sup.a 2800 .times. g 10 Ambient Plasma.sup.b 1400 .times. g
10 Ambient g Gravity. .sup.aExtraction step. .sup.bReconstitution
step.
Plasma Plasma samples used in this Example were stored at
approximately -70.degree. C. before and after analysis. Plasma
samples obtained from male human subjects at 8 hours post-dose were
pooled, including 0.4 g of each sample by weight. The radioactivity
in the pooled sample was determined by liquid scintillation
counting (LSC).
[0112] Approximately 1 g of the pooled plasma sample was combined
with 3 mL of 0.2% (v/v) formic acid (FA) in acetonitrile (ACN),
sonicated, vortex mixed, centrifuged, and the supernatant was
removed. The extraction was repeated, and the respective
supernatants were combined. Duplicate aliquots were analyzed by LSC
to determine the extraction recovery, which was 96.6%.
[0113] The combined supernatants were evaporated to dryness under
nitrogen and reconstituted in 350 .mu.L of reverse osmosis water:
0.2% (v/v) formic acid (FA) in acetonitrile (ACN): methanol:
(4:1:2, v:v:v). Samples were sonicated, vortex mixed, centrifuged,
and duplicate aliquots were analyzed by LSC to determine the
reconstitution recovery, which was 104%. The reconstituted sample
was analyzed by LC-MS, with eluent fractions collected at 10 second
intervals into 96-well plates containing solid scintillant.
Radioactivity in each well was determined using TopCount analysis,
and a radiochemical profile was generated based on radioactivity
counts.
Co-Chromatography of a Plasma Sample with M15 Standard
[0114] An additional plasma sample was prepared by combining 40
.mu.L of the pooled 8 hour plasma sample with 40 .mu.L of the
standard solution of M15 (500 ng/mL). The sample was analyzed by
LC-MS using the following instrumentation and conditions:
TABLE-US-00002 LC-MS Instrumentation Autosampler: Acquity Sample
Manager FTN (10.degree. C.) Binary pump: Acquity I-Class Binary
Solvent Manager Column oven: Acquity Column Manager (25.degree. C.)
Fraction collector: LEAP Technologies PAL HTS-xt (15.degree. C.)
Mass spectrometer: Vion IMS Q-TOF
TABLE-US-00003 LC-MS Conditions Ionization interface: Positive lock
mass electrospray interface HPLC column: Phenomenex, Luna C18(2),
4.6 .times. 250 mm, 5 .mu.m Guard column: Phenomenex, KrudKatcher
Ultra Mobile phase A: 0.1% (v/v) formic acid in water Mobile phase
B: 0.1% (v/v) formic acid in acetonitrile Gradient: Time (minutes)
% A % B 0.0 85 15 0.1 85 15 4.0 85 15 40.0 60 40 44.0 0 100 54.0 0
100 56.0 85 15 64.0 85 15 Flow rate: 1.0 mL/minute; split ratio
1:3, mass spectrometer: fraction collector Survey scan: m/z 50-1000
at 200 ms scan time Auto MS.sup.2 scans: m/z 50-1000 at 300 ms scan
time Capillary voltage: 3500 V Drying gas 450.degree. C.
temperature: Source 120.degree. C. temperature:
Metabolite Identification
[0115] Samples of human plasma were analyzed by using LC-MS, and
metabolite M15 was confirmed to be the same component as the
standard. The structure, parent mass, and characteristic product
ions of M15 from analysis of a plasma sample are presented in Table
2. A summary of representative accurate mass data is presented in
Table 3.
[0116] An extracted ion chromatogram for M15 in a standard solution
is presented in FIG. 2. A radiochromatogram and extracted ion
chromatogram for M15 in a pooled plasma sample is presented in FIG.
3. Extracted ion chromatograms comparing the standard, pooled
plasma sample, and co-injection sample are presented in FIG. 4. To
confirm that M15 and the standard M15 were the same component, the
M15 standard solution and the human plasma sample were analyzed
separately and were co-injected. The retention times of M15 were
26.24 and 26.28 minutes in the M15 standard solution and human
plasma samples, respectively, when analyzed individually (FIG. 1
and FIG. 2). When the M15 standard solution and plasma sample were
co-injected, a single peak was observed with a retention time of
26.28 minutes, as shown in FIG. 4.
[0117] Representative MS precursor and MS/MS product ion mass
spectra of M15, obtained from analysis of a standard solution of
M15, are shown in FIG. 5. The MS precursor ion mass spectrum shows
the protonated molecular ion at m/z 626. The MS/MS product ion mass
spectrum shows fragment ions at m/z 450, 289, 261, and 145.
Representative MS precursor and MS/MS product ion mass spectra of
metabolite M15, obtained from analysis of a study sample, are shown
in FIG. 6, and the MS/MS product ion mass spectrum is virtually
identical to that of the standard. The elemental composition of
metabolite M15 was confirmed using accurate mass analysis and is
shown in Table 3.
TABLE-US-00004 TABLE 2 Retention Characteristic Metabolite Time
Proposed Metabolite Product Ions Designation (Minutes) [M +
H].sup.+ Identification (m/z) Matrix M15 26.28.sup.a 626
##STR00007## 450, 289, 261, 145 Plasma .sup.aRetention time from
analysis of a plasma sample (FIG. 3).
TABLE-US-00005 TABLE 3 Metabolite Measured Theoretical Proposed
Designation Mass Mass Formula .DELTA. mDa .DELTA. ppm M15 626.1600
626.1592 C.sub.27H.sub.27F.sub.3N.sub.3O.sub.11.sup.+ 0.80 1.3
.DELTA. mDa = (Measured mass - Theoretical Mass) * 1000. .DELTA.
ppm = (.DELTA. mDa/Theoretical Mass) * 1000.
Example 4: Confirmation of Chemical Structure of M20
[0118] All times, centrifuge speeds, temperatures, and volumes are
approximate due to the normal accuracy constraints of laboratory
equipment. Unless otherwise noted, centrifugation was conducted at
a speed of approximately 2800.times.g for 10 minutes at room
temperature.
Solutions
[0119] The following solution was used for sample preparation
procedures.
TABLE-US-00006 Solution Name Composition Standard Solution of M20 2
.mu.g/mL in reverse osmosis water: methanol: 0.2% (v/v) formic acid
in acetonitrile (4:2:1, v/v/v)
Plasma
[0120] Plasma samples obtained from male human subjects at 8 hours
post-dose were pooled, including 200 .mu.L of each sample. The
radioactivity in each pooled sample was determined by liquid
scintillation counting (LSC).
[0121] The pooled plasma sample was combined with 3 mL of 0.2%
(v/v) formic acid (FA) in acetonitrile (ACN), sonicated, vortex
mixed, centrifuged, and the supernatants were removed. The
extraction was repeated, and the respective supernatants were
combined. Duplicate aliquots were analyzed by LSC to determine the
extraction recovery, which was 98.6%. The combined supernatants
were evaporated to dryness under nitrogen and reconstituted in 350
.mu.L of reverse osmosis water:methanol:0.2% (v/v) FA in ACN
(4:2:1, v:v:v). Samples were sonicated, vortex mixed, centrifuged,
and duplicate aliquots were analyzed by LSC to determine the
reconstitution recovery, which was 100%. The reconstituted sample
was analyzed by LC-MS with eluent fractions collected at 10-second
intervals into 96-well plates containing solid scintillant.
Radioactivity in each well was determined using TopCount analysis,
and a radiochemical profile was generated based on radioactivity
counts.
Co-Chromatography of Plasma Sample with M20 Standard
[0122] An additional sample was prepared by combining 100 .mu.L of
the reconstituted 8-hour pooled plasma sample with 50 .mu.L of the
standard solution of M20. The resulting sample contained
approximately 1:1 ratio of bictegravir:M20. The sample was analyzed
by LC-MS using the following instrumentation and conditions.
TABLE-US-00007 LC-MS Instrumentation Controller:
Shimadzu/Prominence CBM-20A Pumps: Shimadzu/Nexera LC-30AD
Autoinjector: Shimadzu/Nexera SIL-30ACMP (15.degree. C.) Column
Oven: Shimadzu/Prominence CTO-20AC (25.degree. C.) Degasser:
Shimadzu/Prominence DGU-20A5R Mass spectrometer: Thermo Fisher
Scientific Q Exactive Fraction collector: Leap Technologies PAL
HTC-xt (15.degree. C.)
TABLE-US-00008 LC-MS Conditions Ionization interface: Positive
heated electrospray interface (HESI) HPLC column: Phenomenex, Luna
C18 (2), 4.6 .times. 250 mm, 5 .mu.m Guard column: Phenomenex C18,
3 .times. 4 mm Mobile phase A: 0.1% (v/v) formic acid in water
Mobile phase B: 0.1% (v/v) formic acid in acetonitrile Time
Gradient: (minutes) % A % B 0.0 85 15 4.0 85 15 40.0 60 40 44.0 0
100 54.0 0 100 56.0 85 15 64.0 85 15 Flow rate: 1.00 mL/minute;
split ratio 25:75 mass spectrometer: fraction collector Survey
scan: m/z 150-900 at 70,000 resolution Dependent Scans: MS.sup.2 at
17,500 resolution Source Voltage: +4.5 kV S-Lens RF level 40
Metabolite Identification
[0123] The structure, parent mass, and characteristic product ions
of M20 as a standard and in a plasma sample are presented in Table
5. A summary of representative accurate mass data is presented in
Table 6.
[0124] An extracted ion chromatogram for M20 in a standard solution
is presented in FIG. 7. An extracted ion chromatogram and a
radiochromatogram for M20 in a pooled plasma sample are presented
in FIG. 8. Extracted ion chromatograms comparing the standard,
pooled plasma sample, and co-injection sample are presented in FIG.
9. To confirm that the M20 of the standard solution and M20 were
the same component, the M20 standard solution and the human plasma
sample were analyzed separately and were co-injected. The retention
times of M2 were 36.96 and 37.94 minutes in the M20 standard
solution and human plasma samples, respectively, when analyzed
individually (FIG. 9). When the M20 standard solution and plasma
sample was co-injected, a single peak was observed with a retention
time of 37.29 minutes.
[0125] The protonated molecular ion of M20 was observed at m/z 546
(data not shown). A representative product ion mass spectrum of M20
obtained from analysis of a standard solution of M20 is shown in
FIG. 10. The mass spectrum showed product ions at m/z 466 (loss of
SO.sub.3), 307 (m/z 289 plus water), 289, and 161. A representative
product ion mass spectrum of M20 obtained from the human plasma
sample is shown in FIG. 11, and is virtually identical to that of
the standard. The elemental composition of M20 was confirmed using
accurate mass analysis, as shown in Table 6.
TABLE-US-00009 TABLE 5 Retention Characteristic Metabolite Time
Proposed Metabolite Product Ions Designation (Minutes) [M +
H].sup.+ Identification (m/z) Matrix M20 36.96.sup.a 37.94.sup.b
546 ##STR00008## 466, 307, 289, 161 Plasma .sup.aRetention time
from analysis of a standard solution of M20 (FIG. 7).
.sup.bRetention time from analysis of a plasma sample (FIG. 8).
TABLE-US-00010 TABLE 6 Metabolite Measured Theoretical Proposed
Designation Mass Mass Formula .DELTA. mDa .DELTA. ppm M20 546.0793
546.0789 C.sub.21H.sub.19F.sub.3N.sub.3O.sub.9S.sup.+ 0.40 0.7
.DELTA. mDa = (Measured mass - Theoretical Mass) * 1000. .DELTA.
ppm = (.DELTA. mDa/Theoretical Mass) * 1000.
Example 5: In Vitro Assessment of Human MRP2 Inhibition Potential
of BIC, M15, M20 and M23
Assay Methodology
[0126] Inhibition of the hepatic efflux transporter
multidrug-resistance protein 2 (MRP2; ABCC2) by bictegravir and its
metabolites (M15, M20 and M23) was studied in the following assay.
Cells and experimental conditions for the transporter inhibition
assays are summarized below in Table 7. BIC can be synthesized
according to the procedures described, for example, in WO
2014/100212. M15, M20, and M23 were prepared according to the
procedures described herein. All other materials were purchased by
SOLVO Biotechnology and experiments were conducted according to
SOLVO Standard Operating Procedures (SOPs) of its certified ISO
9001:2008 system. Lot and product information was recorded by SOLVO
Biotechnology.
TABLE-US-00011 TABLE 7 Transporter System Model Substrate Positive
Control MRP2 Membrane Vesicles .sup.3H-E.sub.217.beta.G
Benzbromarone (E.sub.217.beta.G) Estradiol-17beta-glucuronide
Inhibition of Transport in Membrane Vesicles
[0127] Test compounds and positive control was incubated with
membrane vesicle preparations (total protein: 50 .mu.g/well) and
the model substrate in the absence or presence of ATP. Reaction
mixtures were preincubated for 15 minutes at 37.degree. C.
Reactions were started by the addition of 25 .mu.L of 12 mM MgATP
or AMP assay buffer (for background controls) preincubated
separately. Reactions were stopped after 5 min by the addition of
200 .mu.L of ice-cold washing buffer and immediate filtration via
glass fiber filters mounted to a 96-well plate (filter plate). The
filters were washed, dried and the amount of substrate inside the
filtered vesicles determined by liquid scintillation. A positive
control inhibitor was tested in parallel. Control membranes lacking
transporter expression were used as negative control. All assays
were performed in duplicate.
[0128] Fractional transport activities were calculated from the
following equation:
Activity %=(A-B)/(C-D).times.100
where A is translocated amount of substrate in the presence of TA
and ATP, B is translocated amount of substrate in the presence of
TA, C is translocated amount of substrate in the presence of
solvent and ATP, and D is translocated amount of substrate in the
presence of solvent.
IC.sub.50 Determination in Transporter Assays
[0129] IC.sub.50 is defined as the test article concentration
needed to inhibit the maximal transporter specific transport by
50%. IC.sub.50 values were calculated using non-linear fitting of %
inhibition versus concentration to a sigmoidal curve with a
variable Hill Coefficient using GraphPad Prism 5 (GraphPad Software
Inc., San Diego, Calif.). If the % inhibition was less than 50% at
the highest concentration tested, the IC.sub.50 was not determined.
No inhibition observed (NIO) is reported for relative inhibition
results <20% and no concentration dependent transport observed
up to the highest concentration tested.
[0130] Inhibition data are summarized in Table 8. The positive
control benzbromarone at 200 .mu.M showed .gtoreq.99% inhibition in
each assay. BIC showed no inhibition of MRP2-mediated
E.sub.217.beta.G at concentrations up to 100 .mu.M. M15 and M20
showed dose dependent inhibited MRP2 mediated transport of
E217.beta.G with calculated IC.sub.50 values of 256 .mu.M and 45
.mu.M, respectively. Precipitation was observed at the highest
investigated concentration of 300 .mu.M in assay buffer for M20.
M23 showed 43% inhibition of MRP2 mediated transport at highest
test concentration of 100 .mu.M.
TABLE-US-00012 TABLE 8 Uptake Transporter Inhibition Assay Maximum
inhibition observed (% of IC.sub.50 Compound control) (.mu.M) BIC
0% at 100 .mu.M NIO M15 53% at 300 .mu.M 256 M20 98% at 300 .mu.M
45 M23 43% at 100 .mu.M >100
Example 6: In Vitro Assessment of Human OATP Inhibition Potential
of BIC, M15, M20 and M23
Assay Methodology
[0131] The inhibition of the human uptake transporter organic
anion-transporting polypeptide 1B1, 1B3 and 2B1 (OATP1B1, OATP1B3,
OATP2B1; SLC) by BIC and its metabolites (M15, M20 and M23) was
studied in the following assay.
[0132] Cells and experimental conditions for the transporter
inhibition assays are summarized below in Table 9. BIC can be
synthesized according to the procedures described, for example, in
WO 2014/100212. M15, M20, and M23 were prepared according to the
procedures described herein.
TABLE-US-00013 TABLE 9 Model Positive Transporter Test System
Substrate Control OATP1B1/ CHO Cells Fluo-3 Rifampicin OATP1B3
OATP1B1/ HEK293FT Cells .sup.3H-E.sub.217.beta.G Rifampicin OATP1B3
OATP2B1 MDCKII .sup.3H-E3S Fluvastatin (HEK293FT) Fast growing
human embryonic kidney cells transformed with the SV40 large T
antigen (E.sub.217.beta.G) Estradiol-17beta-glucuronide (E3S)
Estrone-3-sulfate (MDCKII) Madin Darby Canine Kidney subclone
II
OATP1B1 and OATP1B3 Inhibition Assay Using Fluo-3 as the Probe
Substrate
[0133] Chinese Hamster Ovary (CHO) cells, either wild type or
transfected with the genes encoding human OATP1B1 or OATP1B3, were
maintained in Dulbecco's Modification of Eagle's Medium (DMEM)
containing 1,000 mg/L D-glucose, L-glutamine, 25 mM HEPES buffer,
and 110 mg/L sodium pyruvate, 1% Pen/Strep, 10% fetal bovine serum,
0.05 mg/mL L-proline and 0.5 mg/mL of geneticin G-418. Cells were
maintained in incubators set at 37.degree. C., 90% humidity and 5%
CO.sub.2. OATP1B1 or OATP1B3 overexpressing CHO cells were seeded
in BioCoat Poly-D-Lysine coated 96 well black cell culture plates
with clear bottoms at a density of 1.times.10.sup.5 cells/well.
Sodium butyrate (10 mM) was added to the OATP1B1 and OATP1B3 cells
to increase the protein expression level, and the cells were grown
to confluence overnight. The assay buffer contained 142 mM NaCl, 5
mM KCl, 1 mM KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, 1.5 mM
CaCl.sub.2), 5 mM Glucose and 12.5 mM HEPES (pH 7.4). After removal
of the media and before adding test compounds, the cells were
washed twice with 37.degree. C. assay buffer followed by a 0.5 h
pre-incubation with assay buffer. Test compounds were serially
diluted in DMSO at 250-fold of final test concentrations to create
the compound spiking solutions. Compounds were then spiked into
assay buffer containing 2 .mu.M Fluo-3 and incubated with cells for
1 h. Following removal of assay buffer containing Fluo-3 and test
compound, cells were washed 3 times with 200 .mu.l of ice cold
assay buffer and then lysed at room temperature for 15 minutes in a
lysis buffer containing 0.05% SDS in a 1 mM CaCl.sub.2) solution.
Substrate accumulations were determined for Fluo-3 fluorescence at
an excitation of 485 nm and emission of 530 nm.
OATP1B1 and OATP1B3 Inhibition Assay Using .sup.3H-E.sub.217.beta.G
as the Probe Substrate
[0134] HEK293FT-Mock and OATP-transfected cells (1.times.10.sup.5
cells each) were seeded 24 hr prior to assay. Pre-rinsed ells were
incubated for 3 minutes with 1 .mu.M .sup.3H-E.sub.217.beta.G in HK
buffer in the presence of various concentrations of test compound
or positive control rifampicin. After the experiment cells were
rinsed with Krebs-Henseleit buffer and lysed with 0.1 M NaOH. The
amount of substrate inside the cells was determined by liquid
scintillation reader.
OATP2B1 Inhibition Assay
[0135] MDCKII-Mock and OATP2B1-transfected cells (1.times.10.sup.5
cells each) were seeded 24 hr prior to assay. Pre-rinsed cells were
incubated for 2 minutes with 0.2 .mu.M .sup.3H-E3S in HK buffer in
the presence of various concentrations of test compound or positive
control fluvastatin. After the experiment cells were rinsed with
Krebs-Henseleit buffer and lysed with 0.1 M NaOH. The amount of
substrate inside the cells was determined by liquid scintillation
reader.
Data Analysis for OATP Inhibition Assays
[0136] Percent inhibition was calculated according to the following
equation:
% inhibition=[1-{[OATP]i-[WT]ni}/{[OATP]ni-[WT]ni}]*100
where: [OATP]i represent the substrate accumulation in the presence
of test compound for either OATP1B1, OATP1B3, or OATP2B1 cells;
[OATP]ni represents the substrate accumulation in the absence of
test compound for either OATP1B1, OATP1B3, or OATP2B1 cells,
respectively; and [WT]ni represents the substrate accumulation in
the absence of test compound for wild type cells or Mock cells,
respectively.
IC.sub.50 Determination in Transporter Assays
[0137] IC.sub.50 was determined according to the procedures
described in Example 5. The positive control inhibitors for each
transporter showed >80% inhibition in each assay. The highest
concentration of BIC, M15, M20 and M23 assayed was 100, 100, 300
and 100 .mu.M, respectively. IC.sub.50 were not determined for
compounds with <20% inhibition or no dose dependent inhibition
observed. Results are reported as NIO (no interaction
observed).
[0138] BIC showed no inhibition of OATP1B1-mediated
estradiol-17beta-glucuronide (E.sub.217.beta.G) transport at
highest test concentration of 100 .mu.M. BIC inhibited 17% of
estrone-3-sulfate uptake by OATP2B1 cells at highest test
concentration 100 .mu.M. Data for BIC is summarized in Table
10.
TABLE-US-00014 TABLE 10 Probe Maximum inhibition Substrate at
highest test conc. IC.sub.50 Transporter Used (% of control)
(.mu.M) OATP1B1 E.sub.217.beta.G 0% inhibition at 100 .mu.M NIO
OATP1B3 E.sub.217.beta.G 6% inhibition at 100 .mu.M NIO OATP2B1 E3S
17% inhibition at 100 .mu.M NIO
[0139] M15 showed 39% inhibition of OATP1B1-mediated and no
inhibition of OATP1B3-mediated transport of Fluo-3 transport at
highest test concentration of 100 .mu.M. Data for M15 is summarized
in Table 11.
TABLE-US-00015 TABLE 11 Probe Maximum inhibition Substrate at
highest test conc. IC.sub.50 Transporter Used (% of control)
(.mu.M) OATP1B1 Fluo-3 39% inhibition at 100 .mu.M >100 OATP1B3
Fluo-3 0% inhibition at 100 .mu.M NIO
[0140] M20 showed 12% inhibition of OATP1B1-mediated and 49% of
OATP1B3-mediated transport of estradiol-17beta-glucuronide
(E.sub.217.beta.G) transport at highest test concentration of 100
.mu.M. M20 inhibited OATP1B1-mediated Fluo-3 transport with
IC.sub.50 value of 90.1 .mu.M and 18% of OATP1B3-mediated Fluo-3
transport at 100 .mu.M. M20 inhibited 26% of estrone-3-sulfate
uptake by OATP2B1 cells at highest test concentration 100 .mu.M.
Data for M20 is summarized in Table 12.
TABLE-US-00016 TABLE 12 Probe Maximum inhibition Substrate at
highest test conc. IC.sub.50 Transporter Used (% of control)
(.mu.M) OATP1B1 E.sub.217.beta.G 12% inhibition at 100 .mu.M NIO*
OATP1B3 E.sub.217.beta.G 49% inhibition at 100 .mu.M >100*
OATP1B1 Fluo-3 54% inhibition at 100 .mu.M 90.1 OATP1B3 Fluo-3 18%
inhibition at 100 .mu.M NIO OATP2B1 E3S 26% inhibition at 100 .mu.M
>100* *Precipitation of test compound observed at 300 .mu.M test
concentration in assay buffer. Data at 300 .mu.M was not used for
determination of IC.sub.50 values.
[0141] M23 inhibited OATP1B1-mediated Fluo-3 transport with
IC.sub.50 value of 99.9 .mu.M and 20% of OATP1B3-mediated transport
of Fluo-3 transport at highest test concentration of 100 .mu.M.
Data for M23 is summarized in Table 13.
TABLE-US-00017 TABLE 13 Probe Maximum inhibition Substrate at
highest test conc. IC.sub.50 Transporter Used (% of control)
(.mu.M) OATP1B1 Fluo-3 51% inhibition at 100 .mu.M 99.9 OATP1B3
Fluo-3 20% inhibition at 100 .mu.M NIO
[0142] Overall, BIC showed no dose dependent inhibition toward
OATP1B1, OATP1B3 and OATP2B1-mediated transport at concentrations
up to 100 .mu.M. M15, M20 and M23 inhibited OATP1B1-mediated Fluo-3
transport with IC.sub.50 of >100, 90.1, and 99.9 .mu.M,
respectively. M15, M20, and M23 showed minimal to no inhibition
with OATP1B3-mediated Fluo-3 transport at concentrations up to 100
.mu.M. M20 showed no dose dependent inhibition of OATP1B1-mediated
E.sub.217.beta.G transport but inhibited both OAT1B3-mediated
E.sub.217.beta.G transport and OATP2B1-mediated estrone-3-sulfate
transport with IC.sub.50 of >100 .mu.M. For inhibition
.ltoreq.20% at highest test concentration, no IC.sub.50 value was
reported.
Example 7: In Vitro Assessment of Inhibition Potential of
Bictegravir and its Metabolites for Human Hepatic Microsomal
Bilirubin Glucuronidation
[0143] In this example, the potential for bictegravir and its
metabolites, M15, M20, and M23 to reduce the catalytic activity of
human hepatic microsomal UGT1A1 was determined, as assayed by
bilirubin glucuronidation. In this assay, The rates of
enzyme-specific metabolite formation from bilirubin substrate were
quantified in the presence and absence of bictegravir and its
metabolites and their IC50 values were determined. This study is
useful for assessing whether there is potential for bictegravir
and/or its metabolites to undergo pharmacokinetic interactions with
other drugs and with endogenous compounds. In this assay, the
inhibitory effects of bictegravir and its metabolites on the
activity of a major human glucuronidation enzyme, uridine
diphosphate glucuronosyltransferase 1A1 (UGT1A1), responsible for
conjugation of bilirubin, was assessed in vitro with the intent of
determining an IC50 value.
Materials
[0144] BIC can be synthesized according to the procedures
described, for example, in WO 2014/100212. M15, M20, and M23 were
prepared according to the procedures described herein. Other
reagents used in the assays were purchased from Sigma-Aldrich (St.
Louis, Mo.) or BD Biosciences (Woburn, Mass.), except for
atazanavir (Toronto Research Chemicals, North York ON). Human
hepatic microsomal fraction was provided by BD Biosciences (Woburn,
Mass.). Bilirubin substrate was prepared fresh immediately before
starting the assay.
Enzyme Inhibition Assays
[0145] Bilirubin is metabolized by UGT1A1 yielding the two acyl
monoglucuronides and the acyl diglucuronide. There is no clear
preference for which of the target propionates (C8 or C12) is
metabolized first. Atazanavir has been demonstrated to be a potent,
selective inhibitor of this activity and is thus an appropriate
positive control. The conditions for the assay were determined to
be linear with respect to microsomal protein concentration and
incubation time. Under the assay conditions the K.sub.M for
bilirubin monoglucuronide formation was determined to be 0.98 .mu.M
and the substrate concentration of 0.8 .mu.M used here is
.ltoreq.K.sub.M. Microsomal UGT1A1 activity was determined in
duplicate. The final reaction mixture was composed of 0.2 mg/mL
hepatic microsomal protein, 100 .mu.g alamethicin/mg microsomal
protein, 5 mM UDP-glucuronic acid, 5 mM magnesium chloride, 5 mM
D-saccharic acid 1,4-lactone (SACLAC), 0.8 .mu.M bilirubin and 0.1
M potassium phosphate buffer pH 7.4. Diluted microsomal fraction
was incubated on ice for 15 minutes with alamethicin, magnesium
chloride and SACLAC. Substrate and inhibitor were then added and
the mixture warmed to 37.degree. C. for 0.5 minute. The reaction
was initiated by the addition of UDP glucuronic acid in potassium
phosphate buffer. The incubation continued at 37.degree. C. with
shaking and no light exposure for 2 minutes. Reactions were
terminated by addition of one volume of 200 mM ascorbic acid in
methanol, containing 200 nM
2-(N-(2-ethylphenyl)methylsulfonamido)-N-(2-(pyridin-2-ylthio)ethyl)aceta-
mide as the internal standard. The samples were centrifuged at 3600
rpm for 5 minutes at 4.degree. C., and aliquots of the supernatant
subject to LC-MS/MS to monitor monoglucuronide formation from
bilirubin.
Liquid Chromatography--Mass Spectrometry (LC-MS)
[0146] A Shimadzu UFLC XR UPLC system was used for analysis. The
column used was a Thermo-Hypersil Gold 1.9 .mu.m C18 column
(30.times.2.1 mm) held at 60.degree. C. Mobile phases were; A:
water containing 0.1% (v/v) formic acid, and B: acetonitrile
containing 0.1% (v/v) formic acid, pumped at 0.7 mL/minute. Elution
was achieved by a series of linear gradients over 2 minutes. The
mass spectrometer was an Applied Biosciences SCIEX QTRAP 5500
triple quadrupole mass spectrometer with an electrospray interface
operating in positive ion mode. Quantification was by
metabolite/internal standard peak area ratio (PAR). Extracted
samples stored in the autosampler exhibited instability of the
bilirubin glucuronide signal. Loss was .about.0.1%/min.
Data Analysis
LC-MS/MS Analysis
[0147] Bilirubin glucuronide standards are not available
commercially so the bilirubin monoglucuronide and diglucuronide
peaks were identified by their MS properties. MS/MS transitions
([M+H]+) of m/z 761.2/475.1 and 937.2/475.1 for the monoglucuronide
and diglucuronide, respectively. The PAR values for the two
monoglucuronides were combined for quantification. The PAR values
in the presence of inhibitors were compared to those of vehicle
controls (no bictegravir, M15, M20, M23, or positive control
inhibitor) and activities expressed as the percentage of control
activity remaining.
IC.sub.50 Determinations
[0148] Reaction velocities were calculated from the rates of
formation of the metabolites and were compared to those seen with
the vehicle control (100% activity). IC.sub.50 values were
calculated by non-linear regression using GraphPad Prism 7.03 and a
sigmoidal three parameter inhibition model. Weak inhibition by the
test compounds necessitated constraining the lower plateau value of
the model (residual activity when UGT1A1 is fully inhibited) to
generate meaningful IC.sub.50 values. During the testing period the
inhibitory potency of atazanavir was determined four times, with
each concentration tested in duplicate in each determination. The
data from all runs were pooled and a global fit performed to
determine the lower plateau value. The Best-fit value was 10.96%
(standard error 2.94%) activity remaining. IC.sub.50 values of
bictegravir and its metabolites were calculated by non-linear
regression with the lower plateau constrained to this value. For
atazanavir, the IC.sub.50 values from the four duplicate runs were
combined to generate a summary geometric mean and multiplicative
standard deviation for this positive control inhibitor.
Results
[0149] Inhibitory effects of bictegravir, M15, M20, and M23 on the
activity of human hepatic microsomal bilirubin monoglucuronidation
were assessed. A summary of the inhibitory potencies and for the
positive control inhibitor, atazanavir, is presented in Table 14.
The positive control inhibitor, atazanavir, reduced UGT1A1 activity
as expected, confirming satisfactory incubation conditions for the
assays (Table 1). The geometric mean IC.sub.50 value for atazanavir
obtained over the four runs was 1.2 .mu.M. Concentrations of
bictegravir and its glucuronide metabolite (M15) up to 300 .mu.M
had little or no inhibitory effect upon UGT1A1 activity (inhibition
<2%). There was modest stimulation of enzyme activity at high
concentrations of these two test compounds, reaching peak increases
of 76% at 200 .mu.M bictegravir and 21% at 200 .mu.M M15Bictegravir
metabolites M20 and M23 were weak inhibitors of human hepatic
bilirubin glucuronidation with IC.sub.50 values of 153 and 256
.mu.M, respectively.
TABLE-US-00018 TABLE 14 Calculated IC.sub.50 (.mu.M) Atazanavir
Activity (Control) Bictegravir M15 M20 M23 Bilirubin 1.2
(1.44).sup.a >300.sup.b (NIO) >300.sup.b (NIO) 153.sup.c
256.sup.c monoglucuronidation NIO No inhibition observed (<2%
inhibition over the concentration range 0-300 .mu.M)
.sup.aGeometric mean and multiplicative standard deviation for four
determinations in duplicate runs. .sup.bFit did not converge.
Maximum concentration tested was 300 .mu.M. .sup.cBest-fit value
using 8 duplicate datapoints
[0150] At concentrations up to 300 .mu.M there was little or no
inhibitory effect of bictegravir or M15 on human hepatic microsomal
bilirubin glucuronidation (an activity catalyzed by UGT1A1). M20
and M23 were weak inhibitors with calculated IC.sub.50 values of
153 .mu.M and 256 .mu.M respectively.
Example 8: Metabolites of BIC Detected In Cyropreserved Hepatocytes
from Different Species
[0151] Cryopreserved hepatocytes were incubated for 4 hours with
radiolabeled BIC to identify metabolites, determine their abundance
and compare nonclinical species with human. The percentage of
parent drug and identified metabolites following incubation with
[.sup.14C]BIC (20 .mu.M) in cryopreserved hepatocytes are
summarized in Table 15 and their proposed identities are shown in
FIG. 12. Metabolic pathways included hydroxylation (3 variants),
N-dealkylation, and direct glucuronidation. All human metabolites
were also observed in nonclinical species. Using the hepatocyte
system where the full range of hepatic metabolic enzymes are
represented, it appeared that the metabolism of BIC was extensive
in monkey and dog but lower in rat and human.
TABLE-US-00019 TABLE 15 Fraction of Radiochromatogram (%) Wister-
Cynomolgus Analyte.sup.a Identity Han Rat Beagle Dog Monkey Human
BIC Parent 91.5 78.7 52.4 93.9 M305 N-dealkylation 1.7 8.7 2.4 1.2
M465a Hydroxylation-1 1.2 1.4 2.7 -- M465b Hydroxylation-2 -- 0.2
11.6 0.6 M465c Hydroxylation-3 -- 3.6 -- -- M611 Glucose
conjugation -- 0.8 4.4 -- M625 Glucuronide conjugation 5.2 6.6 21.7
4.3 M641 Hydroxylation/ -- -- 4.1 -- glucuronidation Total -- 99.6
100 99.3 100 .sup.aAnalyte metabolite identification numbers
correspond to their molecular weight, e.g., M305 = metabolite with
305 Da molecular weight.
Example 9. In Vivo BIC Metabolism in Different Species
[0152] Bictegravir metabolism was determined following a single
oral administration of [.sup.14C]BIC to mouse, rat, monkey, and
human. Pooled plasma, urine, bile, and fecal samples obtained
following in vivo oral administration of [.sup.14C]BIC were
profiled and a comprehensive listing of the identified metabolites
are provided in transgenic mice, Wistar-Han rats, monkeys, and
healthy human subjects. The combined results demonstrate that BIC
is mainly eliminated by hepatic metabolism followed by excretion
into feces and urine. Metabolic pathways included hydroxylation,
oxidative defluorination, direct glucuronidation, and oxidation
followed by phase II conjugation. In the monkey, BIC was
metabolized through the oxidative pathways to a greater extent
compared to rat and human. Results of the plasma profile following
oral administration of [.sup.14C]BIC is shown below in Table
16.
TABLE-US-00020 TABLE 16 % of Total Radioactivity in AUC Pooled
Plasma.sup.a Transgenic Wistar Han Cynomolgus Component Mouse Rat
Monkey Human BIC 95.5 76.5 80.2 67.9 M12 1.86 2.18 ND ND M15 ND ND
0.55 8.6 M20 ND 11.3 0.77 20.1 M21/M22 ND 1.18 ND 2.0 M23 ND 2.36
ND 0.2.sup.c M42 ND ND 12.2 ND Other.sup.b 0.64 2.36 3.44 0.6 Total
98.0 95.9 97.2 99.4 ND = not detected .sup.aAUC pool plasma = area
under the plasma .sup.14C concentration-time curve from time zero
to 48 hours post dose in transgenic mice, from time zero to 168
hours post dose in rats, from time zero to 72 hours post dose in
monkeys, and from time zero to 72 hours dose in human subjects.
.sup.bOther = sum of other metabolites; each component <1% in
mouse; <1.5% in rat, monkey, and human. .sup.cCo-eluted with
M51.
[0153] All references, including publications, patents, and patent
documents are incorporated by reference herein, as though
individually incorporated by reference. The present disclosure
provides reference to various embodiments and techniques. However,
it should be understood that many variations and modifications may
be made while remaining within the spirit and scope of the present
disclosure.
* * * * *