Note: Descriptions are shown in the official language in which they were submitted.
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
ANTIVIRAL THERAPY USE OF P-GLYCOPROTEIN MODULATORS
Field of the Invention
The present invention relates to treatment of viral infections. More
particularly the present invention is directed to the use of certain P-
glycoprotein
modulators to increase the concentration of HIV-protease inhibitors in certain
tissues.
Background and Summary of the Invention
HIV protease inhibitors have proven to be effective in the treatment of
HIV-1 infection. However, the utility of such drugs can be limited due to poor
transport across certain biological membranes. Oral absorption of protease
inhibitors
is often low and variable, and penetration into certain tissues, including the
brain and
testes, is often poor. The resultant non-uniform distribution of the antiviral
drug in
the body leaves certain tissues as sanctuaries for viral proliferation.
P-glycoprotein is an ATP dependent efflux membrane transporter with
broad substrate specificity for a variety of structurally diverse drugs. P-
glycoprotein
is distributed in various normal tissues, including, of particular importance
in drug
disposition, epithelial cells in the gastrointestinal tract, the liver, and
the kidney.
Apical expression of P-glycoprotein in such tissues results in reduced
absorption
(gastrointestinal tract), and enhanced elimination into the bile (liver) and
urine
(kidney) for drugs functioning as P-glycoprotein substrates. In addition,
expression of
P-glycoprotein at the level of the blood-brain barner has been shown to be a
critical
factor in preventing the entry of some drugs into the central nervous system.
Previous
work has shown that various HIV-1 protease inhibitors are substrates of P-
glycoprotein, explaining some of the limits on membrane permeability of these
drugs.
See, for example, Kim, R.B., et al., The Drug Transporter P-Glycoprotein
Limits Oral
Absorption and Brain Entry of HIV-1 Protease Inhibitors, J. Clin. Invest.,
101:289-
294, 1998.
Certain 10,11-methanodibenzosuberane derivatives have been shown
to be pharmaceutically active agents in the treatment of multidrug resistance
in cancer
therapy. See, for example, U.S. Patents Nos. 5,654,304 and 5,874,434. Such
compounds are known to interact with P-glycoprotein.
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-2-
The present invention relates to a use of a 10,11-
methanodibenzosuberanes of formula (I):
R1 R2
N
c~
I N
A
\ 0
R3
wherein: A is -CHzCHz-; -CHzCHRaCHz- where Ra is H, OH or lower acyloxy; or
-CH~CHRbCHR'CHZ- where one of Rb or R' is H, OH, or lower
acyloxy, and the other is H;
R' is H, F, Cl, or Br;
RZ is H, F, Cl, or Br; and
R3 is heteroaryl or phenyl optionally substituted with F, C1, Br, CF3,
CN, NO2, or OCHF2; or a pharmaceutically acceptable salt thereof; for the
manufacture of a medicament for the treatment of HIV in a patient undergoing
treatment with an HIV protease inhibitor. The use increases the concentration
of the
HIV inhibitor in the brain and/or testes of the patient without significantly
increasing
plasma levels of the protease inhibitor. Accordingly, more effective antiviral
therapy
can be achieved without use of increased drug dosages, thereby reducing the
potential
for occurrence of undesirable side effects deriving from drug toxicity. Thus,
one
aspect of this invention relates to a method for increasing the concentration
of an HIV
protease inhibitor in the brain of a patient, the method comprising
administering to an
HIV infected patient an amount of a 10,11-methanodibenzosuberane of formula
(I), or
a pharmaceutically acceptable salt thereof, and co-administering to the
patient a
therapeutically effective amount of the protease inhibitor.
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-3-
Another related aspect of this invention is a method of treatment of an
HIV infected patient. The method comprises administering a compound comprising
a
10,11-methanodibenzosuberane of formula (I) in an amount effective to increase
the
concentration of a co-administered protease inhibitor in the brain and testes
of the
patient.
In another embodiment, the 10,11-methanodibenzosuberane of formula
(I) is administered in combination with a protease inhibitor to increase
concentrations
of the protease inhibitor in the brain.
Still another aspect of this invention is a pharmaceutical composition
comprising a protease inhibitor, most preferably nelfinavir, and a 10,11-
methanodibenzosuberane of formula (I), with a pharmaceutical carrier. In a
preferred
embodiment, the 10,11-methanodibenzosuberane is a compound of formula (II):
F F
N
~N~
II
~ OH
O
O O
Still another aspect of this invention is the use of an HIV protease
inhibitor for the manufacture of a medicament for the treatment of HIV wherein
the
concentration of the protease inhibitor in the brain is increased by co-
administration
with a 10, 11-methanodibenzosuberane of formula (I), or a pharmaceutically
acceptable salt thereof.
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-4-
Additional features of the present invention will become apparent to
those skilled in the art upon consideration of the following detailed
description of
preferred embodiments exemplifying the best mode of carrying out the
invention.
Brief Description of the Drawings
Fig. 1 is a plot of percent inhibition of P-glycoprotein mediated [3H]-
digoxin transport across a Caco-2 cell culture monolayer verses concentration
of
putative inhibitor at varying concentrations of formula (II) (o), nelfinavir
(~),
ritonavir (o), saquinavir (~), and indinavir (o).
Fig. 2 shows tissue levels of ['QC]-nelfinavir in mdrla (+/+) mice given
50 mg/kg of formula (II) (plasma - open symbols, brain - closed symbols) in
divided
doses 30 min prior to and simultaneously with (5 mg/kg) ['4C]-nelfinavir
(triangles) or
vehicle (circles).
Fig. 3 shows the effect of P-glycoprotein inhibitors on tissue:plasma
concentration ratios of ['4C]-nelfinavir in mdrla (+/+) and mdrla (-/-) mice.
Detailed Description of the Invention
The following definitions are set forth to illustrate and define the
meaning and scope of the various terms used to describe the invention herein.
Additional details on the preparation of such compounds, and the meaning and
scope
of the terminology and definitions thereof, aie detailed in U.S. Patent No.
5,654,304.
The term "lower acyloxy" refers to the group --O--C(O)--R' where R' is
lower alkyl.
The term "heteroaryl" refers to a monovalent unsaturated aromatic
carbocyclic radical having at least one hetero atom, such as N, O or S, within
the ring,
such as quinolyl, benzofuranyl and pyridyl.
A "pharmaceutically acceptable salt" may be any salt derived from an
inorganic or organic acid. The term "pharmaceutically acceptable anion" refers
to the
anion of such acid addition salts. The salt and/or the anion are chosen not to
be
biologically or otherwise undesirable.
The term "treatment" or "treating" means any treatment of a disease in
a mammal, including:
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-5-
(i) preventing the disease, that is, causing the clinical symptoms of
the disease not todevelop;
(ii) inhibiting the disease, that is, arresting the development of
clinical symptoms; and/or
(iii) relieving the disease, that is, causing the regression of clinical
symptoms.
The term "effective amount" means a dosage sufficient to provide
treatment for the disease state being treated. This will vary depending on the
patient,
the disease and the treatment being effected.
The term "co-administer" means the administration of more than one
active agent as part of the same treatment regimen, whether they are
administered
simultaneously or at different times.
"Structure of formula (I)" refers to the generic structure of the
compounds of the invention.
The present invention is a method for increasing the concentration of
an HIV protease inhibitor in the brain and testes of a patient, said method
comprising
administering to an HIV-infected patient an amount of a 10,11-
methanodibenzosuberanes of the formula (I):
I
R1 R2
N
N
A
\ 0
R3
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-6-
wherein: A is -CHzCHz-; -CHZCHRaCH2- where Ra is H, OH or lower acyloxy; or
-CHZCHRbCHR'CHz- where one of Rb or R' is H, OH, or lower
acyloxy, and the other is H;
R' is H, F, Cl, or Br;
RZ is H, F, Cl, or Br; and
R3 is heteroaryl or phenyl optionally substituted with F, Cl, Br, CF3,
CN, NOZ, or OCHF2; or a pharmaceutically acceptable salts thereof; and co-
administering to the patient a therapeutically effective amount to the
protease
inhibitor.
In a preferred embodiment, a compound of formula (I) is used wherein
A is -CHZCHRaCH2-. In another preferred embodiment, R' and RZ are F. In still
another preferred embodiment, R3 is an optionally substituted quinolyl,
preferably
quinol-S-yl.
In another preferred embodiment of the present invention, a compound
of formula (II):
F F
N
/ \
~N~
II
~ OH
0
00
is employed with protease inhibitors in the method of the present invention.
Examples of such protease inhibitors contemplated by the present
invention are NELFINAVIR, which is preferably administered as the mesylate
salt at
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
_7_
750 mg three times per day (Agouron Pharmaceuticals (La Jolla, CA)) (U.S.
Patent
No. 5,484,926); RITONAVIR, which is preferably administered at 600 mg twice
daily (Roche Ltd. (Lewes, UK) (U.S. Patent No. 5,484,801); SAQUINAVIR, which
is
preferably administered as the mesylate salt at 1,200 mg three times per day
(Roche
Discovery (Rahway, NJ)) (U.S. Patent No. 5,196,438); INDINAVIR, which is
preferably administered as the sulfate salt at 800 mg three times per day
(Merck
Research Laboratories) (U.S. Patent No. 5,413,999); and AMPRENAVIR, which is
preferably administered at 1,200 mg twice daily (U.5. Patent No. 5,585,397).
The
skilled artisan would recognize that this list is not exhaustive. Additionally
the skilled
artisan would recognize that the protease inhibitor's administration to a
patient may
vary from the preferred.
The HIV-1 virus enters the brain and other organs such as the testes
relatively early after primary infection. Reduction of the viral load in such
organs has
proven to be difficult, as most of the current HIV antiviral agents do not
readily
penetrate into the tissues to provide concentrations effective to prevent
viral
replication. See Groothius, D.R., and Levy, R.M., The entry of antiviral drugs
into
the central nervous system, J. NeuroVirology, 3:387-400, l 997. The low rate
of drug
transport into these pharmacologic sanctuary sites is the consequence of a
functional
barner to drug entry. HIV protease inhibitors have been found to be excellent
substrates for the membrane efflux pump P-glycoprotein, which is localized in
the
apical domain of capillary endothelial cells of the brain and testis. The P-
glycoprotein
pump works to limit drug distribution into these tissues. See, for example,
Kim, R.B.,
et al., The drug transporter P-glycoprotein limits oral absorption and brain
entry of
HIV-1 protease inhibitors, J. Clin. Invest., 101:289-294, 1998; Lee, C.G.L.,
et al.,
HIV-1 protease inhibitors are substrates for the mdrl multidrug transporter,
Biochemistry, 37:3594-3601, 1998; Kim, A.E., et al., Saquinavir, an HIV
protease
inhibitor, is transported by P-glycoprotein, J. Pharmaco. Exp. Ther., 286:1439-
1445,
1998; Thiebaut, F., et al., Cellular localization of the multidrug resistance
gene
product P-glycoprotein in normal human tissue, Proc. Natl. Acad. Sci., U.S.A.,
84:7735-7738, 1987; Gordon-Cardo, C., et al., Multidrug-resistance gene (P-
glycoprotein) is expressed by endothelial cells at blood-brain barner sites,
Proc. Natl.
Acad. Sci., U.S.A., 86:695-698, 1989.
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
_g_
The present invention enables pharmacological inhibition of the
functional activity of the P-glycoprotein transporter on HIV protease
inhibitor
substrates through use of a 10,11-methanodibenzosuberane of formula (I) co-
administered with an HIV protease inhibitor. Such modulation of P-glycoprotein
activity results in significantly enhanced HIV protease inhibitor
concentrations in both
the brain and testes relative to drug concentration in plasma.
The magnitude of the effect of P-glycoprotein inhibition attainable by
administration of the compounds of formula (I) is tissue dependent; for
example, the
tissue:plasma drug concentration ratio is enhanced in the brain to a greater
extent than
in the testes. This difference is believed to be related to the level of P-
glycoprotein
function in the respective tissues. There is about a 30-fold difference in
nelfinavir
concentration in the brain of mdrla(+/+) and mdrla(-l-) mice compared to only
a 4--
fold concentration difference in the testes. P-glycoprotein inhibition using
the
compounds of formula (I) exhibits similar tissue differences. Notably,
however,
1 S nelfinavir concentration differences achieved in both organs indicates a
75 to 90%
absence of P-glycoprotein function based on comparable data in the mdrla(-l-)
mice.
At the highest doses of the compound of formula (II), the concentrations of
nelfinavir
in the brain and testes are equal to or higher than the drug concentration in
plasma.
Co-administration of a 10,11-methanodibenzosuberane of formula (I) with an HIV
protease inhibitor in accordance with this invention minimizes P-glycoprotein
modulated drug concentration differences between plasma and the brain and
testes,
thereby reducing or eliminating these tissues as sanctuaries for viral
proliferation in
patients receiving protease inhibitor therapy.
The present invention provides advantages over use of prior art P-
glycoprotein inhibitors such as quinidine, verapamil, valspodar, and
cyclosporine A,
which are known to interact with drug metabolizing enzymes, in particular,
members
of the cytochrome P4503A subfamily (CYP3A). Inhibitors of P-glycoprotein are
frequently inhibitors of CYP3A and vice-versa. See, for example, Wacher, V.J.,
et
al., Overlapping substrate specificities and tissue distribution of cytochrome
P4503A
and P-glycoprotein: implications for drug delivery and activity in cancer
chemotherapy, Mol. Carcinogen, 13:129-134, 1995; Kim, R.B., et al.,
Interrelationship between substrates and inhibitors of human CYP3A and P-
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-9-
glycoprotein, Pharm. Res., 16:408-44, 1999. Accordingly, with drugs such as
quinidine, verapamil, valspodar, and cyclosporine A, a dual interaction occurs
whereby reduced P-glycoprotein function is associated with increased plasma
levels
of the CYP3A substrate.
Although many P-glycoprotein inhibitors impair CYP3A-mediated
metabolism, this is not an absolute relationship. In fact, the two
characteristics appear
to be independently determined such that some CYP3A inhibitors do not cause
significant impairment of P-glycoprotein function and, more importantly, the
reverse
situation is possible, i.e., effective transporter inhibition with minimal
effect on
CYP3A. See Wandel, C., et al., P-glycoprotein and cytochrome P4503A
inhibition:
dissociation of inhibitory potencies, Cancer Res., in press, 1999. The 10,11-
methanodibenzosuberanes of formula (I) are representative of such drugs. For
example, the affinity of the compound of formula (II) for CYP3A is some 40-
fold less
than that for P-glycoprotein. Shepard, R.L., et al., Selectivity of the potent
P-
glycoprotein modulator, LY335979, Proc. Amer. Assoc. Cancer. Res., 39:362,
1998;
Dantzig, A., J. Pharmco. Exp. Ther., 290:854-862, 1999. This selectivity would
account for the relative small formula (II)-induced changes in nelfinavir's
plasma
level. Thus, the present invention has advantages over prior art P-
glycoprotein
inhibitors, since systemic toxicity from the antiviral agent would not be
expected to
increase following administration of compounds of formula (I).
An additional problem associated with prior art use of P-glycoprotein
modulators has been their limited potency. Because of this limited potency,
effective
levels have been difficult to achieve without adverse effects. The minimal
effects of
quinidine, verapamil; ketoconazole, and cyclosporine A on nelfinavir's
tissue:plasma
ratios are consistent with such low potency as demonstrated by their ICSO
values
relative to digoxin translocation across Caco-2 cells. By contrast, the
compound of
formula (II) , which is at least 50-fold more potent than the other
inhibitors, produced
75% to 90% inhibition of P-glycoprotein transport in both the brain and
testes. This
finding emphasizes the importance of potency in the application of P-
glycoprotein
modulators.
Another issue of selectivity by currently available P-glycoprotein
modulators is related to the inhibition of P-glycoprotein itself versus other
membrane
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-10-
transporters that may also be involved in drug efflux or drug uptake into the
cell. An
increasing number of both types of membrane transporters have been identified
and
characterized in various cells/tissues within the body. Moreover, cross-
inhibition of
different transports appears to occur. For example, a number of P-glycoprotein
inhibitors such as quinidine, verapamil, ketoconazole, and valspodar also
impair drug
uptake by OATP, but at higher concentrations than those required for
inhibition of the
efflux transporter. See Cvetkovic, M., et al., OATP and P-glycoprotein
transporters
mediate the coordinate cellular uptake and excretion of fexofenadine, Drug
Metab.
Disp., 27:866-871, 1999. Because an OATP type of transporter is present in the
brain, (see Noe, B., Isolation of a multispecific organic anion and cardiac
glycoside
transporter from rat brain, Proc. Natl. Acad. Sci., 94:10346-10350, 1997), it
is not
unreasonable to suggest that the observed reduction in nelfinavir's plasma
ratio with
higher doses of cyclosporine A reflects such non-selectivity. A similar effort
with
valspodar has also been observed with another P-glycoprotein substrate -
digoxin. In
contrast, since the brain:plasma ratio continues to increase over the whole
dose range
studied, compound of formula (II) does not appear to inhibit transporters
other than
P-glycoprotein, at least in the brain. See Dantzig, supra.
Thus, the present invention employs the 10,11-
methanodibenzosuberanes of formula (I) to increase HIV protease inhibitor
concentrations in the brain and testes, without an associated increase in
plasma
concentrations.
The 10,11-methanodibenzosuberanes of formula (I) are typically
co-administered with an HIV protease inhibitor, such as nelfinavir,
saquinavir,
indinavir, ritonavir, or amprenavir. In one preferred drug administration
protocol a
patient is pretreated with one or more doses of a compound of formula (I), and
another dose of the P-glycoprotein inhibitor is administered concurrently with
a dose
of the HIV protease inhibitor. Typically, HIV protease inhibitors are
administered
orally in tablet form three times per day, in amounts of 600 to 1200 mg per
dose.
Administration of the compounds of formula (I) can be via any accepted mode of
drug
administration.
Dosage levels of the compound of formula (I) for use in accordance
with this invention range can vary according to patient condition and weight
but
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-11-
generally range from about 0.01 to about 50 mg/kg of patient body weight, more
preferably about 0.1 to 10 mg/kg of body weight, and most preferably about 0.3
to 2.0
mg/kg of body weight per dose.
The administration of the compounds of formula (I) in HIV treatment
protocols with protease inhibitors in accordance with this invention can be
carried out
using any pharmaceutically acceptable mode of drug administration. The
compounds
of formula (I) can be administered either alone or more typically in
combination with
pharmaceutically acceptable excipients, including those used in formulating
solid,
semi-solid, liquid, or aerosol dosage forms, such as, for example, tablets,
capsules,
powders, liquids, suspensions, suppositories, nasal solutions, aerosols or the
like. The
compounds of formula (I) can also be administered in sustained or controlled
release
dosage forms, including depot injections, osmotic pumps, biodegradable
matrices,
transdermal (including electrotransport) patches, and the like, for the
prolonged
administration of the compound at a predetermined rate, preferably in unit
dosage
forms suitable for administration of precise dosages. The compositions will
typically
include a conventional pharmaceutical Garner or excipient and a compound of
formula
(I). In addition, the present compositions may include other medicinal agents,
pharmaceutical agents, carriers, adjuvants, etc., including a suitable dose of
an HIV
protease inhibitor. Generally, depending on the intended mode of
administration, the
pharmaceutically acceptable composition will contain about 0.1 % to 90%,
preferably
about 0.5% to 50%, by weight of a compound or salt of formula (I), the
reminder
being suitable pharmaceutical excipients, Garners, etc.
One manner of administration of the compounds of formula (I) is oral,
using a convenient daily dosage regimen which can be adjusted according to
patient
condition and total antiviral treatment protocol. For oral administration, a
pharmaceutically acceptable composition is formulated by the combination of a
compound of formula (I) and optional protease inhibitor with any of the
normally
employed pharmaceutical excipients, for example, mannitol, lactose, starch,
magnesium stearate, sodium saccharine, talcum, cellulose, sodium cross
carmellose,
glucose, gelatin, sucrose, magnesium carbonate, propylene carbonate, vegetable
oils,
or triglycerides, and the like. Such dosage compositions include solutions,
suspensions, tablets, dispersible tablets, capsules, powders, lozenges,
sustained release
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-12-
formulations and the like. Preferably the compositions for oral administration
will
take the form of a tablet, capsule, or caplet.
Liquid pharmaceutical compositions in accordance with this invention
can be prepared by dissolving, dispersing, etc. an active compound of formula
(I) and
S optional pharmaceutical adjuvants in a carrier, such as, for example, water,
saline,
aqueous dextrose, glycerol, glycols, ethanol, and the like, to form a solution
or
suspension. If desired, the pharmaceutical composition to be administered may
also
contain minor amounts of nontoxic auxiliary substances such as wetting agents,
emulsifying agents, or solubilizing agents, pH buffering agents and the like,
for
example, acetate, citrate, cyclodextrine derivatives, sorbitan monolaurate,
triethanolamine sodium acetate, triethanolamine oleate, etc.
Dosage forms or compositions containing active ingredient in the
range of 0.005% to 95% with the balance made up from non-toxic carrier may be
prepared. Other useful formulations include those set forth in U.S. Pat. Nos.
Re.
28,819 and 4,358,603.
The present invention can also be carned out using formulations for
parenteral administration, i.e, subcutaneous, intramuscular, intrathecal, or
intravenous
administration. Injectable dosage forms of this invention can be prepared as
liquid
solutions or suspensions, solid forms suitable for dissolution or suspension
in liquid
prior to injection, or as emulsions. Suitable excipient carriers are, for
example, water,
saline, dextrose, glycerol, ethanol or the like. In addition, if desired, the
pharmaceutical compositions to be administered may also contain minor amounts
of
non-toxic auxiliary substances such as wetting or emulsifying agents, pH
buffering
agents, solubility enhancers, and the like, such as for example, sodium
acetate,
sorbitan monolaurate, triethanolamine oleate, cyclodextrins, etc. A more
recently
devised approach for parenteral administration employs the implantation of a
slow-release or sustained-release system, such that a more or less constant
rate of drug
release is maintained. See, e.g., U.S. Pat. No. 3,710,795.
The percentage of active compound contained in such parenteral
compositions depends on the specific use and the needs of the subject.
However,
percentages of active ingredient of 0.01 % to 10% in solution are acceptable,
and they
may be higher if the composition is a solid which will be subsequently diluted
to the
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-13-
above percentages. Preferably the composition will comprise 0.2 - 10% of the
active
agent in solution.
EXAMPLES
The following preparations and examples are given to enable those
skilled in the art to more clearly understand and to practice the present
invention.
They should not be considered as limiting the scope of the invention, but
merely as
being illustrative and representative thereof.
Example 1
Inhibition of the P-glycoprotein transport pump was measured as a
function of inhibition of digoxin transport in an in vitro culture system.
Inhibition of
digoxin transport was determined using a polarized monolayer of Caco-2 cells.
Caco-
2 cells were grown and cultured on 0.4 ~m polycarbonate membrane filters as
described in Kim, R.B., et al., The drug transporter P-glycoprotein limits
oral
absorption and brain entry of HIV-1 protease inhibitors, J. Clin. Invest.,
101:289-294,
1998. Transport of [3H]-digoxin (15 Ci/mmol; Dupont-New England Nuclear,
Boston, MA) was determined by its addition to either the basal or apical side
of the
polarized cell monolayer, and the transport over a four hour period of time of
radioactivity into the other compartment was measured in the absence or
presence of
putative inhibitor in both compartments. The extent of inhibition by each
putative
inhibitor was determined using the following equation:
i -i
% inhibition = 1- [ B-A A-B ] x 100
as-A aA-s
where i and a are the percentages of digoxin transport in the presence and
absence of
inhibitor, according to the direction of transport. ICS° values were
estimated from the
Hill equation using the computer program Prism~ (GraphPad Software Inc., San
Diego, CA), and the data represent results obtained from at least 3
preparations on
different days.
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-14-
ICSO values were calculated for various known P-glycoprotein inhibitors;
ketoconazole
(1 .2 ~M), cyclosporine A (1.3 ~M), verapamil (2.1 ~M) and quinidine (2.2 ~M),
were in the low micromolar range. Fig. 1 illustrates the P-glycoprotein
inhibition
observed with various other putative inhibitors. Nelfinavir exhibited
comparable
inhibitory potency (1.4 ~M) to the potency of the known P-glycoprotein
inhibitors.
However, ritonavir (3.8 ~M) and saquinavir (6.5 ~M) were somewhat less potent,
and
the ICSO value for indinavir (44~M) was about an order of magnitude greater
than the
ICSO values for the other HIV protease inhibitors. As shown in Fig. l, the
compound
of formula (II) was by far the most potent of the P-glycoprotein inhibitors
studied,
with an ICSO value (0.024 ~M) over 50-fold lower than cyclosporine A.
Example 2
The tissue distribution of nelfinavir in the absence of any other
putative inhibitor was determined in mdrla(+l+) and mdrla(-l-) mice. Male
mdrla(-l-)
mice (FVB/TacfBR-[KO]mdrlaN7), 6-12 weeks of age and genetically matched male
mdrla(+/+) mice (FVB/MTtacfBR) weighing 20 to 30 g were obtained from Taconic
(Germantown, NY). The animals were cared for in accordance with the USPHS
policy for the Care and Use of Laboratory Animals and the experimental studies
were
approved by the Vanderbilt University Animal Care Committee.
The tissue distribution of ['4C]-nelfinavir (8.5 mCi/mmol, Agouron
Pharmaceuticals, Inc., San Diego, CA) was determined following intravenous
injection (5 mg/kg) of an ethanol/0.9% saline solution over 5 minutes into a
tail vein;
the total volume injected was 4 ~1/g. At specific times after drug
administration and
following anesthesia with isoflurane (Isoflo, Abbott Laboratories, Abbott
Park, IL),
blood was removed by orbital bleeding and the animal sacrificed. Subsequently,
tissues were harvested, weighed, and homogenized with 4% bovine serum albumin
solution. Total radioactivity was determined after the addition of 100 u1
plasma or
500 u1 tissue homogenate to vials containing 4 ml scintillation fluid
(Scintiverse BD*,
Fisher Scientific Co., Fairlawn, NJ). The brain:plasma ratio was 0.06 in the
mdrla(+l+) mice, whereas the brain:plasma ratio was 2.3 in the mdrla(-l-)
mice. The
distribution also varied in the testes, where the mdrla(+l+) mice had a 0.29
testes:plasma ratio, and the mdrla(-l-) mice had a testes:plasma ratio of 2:1.
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-15-
Example 3
The effect of P-glycoprotein inhibitors was investigated in rndrla(+l+)
mice by pretreatment with equally divided doses given by intravenous tail vein
injection, 30 minutes prior to and concurrently with administration of
nelfinavir.
Inhibitors studied included the compound of formula (II) (2 x 0.5 to 25 mg/kg,
Lilly
Research Laboratories, Indianapolis, IN), verapamil (2 x 6.25 mg/kg, Sigma-
Aldrich,
St. Louis, MO) and quinidine (2 x 25 mg/kg, Sigma-Aldrich), each dissolved in
20%
ethanol/0.9% saline; cyclosporine A (2 x 0.5 to 25 mg/kg, Novartis Pharma AG,
Basel, Switzerland) dissolved in 10% ethanol/60% propylene glycol/30% water;
nelfinavir (2 x 25 mg/kg, Agouron Pharmaceuticals Inc., San Diego, CA),
ritonavir (2
x 12.5 mg/kg Abbott Laboratories), saquinavir (2 x 25 mg/kg, Roche Products
Ltd.,
Welwyn, UK), and indinavir (2 x 25 mg/kg, Merck Research Laboratories, West
Point, PA) each dissolved in 10% ethanol/40% propylene glycol/50% 0.9% saline;
and ketoconazole (2 x 25 mg/kg, Sigma-Aldrich) dissolved in 25% 0.2N HC1. All
drugs were injected in total volume of 4 pl/g and appropriate vehicle
solutions were
used in control studies.
Similar tissue distribution studies were also performed to study tissue
distribution of ['4C]-saquinavir (9.8 mCi/mmol, Roche Products Ltd) and ['4C]-
indinavir (8.5 mCi/mmol. Merck Research Laboratories), using the compound of
formula (II) (2 x 25 mg/kg) as the P-glycoprotein inhibitor.
At least 3 mice were studied at each time point and differences in
radioactivity between treated and control groups were analyzed by a two-sided
Student's t-test with p < 0.05 as the limit of statistical significance.
As shown in Fig. 3, pretreatment with 25 mg/kg formula (II), 30
minutes prior to and concurrently with ['4CJ-nelfinavir, markedly altered the
disposition of total radioactivity in mdrla(+l+) mice. The brain concentration-
time
profile in particular was especially affected, as seen in Fig. 2. In untreated
mice,
radioactivity in the brain was more than 17 times lower than that in plasma
with a
mean brain:plasma concentration ratio of 0.06, based on the relative area
under the
concentration-time curves. Formula (II) increased brain levels by 20-fold in
contrast
to those in the plasma, which only changed 2-fold. As a result, formula (II)
treatment
produced an 10-fold increase in nelfinavir's brain:plasma distribution ratio.
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-16-
Subsequent studies, also illustrated in Fig. 3, based on tissue distribution
measured
two hours after nelfinavir administration showed that these changes are dose-
dependent. Moreover, 10- to 15-fold higher brain levels could be achieved
without
affecting nelfinavir plasma concentrations at total dosages between 12.5 mg
and 25
mg/kg. Comparison of these findings with those in mdrla(-l-) mice indicated
that if
all of the effects of formula (II) are accounted for by P-glycoprotein
inhibition, then
the transporter is inhibited by about 75% following a total dose of $0 mg/kg
formula
(II). Similar results were obtained with nelfinavir levels in the testes, with
P-
glycoprotein activity being inhibited by over 90%. Similar findings were also
noted
after intravenous administration of ['4C]-saquinavir, ['4C]-indinavir and
pretreatment
with 50 mg/kg formula (II).
More modest, though statistically significant changes, were produced
by cyclosporine A, ketoconazole, and ritonavir administration, but these
largely
reflected increased nelfinavir plasma concentrations rather than altered
tissue
1$ distribution. Finally, neither quinidine, verapamil, nelfinavir,
saquinavir, or indinavir
produced significant changes in nelfinavir's disposition at the doses studied.
The
results are summarized in Table 1:
Table 1: Tissue levels of radioactivity (ng/g tissue) in wildtype and mdrla(-l-
) mice at
2 hr after intravenous injection of ['4C]-nelfinavir (Smg/kg). Mice were
treated with
varying doses of formula (II) or other known P-glycoprotein inhibitors, 50
mg/kg
(unless otherwise noted) in two divided doses, given 30 min prior to and
simultaneously with ['4C]-nelfinavir. Data are shown as mean ~ stan.dard
deviation.
Brain:Plasma Testes:Plasma
2$ PlasmaBrain Ratio Testes Ratio
mdrla(+/+) Mice
Vehicle Control 98 5.1 0.06 t 31 t 0.29 t
t t 1.9 0.01 5.8 0.02
12
Ritonavir (25 618 22 t 0.08 t 59 t 0.31 f
mg/kg) f 3.8 0.05 3.4 0.14
112
Nelfinavir (50 124 7.9 0.06 t 47 t 0.39 t
mg/kg) t t 1.5 0.02 6.6 0.07
11
Saquinavir (50 117 6.9 0.06 t 48 f 0.43 t
mg/kg) t t 1.9 0.01 11 0.13
14
Indinavir (50 100 7.5 0.08 t 37 t 0.39 f
mg/kg) f t 0.9 0.01 8.1 0.05
6.2
Vehicle Control 99 9.4 0.10 t 41 t 0.38 f
t t 3.0 0.02 6.8 0.06
6.7
Quinidine (50 92 5.1 0.06 t 47 t 0.54 t
mg/kg) t t 1.5 0.01 7.3 0.06
2.5
3S Verapamil (12.5 91 8.4 0.09 t 39 t 0.44 f
mg) t t 2.1 0.02 3.0 0.06
6.1
CA 02378984 2002-02-07
WO 01/10387 PCT/US00/40588
-17-
Brain:Plasma Testes:Plasma
PlasmaBrain Ratio Testes Ratio
Ketoconazole (50 292 57 f 0.20 f 87 t 0.30 t
mg/kg) t 14 0.02 16 0.04
68
Cyclosporine
Vehicle Control 103 9.2 t 0.10 t 66 t 0.42 t
f 1.2 0.03 12 0.04
13
$ l mg/kg 120 11 12.4 0.1010.02 61 17.00.51 10.04
t
6.0
4 mg/kg 322 45 f 0.13 t 128 0.40 t
t 20 0.06 t 10 0.05
14
12.5 mg/kg 698 89 t 0.18 t 195 0.30 t
f I9 0.08 t 54 0.07
189
25 mg/kg 659 190 t 0.30 t 294 0.44 t
f 43 0.08 t 50 0.04
57
50 mg/kg 954 242 f 0.27 t 245 0.25 t
~ 46 0.07 t 55 0.07
132
1~
Formula (II)
Vehicle Control 4 ~ .6 t .08 t 0.027 t .48 t
4.9 1.7 3.7 0.07
1 mg/kg 74 9.4 t 0.11 t 56 t 0.81 t
t 1.7 0.04 1.7 0.13
14
4 mg/kg 7214.82414.5 0.33 ~ 95 1 1.410.33
0.04 18
1$ 12.5 mg/kg 71 6015.4 0.8910.16 108127 1.610.44
1
11
25 mg/kg 89 89 ~ I .1 t 168 2.0 t
t 17 0.28 ~ 61 0.48
8.1
50 mg/kg 171 243 t 1.4 f 0.08187 1.2 t
f 19 t 17 0.19
12
mdrla (-l-) Mice
Vehicle 89 184 t 2.3 t 0.24194 2.1 t
t 20 t 34 0.35
15
Formula (11) (SOmg/kg)161 318 t 1.9 t 0.12207 1.3 t
t 52 t 46 0.13
24
Although the invention has been described in detail with reference to
25 preferred embodiments, variations and modifications exist within the scope
and spirit
of the invention as described and defined in the following claims.
