U.S. patent application number 10/976115 was filed with the patent office on 2005-06-30 for aryl phosphate derivatives of d4t with potent anti-viral activity.
This patent application is currently assigned to Parker Hughes Institute. Invention is credited to Chen, Chun-Lin, Uckun, Fatih, Venkatachalam, Taracad K., Zhu, Zhoa-Hai.
Application Number | 20050143339 10/976115 |
Document ID | / |
Family ID | 27365130 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050143339 |
Kind Code |
A1 |
Uckun, Fatih ; et
al. |
June 30, 2005 |
Aryl phosphate derivatives of D4T with potent anti-viral
activity
Abstract
Methods for increasing the elimination half-life of key
metabolites such as d4T by administering an aryl phosphate
derivative of d4T having an electron withdrawing substituent on the
aryl group and an amino acid substituent on the phosphate group are
described. A preferred aryl phosphate derivative of d4T is HI-113
(d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]). The
administration of HI-113 results in more prolonged systemic
exposure to the key metabolites, Ala-d4T-MP and d4T, than
administration of an equimolar dose of either metabolite. Each
metabolite has a significantly longer elimination half life when
formed in vivo from the administration of HI-113 than when the
metabolite is administered directly.
Inventors: |
Uckun, Fatih; (White Bear
Lake, MN) ; Chen, Chun-Lin; (Roseville, MN) ;
Venkatachalam, Taracad K.; (Maplewood, MN) ; Zhu,
Zhoa-Hai; (Shoreview, MN) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Parker Hughes Institute
St. Paul
MN
|
Family ID: |
27365130 |
Appl. No.: |
10/976115 |
Filed: |
October 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10976115 |
Oct 26, 2004 |
|
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|
10037003 |
Oct 19, 2001 |
|
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6825177 |
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60299885 |
Jun 21, 2001 |
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60244263 |
Oct 30, 2000 |
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Current U.S.
Class: |
514/50 ;
514/86 |
Current CPC
Class: |
A61K 31/7064 20130101;
A61K 31/675 20130101 |
Class at
Publication: |
514/050 ;
514/086 |
International
Class: |
A61K 031/675 |
Claims
1-13. (canceled)
14. A method for extending the half-life of a compound of formula I
in a mammal comprising administering to the mammal: an esterase
inhibitor; and a compound of formula I; wherein the compound of
formula I is: 8where R.sub.1 is an aryl group substituted with an
electron withdrawing group and R.sub.2 is an amino acid residue or
an ester of the amino acid residue, or a pharmaceutically
acceptable salt thereof.
15. The method of claim 14, wherein the aryl group is selected from
the group consisting of phenyl, naphthyl, and anthryl.
16. The method of claim 14, wherein the aryl group is phenyl.
17. The method of claim 14, wherein the electron-withdrawing group
is halo.
18. The method of claim 14, wherein R.sub.1 is
para-bromophenyl.
19. The method of claim 14, wherein R.sub.2 is an .alpha.-amino
acid or ester thereof.
20. The method of claim 14, wherein R.sub.2 is
--NHCH(CH.sub.3)COOCH.sub.3- .
21. The method of claim 14, wherein R.sub.1 is para-bromophenyl and
R.sub.2 is --NHCH(CH.sub.3)COOCH.sub.3.
22. The method of claim 14, wherein the compound of formula I is
administered intraveneously.
23. The method of claim 14, wherein the compound of formula I is
administered orally.
24. The method of claim 14, wherein the esterase inhibitor is
selected from the group of an inhibitor of cholinesterase, an
inhibitor of carboxylesterase, or a combination thereof.
25. The method of claim 24, wherein the inhibitor of cholinesterase
is paraoxon.
26. The method of claim 24, wherein the inhibitor of cholinesterase
is phyostigmine.
27. The method of claim 21, wherein the inhibitor of cholinesterase
is selected from paraoxon and phyostigmine.
28. The method of claim 14, wherein the compound of formula I and
the esterase inhibitor are administered concurrently.
29. The method of claim 14, wherein the compound of formula I and
the esterase inhibitor are administered in a single dosage
form.
30. The method of claim 29, wherein the a single dosage form is a
parenteral dosage form.
31. A pharmaceutical composition comprising: an esterase inhibitor;
and a compound of formula I: 9where R.sub.1 is an aryl group
substituted with an electron withdrawing groups and R.sub.2 is an
amino acid residue or an ester of the amino acid residue, or a
pharmaceutically acceptable salt thereof.
32. The composition of claim 31, wherein the aryl group is selected
from the group consisting of phenyl, naphthyl, and anthryl.
33. The composition of claim 31, wherein the aryl group is
phenyl.
34. The composition of claim 31, wherein the electron-withdrawing
group is halo.
35. The composition of claim 31, wherein R.sub.1 is
para-bromophenyl.
36. The composition of claim 31, additionally comprising a
pharmaceutically acceptable carrier or diluent.
37. The composition of claim 31, wherein R.sub.2 is
--NHCH(CH.sub.3)COOCH.sub.3.
38. The composition of claim 31, wherein R.sub.1 is
para-bromophenyl and R.sub.2 is --NHCH(CH.sub.3)COOCH.sub.3.
39. The composition of claim 31, wherein the esterase inhibitor is
selected from the group of an inhibitor of cholinesterase, an
inhibitor of carboxylesterase, or a combination thereof.
40. The composition of claim 39, wherein the inhibitor of
cholinesterase is paraoxon.
41. The composition of claim 39, wherein the inhibitor of
cholinesterase is phyostigmine.
42. The composition of claim 38, wherein the inhibitor of
cholinesterase is selected from paraoxon and phyostigmine.
43. The composition of claim 31, wherein the composition is adapted
for intravenous administration.
44. The composition of claim 31, wherein the composition is adapted
for intravenous oral administration.
45. A method for extending the half-life of a compound of formula I
comprising administering the pharmaceutical composition of claim
31.
46. The method of claim 45, wherein the composition of claim 31
comprises an esterase inhibitor is an inhibitor of cholinesterase,
an inhibitor of carboxylesterase, or a combination thereof.
47. The method of claim 46, wherein the inhibitor of cholinesterase
is paraoxon.
48. The method of claim 46, wherein the inhibitor of cholinesterase
is phyostigmine.
Description
[0001] This application claims priority from provisional
application Ser. No. 60/299,885, filed Jun. 21, 2001 and
provisional application Ser. No. 60/244,263, filed Oct. 30, 2000,
both of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention is directed to methods of treating viral
infections through the administration of aryl phosphate derivatives
of 2',3'-didehydro-2',3'-dideoxythymidine.
BACKGROUND OF THE INVENTION
[0003] One approach to treating AIDS is by inhibiting the reverse
transcriptase (RT) activity of the human immune deficiency virus
(HIV). Potent inhibitors of HIV RT are known and include
5'-triphosphates of 2',3'-dideoxynucleoside (hereinafter "ddN")
analogues such as 3'-azido-3'-deoxythymidine (hereinafter "AZT")
and 2',3'-didehydro-2',3'-dideoxythymidine (hereinafter "d4T").
These active RT inhibitors are generated intracellularly by the
action of nucleoside kinase and nucleotide kinase.
[0004] The rate-limiting step for the conversion of AZT to its
bioactive metabolite AZT-triphosphate seems to be the conversion of
the monophosphate derivative to the diphosphate derivative, whereas
the rate-limiting step for the intracellular generation of the
bioactive d4T metabolite d4T-triphosphate was reported to be the
conversion of the nucleoside to its monophosphate derivative.
(Balzarini et al., 1989, J. Biol. Chem. 264:6127; McGuigan et al.,
1996, J. Med. Chem. 39:1748).
[0005] In an attempt to overcome the dependence of ddN analogues on
intracellular nucleoside kinase activation, McGuigan et al.
prepared aryl methoxyalaninyl phosphate derivatives of AZT
(McGuigan et al., J. Med. Chem., 36, 1048 (1993); McGuigan et al.,
Antiviral Res. 17, 311(1992)) and d4T (McGuigan et al., J. Med.
Chem., 39, 1748 (1996); McGuigan et al., Bioorg. Med. Chem. Lett.,
6, 1183 (1996)). Such compounds undergo intracellular hydrolysis to
yield monophosphate derivatives that are further phosphorylated by
thymidylate kinase to give the bioactive triphosphate derivatives
in a thymidine kinase (TK)-independent fashion.
[0006] U.S. Pat. No. 6,030,957 (Uckun et al.) disclosed that
substitution of the aryl moiety of an aryl phosphate derivative of
d4T enhances the ability of the compounds to undergo hydrolysis.
Such compounds are potent antiviral agents. A particularly potent
compound is HI-113, (d4T-5'-[p-bromophenyl methoxyalaninyl
phosphate]).
SUMMARY OF THE INVENTION
[0007] The invention is directed to aryl phosphate derivatives of
2',3'-didehydro-2',3'-dideoxythymidine (hereinafter "d4T") that
inhibit human immune deficiency virus reverse transcriptase (HIV
RT) and exhibit potent anti-viral activity. One aspect of the
invention provides methods for treating viral infections by
increasing the elimination half-life of key metabolites such as d4T
in a mammal. The method comprises administering an aryl phosphate
derivative of d4T having an electron withdrawing substituent on the
aryl group and an amino acid substituent on the phosphate group as
in Formula I: 1
[0008] where R.sub.1 is an aryl group substituted with an electron
withdrawing group and R.sub.2 is an amino acid or an ester of an
amino acid. In one embodiment of Formula I, R.sub.1 is a phenyl
substituted with an electron withdrawing group and R.sub.2 is an
ester of an .alpha.-amino acid. Preferably, Formula I is HI-113
(d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]) where R.sub.1 is
phenyl group substituted with bromo at the para position and
R.sub.2 is the methyl ester of alanine.
[0009] The oral or intravenous administration of HI-113 results in
the formation of two key metabolites: alaninyl-d4T-monophosphate
(Ala-d4T-MP) and d4T. The administration of HI-113 results in more
prolonged systemic exposure to Ala-d4T-MP as well as d4T than
administration of an equimolar dose of either metabolite. Each
metabolite has a significantly longer elimination half life when
formed from the administration of HI-113 than when administered
directly.
[0010] Another aspect of the invention provides a method for
treating viral infections comprising extending the elimination
half-life of d4T in a mammal by administering an effective amount
of a compound of Formula IV: 2
[0011] where R.sub.2 is an amino acid or amino acid ester residue.
In one embodiment, R.sub.2 is the methyl ester of alanine.
[0012] The methods of the invention can be used to treat HIV.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows representative HPLC chromatograms of blank
plasma (A), blank plasma spiked with HI-113, d4T and Ala-d4T-MP
(B), and plasma samples 10 minutes after intravenous injection of
100 mg/kg HI-113 (C).
[0014] FIG. 2 is a plot showing the stability of HI-113 in plasma
(A) and in whole blood (B) as a function of time.
[0015] FIG. 3 is a plot showing the stability of HI-113 and the
formation of key metabolites in plasma in presence of paraozon (A),
physostigmine (B), and EDTA (C) as a function of time.
[0016] FIG. 4 is a plot showing the stability of HI-113 and the
formation of key metabolites in liver homogenate as a function of
time.
[0017] FIG. 5 is a plot showing the stability of HI-113 and
formation of key metabolites in gastric fluid (A) and in intestinal
fluid (B) as a function of time.
[0018] FIG. 6 shows plots of the plasma concentrations of total
HI-113 (A), HI-113-A (B) and HI-113-B (C) as a function of time in
Balb/C mice following intravenous injection of HI-113 (100 mg/kg, 4
mice per time-point).
[0019] FIG. 7 shows (A) the pharmacokinetic model for describing
HI-113, Ala-d4T-MP and d4T after intravenous injection of HI-113;
and (B) the plasma concentrations of HI-113, Ala-d4T-MP and d4T as
a function of time in Balb/C mice following intravenous injection
of HI-113 (100 mg/kg, 4 mice per time-point).
[0020] FIG. 8 shows (A) the pharmacokinetic model for describing
Ala-d4T-MP and d4T after intravenous injection of Ala-d4T-MP; and
(B) the plasma concentrations of Ala-d4T-MP and d4T as a function
of time in Balb/C mice following intravenous injection of
Ala-d4T-MP (75 mg/kg, 5 mice per time-point).
[0021] FIG. 9 shows the plasma concentration of d4T as a function
of time in BALB/C mice following intravenous injection of D4T (40
mg/kg, 5 mice per time-point).
[0022] FIG. 10 shows the plasma concentrations of Ala-d4T-MP (A)
and d4T (B) as a function of time in BALB/C mice following oral
administration of HI-113 (100 mg/kg, 4 mice per time-point).
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention is directed to aryl phosphate derivatives of
2',3'-didehydro-2',3'-dideoxythymidine (hereinafter "d4T") that
inhibit human immune deficiency virus reverse transcriptase (HIV
RT) and exhibit potent anti-viral activity. More particularly, the
present invention provides methods for increasing the elimination
half-life of key metabolites such as d4T in a mammal by
administering an aryl phosphate derivative of d4T having an
electron withdrawing substituent on the aryl group and an amino
acid substituent on the phosphate group as in Formula I: 3
[0024] where R.sub.1 is an aryl group substituted with an electron
withdrawing group and R.sub.2 is an amino acid or an ester of an
amino acid. The term "aryl" includes aromatic groups such as, for
example, phenyl, naphthyl, and anthryl. Suitable
electron-withdrawing groups include halo (--Br, --Cl, --I, --F),
--NO.sub.2, --CN, --SO.sub.3H, --COOH, --CHO, --COR (where R is a
(C.sub.1 to C.sub.4) alkyl), and the like.
[0025] The term "amino acid" refers to any of the naturally
occurring amino acids, as well as their opposite enantiomers or
racemic mixture of both enantiomers, synthetic analogs, and
derivatives thereof. The term includes, for example, .alpha.-,
.beta.-, .gamma.-, .delta.-, and .omega.-amino acids. Suitable
naturally occurring amino acids include glycine, alanine, valine,
leucine, isoleucine, proline, threonine, serine, methionine,
cysteine, aspartic acid, asparagine, glutamic acid, glutamine,
arginine, lysine, phenylalanine, tryptophan, tyrosine, and
histidine. Synthetic, or unnatural, amino acids such as, for
example, trifluoroleucine, p-fluorophenylalanine, and
3-triethylalanine can be used. The term amino acid includes esters
of the amino acids. Esters include lower alkyl esters in which the
alkyl group has one to seven carbon atoms, preferably one to four
carbon atom such as, for example, methyl, ethyl, propyl, and butyl.
The amino group of the amino acid or ester thereof is attached to
the phosphate group in Formula I.
[0026] The compounds of Formula I can also be in the form of
pharmaceutically acceptable salts. Pharmaceutically acceptable
salts are formed with organic and inorganic acids. Examples of
suitable acids for salt formation with the amino group of the amino
acid or amino acid ester residue are hydrochloric, sulfuric,
phosphoric, acetic, citric, oxalic, malonic, salicylic, malic,
gluconic, fumaric, succinic, asorbic, maleic, methanesulfonic, and
the like. The salts are prepared by contacting the free base form
with a sufficient amount of the desired acid to produce either a
mono or di, etc. salt in the conventional manner. Suitable bases
for the formation of a salt with the carboxylate group of the amino
acid residue include, for example, sodium hydroxide, sodium
carbonate, sodium bicarbonate, potassium hydroxide, potassium
carbonate, and potassium bicarbonate.
[0027] In one embodiment of Formula I, R.sub.1 is a phenyl group
substituted with an electron withdrawing group and R.sub.2 is an
.alpha.-amino acid or ester thereof as shown in Formula II: 4
[0028] In Formula II, X is an electron-withdrawing group such as
halo (--Br, --Cl, --I, --F), --NO.sub.2, --CN, --SO.sub.3H, --COOH,
--CHO, --COR (where R is a (C.sub.1 to C.sub.4) alkyl), and the
like. R.sub.3 is hydrogen or an alkyl of one to seven carbon atoms.
preferably of one to four carbon atoms such as, for example,
methyl, ethyl, propyl, and butyl. R.sub.4 is hydrogen (e.g., as in
glycine), an alkyl (e.g. as in alanine, valine, leucine,
isoleucine, proline), a substituted alkyl (e.g., as in threonine,
serine, methionine, cysteine, aspartic acid, asparagine, glutamic
acid, glutamine, arginie, and lysine), an arylalkyl (e.g., as in
phenylalanine and tryptophan), a substituted arylalkyl (e.g., as in
tyrosine), or a heteroalkyl (e.g., as in histidine).
[0029] One embodiment of Formula II is HI-113
(d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]) where X is bromo
attached to the phenyl group in the para position, R.sub.4 is
methyl, and R.sub.3 is methyl. HI-113 is shown in Formula III:
5
[0030] Previous in vitro studies showed that an electron
withdrawing group at the para position of the phenyl group enhances
the rate of hydrolysis and thereby enhances production of a key
metabolite alaninyl-d4T-monophosphate (Ala-d4T-MP) relative to the
unsubstituted aryl phosphate derivative (Venkatachalam et al.,
Bioorg. Med. Chem. Lett., 8, 3121 (1998); Vig et al., Antiviral
Chem. Chemother., 9, 445 (1998); and U.S. Pat. No. 6,030,957 (Uckun
et al.)). HI-113 is substantially more potent than d4T in
inhibiting HIV replication in human peripheral blood mononuclear
cells (PBMNC) or thymidine kinase-deficient T-cells. HI-113
inhibits the replication of HIV-2 and zidovudine
(ZVD/AZT)-resistant HIV-1 strains in PBMNC at nanomolar
concentrations.
[0031] The present invention resulted from in vivo studies using
various d4T derivatives. The anti-viral agent HI-113 is quickly
metabolized in vivo to form two metabolites:
2',3'-didehydro-3'-deoxythymidine (d4T) and
alaninyl-d4T-monophosphate (Ala-d4T-MP) as shown in Scheme 1.
Ala-d4T-MP can also be metabolized further to yield d4T. The
metabolite d4T had not been found in the earlier in vitro studies
with cells. 6
[0032] HI-113 readily metabolizes in either plasma or whole blood
to form Ala-d4T-MP (see FIG. 2). Only a small amount of d4T forms.
Ala-d4T-MP is stable both in plasma and in whole blood. These
results indicate that other enzymes (e.g., liver enzymes) are
needed for formation of d4T by hydrolysis of either Ala-d4T-MP or
HI-113. This hypothesis is consistent with the formation of a
significant amount of d4T after incubation of HI-113 with a liver
homogenate (see FIG. 4).
[0033] Paraoxon, an inhibitor of both cholinesterase and
carboxylesterase (Augustinsson, Ann. N.Y. Acad. Sci., 94, 884
(1961); McCracken et al., Biochem. Pharmacol., 46, 1125 (1993);
Madhu et al., J. Pharm. Sci., 86, 971 (1997)), significantly
prevented the hydrolysis of HI-113 to Ala-d4T -MP and d4T,
suggesting that both cholinesterase and carboxylesterase are
important for metabolism of HI-113 (see FIG. 3A). Physostigmine, an
inhibitor of cholinesterase, partially prevented the hydrolysis of
HI-113, which further supports the importance of cholinesterase in
hydrolysis of HI-113 (see FIG. 3B). EDTA, an inhibitor of
arylesterase, did not affect the hydrolysis of HI-113, indicating
that arylesterase is probably not involved in the hydrolysis of
HI-113 (see FIG. 3C).
[0034] The elimination half-life of intravenously administered d4T
was fairly similar to the elimination half-life of d4T formed after
intravenous administration of Ala-d4T-MP (t.sub.1/2 of 30.3 minutes
vs. 34.0 minutes). In contrast, the elimination half-life for d4T
formed after intravenous administration of HI-113 was significantly
prolonged (t.sub.1/2 of 114.8 minutes). Similarly, the elimination
half-life for Ala-d4T-MP formed from HI-113 was significantly
longer than the t.sub.1/2 for Ala-d4T-MP administered intravenously
(t.sub.1/2 of 129.2 minutes vs. 28.5 minutes). The intravenous
administration of HI-113 results in prolonged systemic exposure to
both Ala-d4T-MP and d4T compared to administration of equimolar
dose of Ala-d4T-MP or d4T due to apparently longer elimination
half-lives of HI-113-derived metabolites.
[0035] Following intravenous administration, the elimination
half-life (t.sub.1/2) of HI-113 was 3.5 minutes with a systemic
clearance (CL) of 160.9 ml/min/kg. Different estimates for systemic
clearance (CL) values were obtained for the two diastereomers of
HI-113 (HI-113A is 208.2 ml/min/kg and HI-113B is 123.9 ml/min/kg),
but both were completely metabolized within 30 minutes. HI-113 was
converted to the active metabolites Ala-d4T-MP (23%) and d4T (24%).
The t.sub.max values for Ala-d4T-MP and d4T formed from
intravenously administered HI-113 were 5.9 minutes and 18.7
minutes, respectively.
[0036] Intravenous administration of Ala-d4T-MP results in
formation of d4T (15%). Ala-d4T-MP can also be used as a d4T
prodrug. The invention provides a method for treating viral
infections comprising extending the elimination half-life of d4T in
a mammal by administering an effective amount of a compound of
Formula IV: 7
[0037] where R.sub.2 is an amino acid or esterified thereof.
[0038] The compounds of Formula IV can also be in the form of
pharmaceutically acceptable salts. Pharmaceutically acceptable
salts are formed with organic and inorganic acids. Examples of
suitable acids for salt formation with the amino group of the amino
acid or amino acid ester residue are hydrochloric, sulfuric,
phosphoric, acetic, citric, oxalic, malonic, salicylic, malic,
gluconic, fumaric, succinic, asorbic, maleic, methanesulfonic, and
the like. The salts are prepared by contacting the free base form
with a sufficient amount of the desired acid to produce either a
mono or di, etc. salt in the conventional manner. Suitable bases
for the formation of a salt with the carboxylate group of the amino
acid residue include, for example, sodium hydroxide, sodium
carbonate, sodium bicarbonate, potassium hydroxide, potassium
carbonate, and potassium bicarbonate.
[0039] Orally administered HI-113 also yielded Ala-d4T-MP and d4T
as the major metabolites. No parent HI-113 was detectable in the
blood after oral administration. Although HI-113 is stable in
gastric fluid and can be absorbed in the stomach, it can quickly
hydrolyze in blood. On the other hand, HI-113 decomposes readily in
intestinal fluid to form Ala-d4T-MP. This metabolite can be
absorbed in the intestine and then further metabolized to yield d4T
in the blood. The t.sub.max and t.sub.1/2 values for d4T in mice
were longer when derived from orally administered HI-113 (42.4
minutes and 99.0 minutes, respectively) than from orally
administered d4T (5 minutes and 18 minutes, respectively). The
t.sub.max value is higher but the t.sub.1/2 value is lower for
orally administered HI-113 compared to intravenously administered
HI-113. The estimated bioavailabilities of Ala-d4T-MP and d4T were
approximately 12% and 48%, respectively, after oral administration
of HI-113. However, the bioavailability of d4T metabolized from
HI-113 (48%) was lower than that of orally administered D4T
(98%).
[0040] The methods of the invention can be used, for example, to
treat HIV.
[0041] Administration Methods
[0042] Compounds of Formulas I to IV can be formulated as
pharmaceutical compositions and administered to a mammalian host,
including a human patient in a variety of forms adapted to the
chosen route of administration. The compounds are typically
administered in combination with a pharmaceutically acceptable
carrier, and can be combined with specific delivery agents,
including targeting antibodies or cytokines.
[0043] The compounds can be administered orally, parentally
(including subcutaneous injection, intravenous, intramuscular,
intrastemal or infusion techniques), by inhalation spray,
topically, by absorption through a mucous membrane, or rectally, in
dosage unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants or vehicles.
Pharmaceutical compositions of the invention can be in the form of
suspensions or tablets suitable for oral administration, nasal
sprays, creams, sterile injectable preparations, such as sterile
injectable aqueous or oleagenous suspensions or suppositories.
[0044] For oral administration as a suspension, the compositions
can be prepared according to techniques well-known in the art of
pharmaceutical formulation. The compositions can contain
microcrystalline cellulose for imparting bulk, alginic acid or
sodium alginate as a suspending agent, methylcellulose as a
viscosity enhancer, and sweeteners or flavoring agents. As
immediate release tablets, the compositions can contain
microcrystalline cellulose, starch, magnesium stearate and lactose
or other excipients, binders, extenders, disintegrants, diluents
and lubricants known in the art.
[0045] For administration by inhalation or aerosol, the
compositions can be prepared according to techniques well-known in
the art of pharmaceutical formulation. The compositions can be
prepared as solutions in saline, using benzyl alcohol or other
suitable preservatives, absorption promoters to enhance
bioavailability, fluorocarbons or other solubilizing or dispersing
agents known in the art.
[0046] For administration as injectable solutions or suspensions,
the compositions can be formulated according to techniques
well-known in the art, using suitable dispersing or wetting and
suspending agents, such as sterile oils, including synthetic mono-
or diglycerides, and fatty acids, including oleic acid.
[0047] For rectal administration as suppositories, the compositions
can be prepared by mixing with a suitable non-irritating excipient,
such as cocoa butter, synthetic glyceride esters or polyethylene
glycols, which are solid at ambient temperatures, but liquefy or
dissolve in the rectal cavity to release the drug.
[0048] Solutions or suspensions of the compounds can be prepared in
water, isotonic saline (PBS) and optionally mixed with a nontoxic
surfactant. Dispersions may also be prepared in glycerol, liquid
polyethylene, glycols, DNA, vegetable oils, triacetin and mixtures
thereof. Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the growth of
microorganisms.
[0049] The pharmaceutical dosage form suitable for injection or
infusion use can include sterile, aqueous solutions or dispersions,
or sterile powders comprising an active ingredient which are
adapted for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions. In all cases, the ultimate
dosage form should be sterile, fluid and stable under the
conditions of manufacture and storage. The liquid carrier or
vehicle can be a solvent or liquid dispersion medium comprising,
for example, water, ethanol, a polyol such as glycerol, propylene
glycol, or liquid polyethylene glycols and the like, vegetable
oils, nontoxic glyceryl esters, and suitable mixtures thereof. The
proper fluidity can be maintained, for example, by the formation of
liposomes, by the maintenance of the required particle size, in the
case of dispersion, or by the use of nontoxic surfactants. The
prevention of the action of microorganisms can be accomplished by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be desirable to include isotonic agents, for
example, sugars, buffers, or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the
inclusion in the composition of agents delaying absorption--for
example, aluminum monosterate hydrogels and gelatin.
[0050] Sterile injectable solutions are prepared by incorporating
the conjugates in the required amount in the appropriate solvent
with various other ingredients as enumerated above and, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying techniques, which yield a powder of the active
ingredient plus any additional desired ingredient present in the
previously sterile-filtered solutions.
EXAMPLES
[0051] The synthetic procedures for the preparation of HI-113,
Ala-d4T-MP and d4T have been previously described in detail
(Venkatachalam et al., Bioorg. Med Chem. Lett., 8, 3121 (1998); Vig
et al., Antiviral Chem. Chemother., 9, 445, (1998); U.S. Pat. No.
6,030,957 (Uckun et al.)) and are incorporated herein by
reference.
Example 1
Quantitative HPLC For Detection of HI-113 and Its Metabolites
[0052] The HPLC system used for these studies was a Hewlett Packard
(Palo Alto, Calif.) series 1100 instrument equipped with a
quaternary pump, an autosampler, an automatic electronic degasser,
an automatic thermostatic column compartment, a diode array
detector and a computer with Chemstation software for data analysis
(Chen et al., J. Chromatogr. B., 724, 157 (1999); Chen et al., J.
Chromatogr. B., 727,205 (1999); and Chen et al., J. Liq.
Chromatogr., 22, 1771 (1999)). The analytical column used was a
Zobax SB-Phenyl (5 .mu.m, Hewlett Packard, Inc.) column attached to
a guard column (Hewlett Packard, Inc.). The column was equilibrated
prior to data collection. The linear gradient mobile phase (flow
rate=1.0 mL/min) was: 100% A/0% B at 0 minutes, 88% A/12% B at 20
minutes, 8% A/92% B at 30 minutes (A: 10 mM ammonium phosphate
buffer, pH 3.7; B: acetonitrile). The detection wavelength was 268
nm, the peakwidth was less than 0.03 minutes, the response time was
0.5 seconds, and the slit was 4 nm.
[0053] HPLC-grade reagents and deionized, distilled water were used
in this study. Acetonitrile was purchased from Burdick &
Jackson (Allied Signal Inc., Muskegon, Mich.). Acetic acid was
purchased from Fisher Chemicals (Fair Lawn, N.J.). Ammonium
phosphate and phosphoric acid were purchased from Sigma-Aldrich
(St. Louis, Mo.).
[0054] Plasma samples (200 .mu.L) were mixed 1:4 with acetone (800
.mu.L) and vortexed for at least 30 seconds. Following
centrifugation, the supernatant was transferred into a clean tube
and was dried under nitrogen. A 50 .mu.L solution of 50% methanol
in 200 mM HCl was used to reconstitute the extraction residue, and
40 .mu.L was injected into the HPLC.
[0055] The chromatographic retention times (RT) measured for HI-113
and its metabolites in spiked samples were 28.7+0.02 minutes
(HI-113A; n=13; FIG. 1B), 28.9.+-.0.02 minutes (HI-113B; n=13; FIG.
1C), 15.3.+-.0.2 minutes (Ala-d4T-MP; n--30) and 18.5.+-.0.1
minutes (d4T n=30). HI-113A and HI-113B are diastereomers of
HI-113. At these retention times, no significant interference peaks
from the blank plasma were observed (FIGS. 1A and 1B).
[0056] The hydrochloric acid component of the reconstituted
solutions played a key role in the chromatography of HI-113 and its
metabolites. The acid protonated Ala-d4T-MP and no peak appeared in
the chromatogram for this metabolite if there was no hydrocholoric
acid in the reconstituted solution. The acidic solution decreased
the stability of Ala-d4T-MP, however. Therefore, all of the
extracted samples were analyzed immediately after
reconstitution.
Example 2
Stability of HI-113 and Ala-d4T-MP in Whole Blood and Plasma
[0057] Whole blood and plasma samples were spiked with HI-113 and
Ala-d4T-MP to yield final concentrations of 250 .mu.M HI-113 and
100 .mu.M Ala-d4T-MP, respectively. The whole blood samples were
placed in a 37.degree. C. water bath, while plasma samples were
stored at -20.degree. C. At a predetermined time, an aliquot (100
.mu.l) of spiked whole blood or plasma was extracted by adding 400
.mu.l of acetone to induce precipitation of proteins, as described
above. The absolute peak area was used to evaluate the stability of
HI-113 and Ala-d4T-MP.
[0058] The results shown in FIGS. 2A and 2B indicate that HI-113 is
very unstable in plasma and in whole blood. Following incubation
with plasma, over 95% of the HI-113 decomposed after 5 minutes. In
the whole blood samples, 68%, 87% and 92% of the HI-113 decomposed
in samples taken at 5, 10 and 15 minutes, respectively. In both the
plasma and whole blood samples, decomposition of HI-113 was
complete within 30 minutes. (see Table 1 for plasma data). Thus,
extraction of the samples was required immediately after the
samples were obtained. In contrast, Ala-d4T-MP was stable in both
whole blood and plasma for 1 day.
Example 3
[0059] Stability of HI-113 in Plasma in the Presence of Selective
Esterase Inhibitors Plasma samples were pre-incubated with the
esterase inhibitors paraoxon (final concentration of 0.1 mM),
physostigmine (final concentration of 0.1 mM), and EDTA (final
concentration of 1M) at 37.degree. C. for 30 minutes. Then HI-113
was added to yield final concentrations of 250 .mu.M. At a
predetermined time, an aliquot (100 .mu.l) of spiked plasma was
extracted by adding 400 .mu.l of acetone to induce precipitation of
proteins, as described above. Decomposition of HI-113 in plasma was
significantly inhibited by paraoxon, partially inhibited by
physostigmine, but not affected by EDTA (see FIGS. 3A, 3B, and 3C
as well as Table 1). The data in Table 1 was the mean percent
hydrolysis from two experiments.
1TABLE 1 Effect of Selective Esterase Inhibitors on Metabolism of
HI-113 in Plasma Paraoxon (0.1 mM) Physostigmine EDTA No
cholinesterase & (0.1 mM) (1 mM) Specificity inhibitor
carboxylesterase cholinesterase arylesterase 5 min 95% 0% 43% 99%
10 min 98% 2% 65% 100% 15 min 99% 2% 76% 100% 30 min 100% 2% 89%
100% 60 min 100% 3% 100% 100% 120 min 100% 24% 100% 100%
Example 4
Stability of HI-113 in Murine Liver Homogenates
[0060] Fresh mouse liver was obtained from Balb/c mice and
homogenated in 1.times. PBS (1:1, W/V) using a Polytron (PT-MR2000)
homogenizer (Kinematical AG, Littau, Switzerland). HI-113 was added
to the liver homogenate to yield a final concentration of 100
.mu.M. At a predetermined time, an aliquot (100 .mu.l) of spiked
liver homogenate was extracted by adding 400 .mu.l of acetone to
induce precipitation of proteins, as described above.
[0061] Like in plasma, HI-113 decomposed after incubation with the
liver homogenate within 30 minutes (FIG. 4). However, unlike in
plasma, significant amounts of d4T were detected after incubation
with the liver homogenate.
Example 5
Stability of HI-113 and Ala-d4T-MIP in Gastric and Intestinal
Fluids
[0062] The simulated gastric and intestinal fluids were prepared
following USP XXII and were spiked with HI-113 and Ala-d4T-MP to
yield a solution with a final concentration of 100 .mu.M of each
compound. The spiked fluids were then placed in a 37.degree. C.
water bath. At a predetermined time, 100 .mu.l aliquots of the
spiked gastric or intestinal fluid were extracted by adding 400
.mu.l of acetone as discussed above.
[0063] HI-113 is relatively stable in gastric fluid for 8 hours,
but it is not stable in intestinal fluid (FIGS. 5A & 5B).
HI-113 quickly decomposed to yield Ala-d4T-MP in intestinal fluid
(approximately 94% of the HI-113 had decomposed within 2 hours).
Ala-d4T-MP was stable in intestinal fluid; only a trace amount of
d4T was detected in the intestinal fluid.
Example 6
Pharmacokinetic Studies in Mice
[0064] Female Balb/c mice (6-8 weeks old) from Taconic (Germantown,
N.Y.) were housed in a controlled environment (12-hours of
light/12-hours of dark, 22.+-.1.degree. C., 60.+-.10% relative
humidity), which is fully accredited by the USDA. All rodents were
housed in microisolator cages (Lab Products, Inc., N.J.) containing
autoclaved bedding. The mice were allowed free access to autoclaved
pellet food and tap water throughout the study. All animal care
procedures conformed to the Guide for the Care and Use of
Laboratory Animals (National Research Council, National Academy
Press, Washington D.C. 1996).
[0065] A solution (50 .mu.l) of HI-113 (100 mg/kg) dissolved in
DMSO was administered intravenously via the tail vein. This volume
of DMSO is well-tolerated by mice when administrated by rapid
intravenous or extravascular injection (Rosenkrantz et al., Cancer
Chemother. Rep., 31, 7 (1963); Wilson et al., Toxicol. Appl.
Pharmacol., 7, 104 (1965)). Blood samples (.about.500 .mu.L) were
obtained from the ocular venous plexus by retro-orbital
venipuncture at 0, 2, 5, 10, 15, 30, 45, 60, 120, 240 and 360
minutes after intravenous injection. In order to study the
pharmacokinetics of Ala-d4T-MP and d4T following systemic
administration of these compounds, mice were injected with 75 mg/kg
Ala-d4T-MP and 40 mg/kg d4T, respectively (these doses are
equimolar to the 100 mg/kg HI-113).
[0066] In order to determine the pharmacokinetics of HI-113
following oral administration, 12 hour fasted mice were given a
bolus dose of 100 mg/kg HI-113 via gavage using a #21
stainless-steel ball-tipped feeding needle. Sampling time points
were 0, 2, 5, 10, 15, 30, 45, 60, 120, 240 and 360 minutes after
the gavage.
[0067] All collected blood samples were heparinized and centrifuged
at 7000.times.g for 5 minutes to separate the plasma fraction from
the whole blood. The plasma samples were then processed immediately
using the extraction procedure described above.
[0068] Pharmacokinetic modeling and parameter calculations were
carried out using the WinNonlin Professional Version 3.0
(Pharsight, Inc., Mountain, Calif.) pharmacokinetics software (Chen
et al., Pharm. Res., 16, 1003 (1999); Chen et al., Pharm. Res., 16,
117 (1999); Chen et al., J. Clin. Pharmacol., 39, 1248 (1999);
Uckun et al., Clin. Cancer Res., 5, 2954 (1999); and Uckun et al.,
J. Pharmacol. Exp. Ther., 291, 1301 (1999)). An appropriate model
was chosen on the basis of the lowest sum of weighted squared
residuals, the lowest Schwartz Criterion (SC), the lowest Akaike's
Information Criterion (AIC) value, the lowest standard errors of
the fitted parameters, and the dispersion of the residuals. The
elimination half-life was estimated by linear regression analysis
of the terminal phase of the plasma concentration-time profile. The
area under the concentration-time curve (AUC) was calculated
according to the linear trapezoidal rule between the first sampling
time (0 hours) and the last sampling time plus C/k, where C is the
concentration of the last sampling and k is the elimination rate
constant. The systemic clearance (CL) was determined by dividing
the dose by the AUC. The metabolic clearance of the parent drug,
the formation clearance of the metabolite, the clearance
elimination of the metabolite, and the distribution clearance of
the metabolite were estimated by simultaneous fitting of the
concentration of parent drug and metabolites as a function of time
curve to pharmacokinetic models (see FIGS. 7A & 8A) specified
as a system of differential equations (Gabrielsson & Weiner,
Phamacokinetic/Phamacodynam- ic Data Analysis: Concepts and
Applications, Swedish Pharmaceutical Press (1997)). The fraction of
HI-113 converted to a metabolite (f.sub.m) was calculated as the
ratio of the AUC for the metabolite after administration of the
parent drug [(AUC.sub.m).sub.p] to the AUC after administration of
an equimolar dose of the metabolite [(AUC.sub.m).sub.m] (Gibaldi
& Perrier, 1982):
f.sub.m=[(AUC.sub.m).sub.p/D.sub.p].times.[D.s-
ub.m/(AUC.sub.m).sub.m]=(AUC.sub.m).sub.p.circle-solid.CL.sub.m/D.sub.p
Example 7
Metabolism and Pharmacokinetic Profile of HI-113 Following
Intravenous Administration
[0069] Following intravenous administration, HI-113 (100 mg/kg) was
metabolized to yield HI-113-M1 (R.sub.T=15.3 minutes) and HI-213-M2
(R.sub.T=18.5 minutes) (FIG. 1C). HI-113-M1 had the same retention
time as Ala-d4T-MP, whereas HI-113-M2 had the same retention time
as d4T (FIGS. 1B and 1C). The UV spectra of these two metabolites
were identical to those of Ala-d4T-MP and d4T, respectively.
[0070] After intravenous administration of 100 mg/kg HI-113, the
plasma concentration of HI-113 as a function of time was described
by a one-compartment model (FIG. 6A). The estimated pharmacokinetic
parameter values are presented in Table 2. HI-113 had a C.sub.max
of 224.2 .mu.M and an AUC of 1142.0 .mu.M.circle-solid.min. The
systemic clearance of HI-113 was moderately fast with a CL of 160.9
mL/min/kg, which is approximately twice the rate of blood flow to
the kidney or the liver (Davies et al., Pharm. Res., 10, 1093
(1993)). HI-113 had a moderate size volume of distribution with a
V.sub.ss of 819.9 ml/kg, which is roughly equal to the total volume
of water in the body. HI-113 had a short elimination half-life
(t.sub.1/2=3.5 minutes), however, because of its rapid
metabolism.
[0071] The diastereomers of HI-113 were separated using the HPLC
conditions described above (the retention times were 28.7 and 28.9
minutes). One of the diastereomers (HI-113-A, retention time=28.7
minutes) was metabolized more quickly than the other (HI-113-B,
retention time 28.9 minutes; FIG. 1C). The pharmacokinetic features
of these two diastereomers are summarized in Table 2. HI-113-B had
a higher AUC (741.2 vs. 441.5 .mu.M.circle-solid.min) and C.sub.max
(125.7 vs. 107.9 .mu.M) than HI-113-A (FIGS. 6B and 6C). HI-113-B
also had a slightly longer elimination half-life than the HI-113-A
diastereomer (4.1 minutes vs. 2.8 minutes), which can be due to the
faster clearance of HI-113-A relative to that of HI-113-B (208.2
vs. 123.9 ml/min/kg). However, both of the HI-113 diastereomers
were completely metabolized within 30 minutes.
[0072] Following intravenous injection, HI-113 was rapidly
metabolized to yield Ala-d4T-MP (t.sub.max=5.9 minutes;
C.sub.max=67.4 .mu.M; t.sub.1/2=129.2 minutes) and d4T
(t.sub.max=18.7 minutes; C.sub.max=15.7 .mu.M; t.sub.1/2=114.8
minutes) as shown in Table 2.
2TABLE 2 Estimated Pharmacokinetic Parameter Values for HI-113 and
Its Metabolites in Balb/C Mice V.sub.ss AUC C.sub.max t.sub.1/2 CL
t.sub.max Measured (ml/kg) (.mu.M .multidot. min) (.mu.M) (min)
(ml/min/kg) (min) Total HI- 819.9 1142.0 224.2 3.5 160.9 ND 113
(920 .+-. 127.4) (1071.8 .+-. 81.8) (211.6 .+-. 29.3) (3.6 .+-.
0.3) (174.5 .+-. 13.2) HI-113-A 852.1 441.5 107.9 2.8 208.2 ND
(1005.3 .+-. 134.0) (359.7 .+-. 43.9) (96.5 .+-. 12.8) (2.6 .+-.
0.1) (266.5 .+-. 30.2) HI-113-B 731.1 741.2 125.7 4.1 123.9 ND
(791.8 .+-. 113.1) (730.8 .+-. 45.7) (123.1 .+-. 16.8) (4.3 .+-.
0.4) (127.3 .+-. 8.2) Ala-d4T- ND 2854.8 67.4 129.2 ND 5.9 MP
(2795.9 .+-. 361.2) (69.3 .+-. 4.1) (138.8 .+-. 40.2) (5.1 .+-.
0.7) d4T ND 2915.2 15.7 114.8 ND 18.7 (2858.1 .+-. 182.2) (15.6
.+-. 1.2) (116.2 .+-. 11.9) (17.4 .+-. 2.6)
[0073] Pharmacokinetic parameters in Balb/c mice (N=4 mice per
time-point) are presented as the average values estimated from
composite plasma concentration-time curves of pooled data. The
mean.+-.S.E.M values are indicated in parentheses. ND means the
value was not determined.
[0074] The model depicted in FIGS. 7A and 7B describes the
metabolite pharmacokinetics of Ala-d4T-MP and d4T after intravenous
injection of HI-113. According to this model, HI-113 is
biotransformed to produce Ala-d4T-MP (CL.sub.m1) and d4T
(CL.sub.m2), respectively. Ala-d4T-MP derived from HI-113 can be
further metabolized to form D4T (CL.sub.m3) or distributed to the
extravascular compartment (Cl.sub.m1d). D4T produced from either
HI-113 or Ala-d4T-MP is finally eliminated from the body
(CL.sub.me2). The pharmacokinetic parameters estimated for these
two metabolites are presented in Table 3.
3TABLE 3 Estimated Metabolite Pharmacokinetic Parameter Values
Phamacokinetic Parameter ml/min/kg CL.sub.m1 83.9 (21.5%) CL.sub.m2
87.4 (24.4%) CL.sub.m3 36.1 (85.9%) CL.sub.m1d 62.0 (69.8%)
CL.sub.me2 47.1 (74.1%)
[0075] The values in parenthesis are the C.V. of modeling. The
metabolic clearance of HI-113 and the formation clearance of the
metabolites were 83.9 ml/min/kg for Ala-d4T-MP and 87.4 ml/min/kg
for d4T, respectively. The metabolic clearance of Ala-d4T-MP and
the formation clearance of its metabolite, d4T, were 36.1
ml/min/kg, and a small portion of Ala-d4T-MP was distributed to the
extravascular compartment with a CL.sub.m1d of 47.1 ml/min/kg.
Finally, d4T was eliminated with a CL.sub.me2 of 62.0
ml/min/kg.
Example 8
Pharmacokinetic Profile of Ala-d4T-MP Following Intravenous
Administration
[0076] Following intravenous injection of Ala-d4T-MP (75 mg/kg, a
dose equimolar to the 100 mg/kg dose of HI-113 discussed above),
Ala-d4T-MP was quickly metabolized to yield d4T (t.sub.max=4.4
minutes; t.sub.1/2=34.0 minutes) (FIGS. 8A & 8B, Table 4). The
concentration of Ala-d4T-MP as a function of time can be described
using a two-compartment model, while a one-compartment model best
fits the concentration of its metabolite, d4T, as a function of
time (FIG. 8B). The C.sub.max values for Ala-d4T-MP and d4T were
1206.6 .mu.M and 35.2 .mu.M, respectively. The AUC was 11648.7
.mu.M.circle-solid.min for Ala-d4T-MP and 1888.0
.mu.M.circle-solid.min for d4T. The systemic clearance of
Ala-d4T-MP was only 15.8 mL/min/kg (Table 4), which is much less
than the blood flow to either the kidney or the liver (Davies et
al., Pharm. Res., 10, 1093 (1993)). Ala-d4T-MP also had a small
volume of distribution (V.sub.ss=275.5 ml/kg) that is less than the
total volume of water in the body. Nevertheless, the elimination
half-life of Ala-d4T-MP was short (t.sub.1/2=28.5 minutes) due to
its rapid metabolism.
4TABLE 4 Estimated Pharmacokinetic Parameter Values for Ala-d4T-MP
and Its Metabolite in Balb/C Mice V.sub.SS AUC C.sub.max t.sub.1/2
CL t.sub.max Measured (ml/kg) (.mu.M .multidot. min) (.mu.M) (min)
(ml/min/kg) (min) Ala-d4T- 275.5 11648.7 1206.6 28.5 15.8 ND MP
(412.6 .+-. 126.3) (11761.5 .+-. 447.2) (1658.1 .+-. 544.9) (91.5
.+-. 54.5) (15.7 .+-. 0.6) d4T ND 1888.0 35.2 34.0 ND 4.4 (1818.2
.+-. 42.9) (35.9 .+-. 3.7) (32.4 .+-. 2.2) (5.0 .+-. 1.2)
[0077] Pharmacokinetic parameters in Balb/c mice (N=5 mice per
time-point) are presented as the average values estimated from
composite plasma concentration-time curves of pooled data. The
mean.+-.S.E.M values are indicated in parentheses. ND means the
value was not determined.
[0078] The model depicted in FIG. 8A best described the metabolite
pharmacokinetics after intravenous injection of Ala-d4T-MP.
According to this model, Ala-d4T-MP can either be metabolized to
form d4T (CL.sub.m1) or distributed to the extravascular
compartment (Cl.sub.pd). D4T derived from Ala-d4T-MP is eliminated
from the body (CL.sub.me). By simultaneous fitting of the parent
Ala-d4T-MP and d4T concentration values as a function of time to
the described model, the metabolic clearance of Ala-d4T-MP and the
formation clearance of d4T (CL.sub.m1) were estimated to be 15.6
ml/min/kg as shown in Table 5.
5TABLE 5 Estimated Metabolite Pharmacokinetic Parameter Values
Pharmacokinetic Parameter ml/min/kg CL.sub.m1 15.6 (16.6%)
CL.sub.me 88.4 (13.0%) CL.sub.pd 4.7 (44.7%)
[0079] The data in parentheses are the C.V. of modeling. A small
portion of Ala-d4T-MP was distributed to extravascular compartment
with a CL.sub.pd of 4.7 ml/min/kg and d4T derived from Ala-d4T-MP
was finally eliminated with a relatively high CL.sub.me of 88.4
ml/min/kg. The CL.sub.m1 of 15.6 ml/min/kg accounts for 99% of the
total systemic clearance (CL=15.8 ml/min/kg) (see Table 4),
indicating that most of Ala-d4T-MP is biotransformed to form
d4T.
Example 9
Pharmacokinetic Profile of d4T Following Intravenous
Administration
[0080] Following intravenous injection at a dose level of 40 mg/kg,
a dose equimolar to the 100 mg/kg dose of HI-113, the concentration
of d4T as a function of time was described using a one-compartment
model (FIG. 9). The estimated pharmacokinetic parameter values are
presented in Table 6. The estimated C.sub.max and AUC values for
D4T were 279.5 .mu.M and 12227.1 .mu.M.circle-solid.min,
respectively. D4T had a short elimination half-life (30.3 minutes).
The systemic clearance of d4T was slow with a CL of only 15.0
ml/min/kg, which is much lower than the blood flow to either the
kidney or the liver (Davies et al., Pharm. Res., 10, 1093 (1993)).
D4T had a moderately large volume of distribution (V.sub.ss=657.8
ml/kg) that is approximately equal to the volume of water in the
body.
6TABLE 6 Estimated Pharmacokinetic Parameter Values for D4T in
Balb/C Mice V.sub.SS AUC C.sub.max t.sub.1/2 CL Measured (ml/kg)
(.mu.M .multidot. min) (.mu.M) (min) (ml/min/kg) d4T 657.8 12227.1
279.5 30.3 15.0 (581.8 .+-. 62.8) (12173.6 .+-. 559.5) (318.9 .+-.
15.7) (26.6 .+-. 1.2) (15.2 .+-. 0.7)
[0081] Pharmacokinetic parameters in Balb/c mice (N=5 mice per
time-point) are presented as the average values estimated from
composite plasma concentration-time curves of pooled data. The
mean.+-.S.E.M values are indicated in parentheses.
Example 10
Pharmacokinetic Profile of HI-113 Following Oral Administration
[0082] The pharmacokinetic behavior of orally administered HI-113
(100 mg/kg) was also examined. Both metabolites (Ala-d4T-MP and
d4T) were detected, but the concentration of the parent HI-113 was
below the detection limit (0.5 .mu.M). The t.sub.max values are
10.3 minutes for Ala-d4T-MP and 42.4 minutes for d4T. A
one-compartment pharmacokinetic model was used to describe both the
Ala-d4T-MP and the d4T concentration changes as a function of time
(FIGS. 10A and 10B). The estimated values for the pharmacokinetic
parameters are presented in Table 7. The maximum concentrations
(C.sub.max) for Ala-d4T-MP and D4T are 12.7 .mu.M and 30.7 .mu.M,
respectively. The elimination half-lives were 66.4 minutes and 99.0
minutes for Ala-d4T-MP and d4T, respectively.
7TABLE 7 Estimated Pharmacokinetic Parameter Values Following Oral
Administration of HI-113 in Balb/C Mice Mea- AUC C.sub.max
t.sub.1/2 t.sub.max sured (.mu.M .multidot. min) (.mu.M) (min)
(min) Ala- 1350.5 12.7 66.4 10.3 d4T-MP (1355.4 .+-. 88.2) (15.6
.+-. 4.1) (56.1 .+-. 8.5) (9.3 .+-. 0.9) d4T 5905.3 30.7 99.0 42.4
(5928.4 .+-. 294.6) (29.5 .+-. 0.3) (102.6 .+-. 3.8) (45.2 .+-.
5.2)
[0083] Pharmacokinetic parameters in Balb/c mice (N=4 mice per
time-point) are presented as the average values estimated from
composite plasma concentration-time curves of pooled data. The
mean.+-.S.E.M values are indicated in parentheses.
[0084] While a detailed description of the present invention has
been provided above, the invention is not limited thereto. The
invention described herein can be modified to include alternative
embodiments, as will be apparent to those skilled in the art. All
such alternatives should be considered within the spirit and scope
of the invention, as claimed below.
* * * * *