U.S. patent application number 15/875101 was filed with the patent office on 2018-05-24 for compounds and methods for delivery of prostacyclin analogs.
This patent application is currently assigned to United Therapeutics Corporation. The applicant listed for this patent is United Therapeutics Corporation. Invention is credited to Ken Phares.
Application Number | 20180141891 15/875101 |
Document ID | / |
Family ID | 34079029 |
Filed Date | 2018-05-24 |
United States Patent
Application |
20180141891 |
Kind Code |
A1 |
Phares; Ken |
May 24, 2018 |
COMPOUNDS AND METHODS FOR DELIVERY OF PROSTACYCLIN ANALOGS
Abstract
This invention pertains generally to prostacyclin formulations
and methods for their use in promoting vasodilation, inhibiting
platelet aggregation and thrombus formation, stimulating
thrombolysis, inhibiting cell proliferation (including vascular
remodeling), providing cytoprotection, preventing atherogenesis and
inducing angiogenesis.
Inventors: |
Phares; Ken; (Chapel Hill,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
United Therapeutics Corporation |
Silver Spring |
MD |
US |
|
|
Assignee: |
United Therapeutics
Corporation
Silver Spring
MD
|
Family ID: |
34079029 |
Appl. No.: |
15/875101 |
Filed: |
January 19, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15239014 |
Aug 17, 2016 |
9878972 |
|
|
15875101 |
|
|
|
|
14881379 |
Oct 13, 2015 |
9422223 |
|
|
15239014 |
|
|
|
|
14710694 |
May 13, 2015 |
9278901 |
|
|
14881379 |
|
|
|
|
14490014 |
Sep 18, 2014 |
9199908 |
|
|
14710694 |
|
|
|
|
13906585 |
May 31, 2013 |
9050311 |
|
|
14490014 |
|
|
|
|
13558757 |
Jul 26, 2012 |
8536363 |
|
|
13906585 |
|
|
|
|
12078955 |
Apr 8, 2008 |
8252839 |
|
|
13558757 |
|
|
|
|
11603124 |
Nov 22, 2006 |
7384978 |
|
|
12078955 |
|
|
|
|
10851481 |
May 24, 2004 |
7417070 |
|
|
11603124 |
|
|
|
|
60472407 |
May 22, 2003 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
C07C 229/08 20130101; A61P 11/00 20180101; A61P 9/04 20180101; A61P
9/12 20180101; A61P 9/08 20180101; A61P 9/10 20180101; C07C 51/412
20130101; C07C 2603/10 20170501; A61P 15/00 20180101; C07C 59/13
20130101; C07C 69/712 20130101; A61K 31/235 20130101; C07C 235/06
20130101; C07C 259/06 20130101; C07F 9/117 20130101; A61K 31/192
20130101; A61K 9/0053 20130101; A61P 35/00 20180101; A61P 13/12
20180101; A61K 9/0019 20130101; C07C 59/70 20130101; A61P 27/02
20180101; C07F 9/091 20130101; A61P 7/02 20180101; A61P 17/06
20180101; A61P 9/14 20180101; A61P 17/00 20180101; C07C 235/08
20130101; A61P 9/00 20180101; A61K 31/223 20130101; A61P 29/00
20180101; A61K 31/216 20130101; A61P 25/00 20180101 |
International
Class: |
C07C 59/13 20060101
C07C059/13; A61K 9/00 20060101 A61K009/00; A61K 31/216 20060101
A61K031/216; A61K 31/192 20060101 A61K031/192; A61K 31/223 20060101
A61K031/223; A61K 31/235 20060101 A61K031/235; C07C 51/41 20060101
C07C051/41; C07F 9/117 20060101 C07F009/117; C07F 9/09 20060101
C07F009/09; C07C 259/06 20060101 C07C259/06; C07C 235/08 20060101
C07C235/08; C07C 235/06 20060101 C07C235/06; C07C 229/08 20060101
C07C229/08; C07C 69/712 20060101 C07C069/712; C07C 59/70 20060101
C07C059/70 |
Claims
1. A method of treating pulmonary hypertension, treating peripheral
vascular disease, promoting vasodilation, inhibiting platelet
aggregation and thrombus formation, stimulating thrombolysis,
inhibiting cell proliferation, providing cytoprotection, preventing
atherogenesis, or inducing angiogenesis comprising administering to
a patient in need thereof a pharmaceutically effective amount of a
prodrug of treprostinil, wherein the prodrug converts to
treprostinil upon said administration.
2. The method of claim 1, wherein the prodrug has no activity
before being converted to treprostinil.
3. The method of claim 1, wherein the prodrug is administered by
oral administration.
4. The method of claim 1, wherein the prodrug is administered by
ingestion.
5. The method of claim 1, wherein the prodrug is administered by
transmucosal administration.
6. The method of claim 1, wherein the prodrug is administered by
parenteral administration.
7. The method of claim 1, wherein the prodrug is administered by
subcutaneous administration.
8. The method of claim 1, wherein the prodrug is administered by
injection.
9. The method of claim 8, wherein the injection is a bolus
injection.
10. The method of claim 8, wherein the injection is continuous
infusion.
11. The method of claim 8, wherein the injection is an intravenous
injection.
12. The method of claim 1, wherein the prodrug is administered by
duodenal administration.
13. The method of claim 1, which is a method of treating pulmonary
hypertension.
14. The method of claim 1, wherein the prodrug is an ester of
treprostinil.
15. The method of claim 14, which is a method of treating pulmonary
hypertension.
16. The method of claim 1, wherein the prodrug provides a Cmax up
to 240 minutes post-dosing.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/239,014, filed Aug. 17, 2016, which is a continuation of
U.S. application Ser. No. 14/881,379, filed Oct. 13, 2015, which is
a divisional of U.S. application Ser. No. 14/710,694, filed May 13,
2015, which is a continuation of U.S. application Ser. No.
14/490,014, filed Sep. 18, 2014, which is a Continuation of U.S.
application Ser. No. 13/906,585, filed May 31, 2013, which is a
divisional of U.S. application Ser. No. 13/558,757, filed Jul. 26,
2012, which is a continuation of U.S. application Ser. No.
12/078,955, filed Apr. 8, 2008, which is a divisional of U.S.
application Ser. No. 11/603,124, filed Nov. 22, 2006, which is a
continuation of U.S. application Ser. No. 10/851,481, filed May 24,
2004, which claims benefit of U.S. Provisional Application Ser. No.
60/472,407, filed on May 22, 2003, the entire contents of which
applications are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] This invention pertains generally to prostacyclin analogs
and methods for their use in promoting vasodilation, inhibiting
platelet aggregation and thrombus formation, stimulating
thrombolysis, inhibiting cell proliferation (including vascular
remodeling), providing cytoprotection, preventing atherogenesis and
inducing angiogenesis. Through these prostacyclin-mimetic
mechanisms, the compounds of the present invention may be used in
the treatment of/for: pulmonary hypertension, ischemic diseases
(e.g., peripheral vascular disease, Raynaud's phenomenon,
Scleroderma, myocardial ischemia, ischemic stroke, renal
insufficiency), heart failure (including congestive heart failure),
conditions requiring anticoagulation (e.g., post MI, post cardiac
surgery), thrombotic microangiopathy, extracorporeal circulation,
central retinal vein occlusion, atherosclerosis, inflammatory
diseases (e.g., COPD, psoriasis), hypertension (e.g.,
preeclampsia), reproduction and parturition, cancer or other
conditions of unregulated cell growth, cell/tissue preservation and
other emerging therapeutic areas where prostacyclin treatment
appears to have a beneficial role. These compounds may also
demonstrate additive or synergistic benefit in combination with
other cardiovascular agents (e.g., calcium channel blockers,
phosphodiesterase inhibitors, endothelial antagonists, antiplatelet
agents).
BACKGROUND OF THE INVENTION
[0003] Many valuable pharmacologically active compounds cannot be
effectively administered orally for various reasons and are
generally administered via intravenous or intramuscular routes.
These routes of administration generally require intervention by a
physician or other health care professional, and can entail
considerable discomfort as well as potential local trauma to the
patient.
[0004] One example of such a compound is treprostinil, a chemically
stable analog of prostacyclin. Although treprostinil sodium
(Remodulin.RTM.) is approved by the Food and Drug Administration
(FDA) for subcutaneous administration, treprostinil as the free
acid has an absolute oral bioavailability of less than 10%.
Accordingly, there is clinical interest in providing treprostinil
orally.
[0005] Thus, there is a need for a safe and effective method for
increasing the systemic availability of treprostinil via
administration of treprostinil or treprostinil analogs.
SUMMARY OF THE INVENTION
[0006] In one embodiment, the present invention provides a compound
having structure I:
##STR00001##
wherein,
[0007] R.sup.1 is independently selected from the group consisting
of H, substituted and unsubstituted benzyl groups, and groups
wherein OR.sup.1 are substituted or unsubstituted glycolamide
esters;
[0008] R.sup.2 and R.sup.3 may be the same or different and are
independently selected from the group consisting of H, phosphate
and groups wherein OR.sup.2 and OR.sup.3 form esters of amino acids
or proteins, with the proviso that all of R.sup.1, R.sup.2 and
R.sup.3 are not H;
[0009] an enantiomer of the compound;
[0010] and pharmaceutically acceptable salts of the compound and
polymorphs.
[0011] In some of these embodiments, R.sup.1 is a substituted or
unsubstituted benzyl group, such as CH.sub.2C.sub.6H.sub.5. In
other embodiments, OR.sup.1 is a substituted or unsubstituted
glycolamide ester, R.sup.1 is --CH.sub.2CONR.sup.4R.sup.5, R.sup.4
and R.sup.5 may be the same or different and are independently
selected from the group consisting of H, OH, substituted and
unsubstituted alkyl groups, --(CH.sub.2).sub.mCH.sub.3,
--CH.sub.2OH, and --CH.sub.2(CH.sub.2).sub.nOH, with the proviso
that m is 0, 1, 2, 3 or 4, and n is 0, 1, 2, 3 or 4. In certain of
these embodiments one or both of R.sup.4 and R.sup.5 are
independently selected from the group consisting of H, --OH,
--CH.sub.3, or --CH.sub.2CH.sub.2OH. In any of the previously
discussed embodiments, one or both of R.sup.2 and R.sup.3 can be H.
In some enantiomers of the compound R.sup.1=R.sup.2=R.sup.3=H, or
R.sup.2=R.sup.3=H and R.sup.1=valinyl amide.
[0012] In still further embodiments of the present compounds
R.sup.2 and R.sup.3 are independently selected from phosphate and
groups wherein OR.sup.2 and OR.sup.3 are esters of amino acids,
dipeptides, esters of tripeptides and esters of tetrapeptides. In
some compounds only one of R.sup.2 or R.sup.3 is a phosphate group.
In other compounds R.sup.2 and R.sup.3 are independently selected
from groups wherein OR.sup.2 and OR.sup.3 are esters of amino
acids, such as esters of glycine or alanine. In any of the above
embodiments, one of R.sup.2 and R.sup.3 are H. In certain of the
present compounds, the oral bioavailability of the compound is
greater than the oral bioavailability of treprostinil, such as at
least 50% or 100% greater than the oral bioavailability of
treprostinil. The above compounds can further comprise an inhibitor
of p-glycoprotein transport. Any of these compounds can also
further comprise a pharmaceutically acceptable excipient.
[0013] The present invention also provides a method of using the
above compounds therapeutically of/for: pulmonary hypertension,
ischemic diseases, heart failure, conditions requiring
anticoagulation, thrombotic microangiopathy, extracorporeal
circulation, central retinal vein occlusion, atherosclerosis,
inflammatory diseases, hypertension, reproduction and parturition,
cancer or other conditions of unregulated cell growth, cell/tissue
preservation and other emerging therapeutic areas where
prostacyclin treatment appears to have a beneficial role. A
preferred embodiment is a method of treating pulmonary hypertension
and/or peripheral vascular disease in a subject comprising orally
administering a pharmaceutically effective amount of a compound of
structure II:
##STR00002##
wherein,
[0014] R.sup.1 is independently selected from the group consisting
of H, substituted and unsubstituted alkyl groups, arylalkyl groups
and groups wherein OR.sup.1 form a substituted or unsubstituted
glycolamide ester;
[0015] R.sup.2 and R.sup.3 may be the same or different and are
independently selected from the group consisting of H, phosphate
and groups wherein OR.sup.2 and OR.sup.3 form esters of amino acids
or proteins, with the proviso that all of R.sup.1, R.sup.2 and
R.sup.3 are not H;
[0016] an enantiomer of the compound; and
[0017] a pharmaceutically acceptable salt or polymorph of the
compound.
[0018] In some of these methods, when OR.sup.1 forms a substituted
or unsubstituted glycolamide ester, R.sup.1 is
--CH.sub.2CONR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5 may be the
same or different and are independently selected from the group
consisting of H, OH, substituted and unsubstituted alkyl groups,
--(CH.sub.2).sub.mCH.sub.3, --CH.sub.2OH, and
--CH.sub.2(CH.sub.2).sub.nOH, with the proviso that m is 0, 1, 2, 3
or 4, and n is 0, 1, 2, 3 or 4. In other methods R.sup.1 is a
C.sub.1-C.sub.4 alkyl group, such as methyl, ethyl, propyl or
butyl. In the disclosed methods, R.sup.1 can also be a substituted
or unsubstituted benzyl group. In other methods, R.sup.1 can be
--CH.sub.3 or --CH.sub.2C.sub.6H.sub.5. In still other methods
R.sup.4 and R.sup.5 are the same or different and are independently
selected from the group consisting of H, OH, --CH.sub.3, and
--CH.sub.2CH.sub.2OH. In yet other methods, one or both of R.sup.2
and R.sup.3 are H. Alternatively, one or both of R.sup.2 and
R.sup.3 are not H and R.sup.2 and R.sup.3 are independently
selected from phosphate and groups wherein OR.sup.2 and OR.sup.3
are esters of amino acids, dipeptides, esters of tripeptides and
esters of tetrapeptides. In some methods, only one of R.sup.2 or
R.sup.3 is a phosphate group. In additional methods, R.sup.2 and
R.sup.3 are independently selected from groups wherein OR.sup.2 and
OR.sup.3 are esters of amino acids, such as esters of glycine or
alanine. In further methods one of R.sup.1 and R.sup.2 is H. In
some methods, enantiomers of the compound where
R.sup.1=R.sup.2=R.sup.3=H, or R.sup.2=R.sup.3=H and R.sup.1=valinyl
amide are used.
[0019] In various methods the oral bioavailability of the compound
is greater than the oral bioavailability of treprostinil, such as
at least 50% or 100% greater than the oral bioavailability of
treprostinil. The present methods can also comprise administering
pharmaceutically effective amount of a p-glycoprotein inhibitor,
simultaneously, sequentially, or prior to administration of the
compound of structure II. In some embodiments the p-glycoprotein
inhibitor is administered orally or intravenously. The disclosed
methods can be used to treat pulmonary hypertension.
[0020] The present invention also provides a method of increasing
the oral bioavailability of treprostinil or pharmaceutically
acceptable salt thereof, comprising administering a
pharmaceutically effective amount of a p-glycoprotein inhibitor and
orally administering a pharmaceutically effective amount of
treprostinil to a subject. In certain of these embodiments the
p-glycoprotein inhibitor is administered prior to or simultaneously
with the treprostinil. The route of the p-glycoprotein inhibitor
administration can vary, such as orally or intravenously. The
present invention also provides a composition comprising
treprostinil or a pharmaceutically acceptable salt thereof and a
p-glycoprotein inhibitor.
[0021] The present compound can also be administered topically or
transdermally.
[0022] Pharmaceutical formulations according to the present
invention are provided which include any of the compounds described
above in combination with a pharmaceutically acceptable
carrier.
[0023] The compounds described above can also be used to treat
cancer.
[0024] Further objects, features and advantages of the invention
will be apparent from the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIGS. 1A and 1B respectively show plasma concentration
versus time curves for intravenous and intraportal dosing of
treprostinil diethanolamine salt in rats as described in Example
1;
[0026] FIGS. 2A, 2B and 2C respectively show plasma concentration
versus time curves for intraduodenal, intracolonic and oral dosing
of treprostinil diethanol amine salt in rats as described in
Example 1;
[0027] FIG. 3 shows on a logarithmic scale the average plasma
concentration versus time curves for the routes of administration
described in Example 1;
[0028] FIG. 4 is a graphical representation of the plasma
concentration versus time curve for treprostinil in rat following
oral administration in rats of treprostinil methyl ester as
described in Example 2;
[0029] FIG. 5 is a graphical representation of the plasma
concentration versus time curve for treprostinil in rat following
oral administration in rats of treprostinil benzyl ester as
described in Example 2;
[0030] FIG. 6 is a graphical representation of the plasma
concentration versus time curve for treprostinil in rat following
oral administration in rats of treprostinil diglycine as described
in Example 2;
[0031] FIG. 7 is a graphical representation of the plasma
concentration versus time curve for treprostinil in rat following
oral administration in rates of treprostinil benzyl ester (0.5
mg/kg) and treprostinil diglycine (0.5 mg/kg) as described in
Example 2 compared to treprostinil (1 mg/per kg).
[0032] FIG. 8 is a graphical representation of the plasma
concentration versus time curve for treprostinil in rat following
intraduodenal administration of treprostinil monophosphate (ring)
as described in Example 3;
[0033] FIG. 9 is a graphical representation of the plasma
concentration versus time curve for treprostinil in rat following
intraduodenal administration of treprostinil monovaline (ring) as
described in Example 3;
[0034] FIG. 10 is a graphical representation of the plasma
concentration versus time curve for treprostinil in rat following
intraduodenal administration of treprostinil monoalanine (ring) as
described in Example 3;
[0035] FIG. 11 is a graphical representation of the plasma
concentration versus time curve for treprostinil in rat following
intraduodenal administration of treprostinil monoalanine (chain) as
described in Example 3; and
[0036] FIG. 12 is a graphical representation of the average plasma
concentration versus time curve for each prodrug compared to
treprostinil alone from Example 1, as described in Example 3.
Treprostinil was dosed at 1 mg/kg whereas the prodrugs were dosed
at 0.5 mg/kg.
[0037] FIGS. 13A-13D respectively show doses, administered every
two hours for four doses, for either 0.05 mg per dose (total=0.2
mg), 0.125 mg per dose (total=0.5 mg), 0.25 mg per dose (total=1.0
mg), or 0.5 mg per dose (total=2.0 mg).
[0038] FIG. 14 shows pharmacokinetic profiles of UT-15C sustained
release tablets and sustained release capsules, fasted and fed
state.
[0039] FIG. 15 shows an X ray powder diffraction spectrum of the
polymorph Form A.
[0040] FIG. 16 shows an IR spectrum of the polymorph Form A.
[0041] FIG. 17 shows a Raman spectrum of the polymorph Form A.
[0042] FIG. 18 shows thermal data of the polymorph Form A.
[0043] FIG. 19 shows moisture sorption data of the polymorph Form
A.
[0044] FIG. 20 shows an X ray powder diffraction spectrum of the
polymorph Form B.
[0045] FIG. 21 shows thermal data of the polymorph Form B.
[0046] FIG. 22 shows moisture sorption data of the polymorph Form
B.
DETAILED DESCRIPTION OF THE INVENTION
[0047] Unless otherwise specified, "a" or "an" means "one or more".
The present invention provides compounds and methods for inducing
prostacyclin-like effects in a subject or patient. The compounds
provided herein can be formulated into pharmaceutical formulations
and medicaments that are useful in the methods of the invention.
The invention also provides for the use of the compounds in
preparing medicaments and pharmaceutical formulations and for use
of the compounds in treating biological conditions related to
insufficient prostacyclin activity as outlined in the Field of
Invention. The present invention also provides compounds and
methods for the treatment of cancer and cancer related
disorders.
[0048] In some embodiments, the present compounds are chemical
derivatives of (+)-treprostinil, which has the following
structure:
##STR00003##
[0049] Treprostinil is a chemically stable analog of prostacyclin,
and as such is a potent vasodilator and inhibitor of platelet
aggregation. The sodium salt of treprostinil,
(1R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-Hexahydro-2-hydroxy-1-[(3S)-3-hydroxyocty-
l]-1H-benz[f]inden-5-yl] oxy]acetic acid monosodium salt, is sold
as a solution for injection as Remodulin.RTM. which has been
approved by the Food and Drug Administration (FDA) for treatment of
pulmonary hypertension. In some embodiments, the present compounds
are derivatives of (-)-treprostinil, the enantiomer of
(+)-treprostinil. A preferred embodiment of the present invention
is the diethanolamine salt of treprostinil. The present invention
further includes polymorphs of the above compounds, with two forms,
A and B, being described in the examples below. Of the two forms, B
is preferred. A particularly preferred embodiment of the present
invention is form B of treprostinil diethanolamine.
[0050] In some embodiments, the present compounds are generally
classified as prodrugs of treprostinil that convert to treprostinil
after administration to a patient, such as through ingestion. In
some embodiments, the prodrugs have little or no activity
themselves and only show activity after being converted to
treprostinil. In some embodiments, the present compounds were
produced by chemically derivatizing treprostinil to make stable
esters, and in some instances, the compounds were derivatized from
the hydroxyl groups. Compounds of the present invention can also be
provided by modifying the compounds found in U.S. Pat. Nos.
4,306,075 and 5,153,222 in like manner.
[0051] In one embodiment, the present invention provides compounds
of structure I:
##STR00004##
wherein,
[0052] R.sup.1 is independently selected from the group consisting
of H, substituted and unsubstituted benzyl groups and groups
wherein OR.sup.1 are substituted or unsubstituted glycolamide
esters;
[0053] R.sup.2 and R.sup.3 may be the same or different and are
independently selected from the group consisting of H, phosphate
and groups wherein OR.sup.2 and OR.sup.3 form esters of amino acids
or proteins, with the proviso that all of R.sup.1, R.sup.2 and
R.sup.3 are not H;
[0054] enantiomers of the compound; and
[0055] pharmaceutically acceptable salts of the compound.
[0056] In some embodiments wherein OR.sup.1 are substituted or
unsubstituted glycolamide esters, R.sup.1 is
--CH.sub.2CONR.sup.4R.sup.5 and R.sup.4 and R.sup.5 may be the same
or different and are independently selected from the group
consisting of H, OH, substituted and unsubstituted alkyl groups,
--(CH.sub.2).sub.mCH.sub.3, --CH.sub.2OH, and
--CH.sub.2(CH.sub.2).sub.nOH, with the proviso that m is 0, 1, 2, 3
or 4, and n is 0, 1, 2, 3 or 4.
[0057] One skilled in the art will also readily recognize that
where members are grouped together in a common manner, such as in a
Markush group or the groups described in the R of structures I and
II above and below, the present invention encompasses not only the
entire group listed as a whole, but each member of the group
individually and all possible subgroups of the main group.
Accordingly, for all purposes, the present invention encompasses
not only the main group, but also the main group absent one or more
of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in
the claimed invention. For example, R.sup.1 can specifically
exclude H, substituted and unsubstituted benzyl groups, or groups
wherein OR.sup.1 are substituted or unsubstituted glycolamide
esters.
[0058] In some embodiments, R.sup.1 is a substituted or
unsubstituted benzyl groups, such as --CH.sub.2C.sub.6H.sub.5,
--CH.sub.2C.sub.6H.sub.4NO.sub.2,
--CH.sub.2C.sub.6H.sub.4OCH.sub.3, --CH.sub.2C.sub.6H.sub.4Cl,
--CH.sub.2C.sub.6H.sub.4(NO.sub.2).sub.2, or
--CH.sub.2C.sub.6H.sub.4F. The benzyl group can be ortho, meta,
para, ortho/para substituted and combinations thereof. Suitable
substituents on the aromatic ring include halogens (fluorine,
chlorine, bromine, iodine), --NO.sub.2 groups, --OR.sup.16 groups
wherein R.sup.16 is H or a C.sub.1-C.sub.4 alkyl group, and
combinations thereof.
Alternatively, when R.sup.1 is --CH.sub.2CONR.sup.4R.sup.5 then
R.sup.4 and R.sup.5 may be the same or different and are
independently selected from the group consisting of H, OH,
--CH.sub.3, and --CH.sub.2CH.sub.2OH. In these compounds where
R.sup.1 is not H, generally one or both of R.sup.2 and R.sup.3 are
H.
[0059] In some embodiment one or both of R.sup.2 and R.sup.3 are H
and R.sup.1 is --CH.sub.2CONR.sup.4R.sup.5, and one or both of
R.sup.4 and R.sup.5 are H, --OH, --CH.sub.3,
--CH.sub.2CH.sub.2OH.
[0060] In compounds where one or both of R.sup.2 and R.sup.3 are
not H, R.sup.2 and R.sup.3 can be independently selected from
phosphate and groups wherein OR.sup.2 and OR.sup.3 are esters of
amino acids, dipeptides, esters of tripeptides and esters of
tetrapeptides. In some embodiments, only one of R.sup.2 or R.sup.3
is a phosphate group. In compounds where at least one of R.sup.2
and R.sup.3 is not H, generally R.sup.1 is H. In additional
embodiments, one of R.sup.2 and R.sup.3 are H and thus the compound
of structure I is derivatized at only one of R.sup.2 and R.sup.3.
In particular compounds, R.sup.2 is H and R.sup.3 is defined as
above. In additional embodiments, R.sup.1 and R.sup.3 are H and
R.sup.2 is a group wherein OR.sup.2 is an ester of an amino acid or
a dipeptide. In further embodiments, R.sup.1 and R.sup.2 are H and
R.sup.3 is a group wherein OR.sup.3 is an ester of an amino acid or
a dipeptide.
[0061] When one or both of the OR.sup.2 and OR.sup.3 groups form
esters of amino acids or peptides, i.e., dipeptides, tripeptides or
tetrapeptides, these can be depicted generically as
--COCHR.sup.6NR.sup.7R.sup.8 wherein R.sup.6 is selected from the
group consisting of amino acid side chains, R.sup.7 and R.sup.8 may
be the same or different and are independently selected from the
group consisting of H, and --COCHR.sup.9NR.sup.10R.sup.11.
Generally, reference to amino acids or peptides refers to the
naturally occurring, or L-isomer, of the amino acids or peptides.
However, the present compounds and methods are not limited thereto
and D-isomer amino acid residues can take the place of some or all
of L-amino acids. In like manner, mixtures of D- and L-isomers can
also be used. In the embodiments wherein the amino acid is proline,
R.sup.7 together with R.sup.6 forms a pyrrolidine ring structure.
R.sup.6 can be any of the naturally occurring amino acid side
chains, for example --CH.sub.3 (alanine),
--(CH.sub.2).sub.3NHCNH.sub.2NH (arginine), --CH.sub.2CONH.sub.2
(asparagine), --CH.sub.2COOH (aspartic acid), --CH.sub.2SH
(cysteine), --(CH.sub.2).sub.2CONH.sub.2 (glutamine),
--(CH.sub.2).sub.2COOH (glutamic acid), --H (glycine),
--CHCH.sub.3CH.sub.2CH.sub.3 (isoleucine),
--CH.sub.2CH(CH.sub.3).sub.2 (leucine), --(CH.sub.2).sub.4NH.sub.2
(lysine), --(CH.sub.2).sub.2SCH.sub.3 (methionine), --CH2Ph
(phenylalanine), --CH.sub.2OH (serine), --CHOHCH.sub.3 (threonine),
--CH(CH.sub.3).sub.2 (valine),
##STR00005##
--(CH.sub.2).sub.3NHCONH.sub.2 (citrulline) or
--(CH.sub.2).sub.3NH.sub.2 (ornithine). Ph designates a phenyl
group.
[0062] In the above compounds, R.sup.7 and R.sup.8 may be the same
or different and are selected from the group consisting of H, and
--COCHR.sup.9NR.sup.10R.sup.11, wherein R.sup.9 is a side chain of
amino acid, R.sup.10 and R.sup.11 may be the same or different and
are selected from the group consisting of H, and
--COCHR.sup.12NR.sup.13R.sup.14, wherein R.sup.12 is an amino acid
side chain, R.sup.13 and R.sup.14 may be the same or different and
are independently selected from the group consisting of H, and
--COCHR.sup.15NH.sub.2. One skilled in the art will realize that
the peptide chains can be extended on the following scheme to the
desired length and include the desired amino acid residues.
[0063] In the embodiments where either or both of OR.sup.2 and
OR.sup.3 groups form an ester of a peptide, such as dipeptide,
tripeptide, tetrapeptide, etc. the peptides can be either
homopeptides, i.e., repeats of the same amino acid, such as
arginyl-arginine, or heteropeptides, i.e., made up of different
combinations of amino acids. Examples of heterodipeptides include
alanyl-glutamine, glycyl-glutamine, lysyl-arginine, etc.
[0064] As will be understood by the skilled artisan when only one
R.sup.7 and R.sup.8 includes a peptide bond to further amino acid,
such as in the di, tri and tetrapeptides, the resulting peptide
chain will be linear. When both R.sup.7 and R.sup.8 include a
peptide bond, then the peptide can be branched.
[0065] In still other embodiments of the present compounds R.sup.1
is H and one of R.sup.2 or R.sup.3 is a phosphate group or H while
the other R.sup.2 or R.sup.3 is a group such the OR.sup.2 or
OR.sup.3 is an ester of an amino acid, such as an ester of glycine
or alanine.
[0066] Pharmaceutically acceptable salts of these compounds as well
as pharmaceutical formulation of these compounds are also
provided.
[0067] Generally, the compounds described herein have enhanced oral
bioavailability compared to the oral bioavailability of
treprostinil, either in free acid or salt form. The described
compounds can have oral bioavailability that is at least 25%, 50%
100%, 200%, 400% or more compared to the oral bioavailability of
treprostinil. The absolute oral bioavailability of these compounds
can range between 10%, 15%, 20%, 25%, 30% and 40%, 45%, 50%, 55%,
60% or more when administered orally. For comparison, the absolute
oral bioavailability of treprostinil is on the order of 10%,
although treprostinil sodium has an absolute bioavailability
approximating 100% when administered by subcutaneous infusion.
[0068] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein, and in particular the
bioavailability ranges described herein also encompass any and all
possible subranges and combinations of subranges thereof. As only
one example, a range of 20% to 40%, can be broken down into ranges
of 20% to 32.5% and 32.5% to 40%, 20% to 27.5% and 27.5% to 40%,
etc. Any listed range can be easily recognized as sufficiently
describing and enabling the same range being broken down into at
least equal halves, thirds, quarters, fifths, tenths, etc. As a
non-limiting example, each range discussed herein can be readily
broken down into a lower third, middle third and upper third, etc.
As will also be understood by one skilled in the art all language
such as "up to," "at least," "greater than," "less than," "more
than" and the like include the number recited and refer to ranges
which can be subsequently broken down into subranges as discussed
above. In the same manner, all ratios disclosed herein also include
all subratios falling within the broader ratio.
[0069] Administration of these compounds can be by any route by
which the compound will be bioavailable in effective amounts
including oral and parenteral routes. The compounds can be
administered intravenously, topically, subcutaneously,
intranasally, rectally, intramuscularly, transdermally or by other
parenteral routes. When administered orally, the compounds can be
administered in any convenient dosage form including, for example,
capsule, tablet, liquid, suspension, and the like.
[0070] Testing has shown that that treprostinil can be irritating
upon skin contact. In contrast, some of the compounds disclosed
herein, generally as prodrugs of treprostinil, are not irritating
to the skin. Accordingly, the present compounds are well suited for
topical or transdermal administration.
[0071] When administered to a subject, the above compounds, and in
particular the compounds of structure I, are prostacyclin-mimetic
and are useful in treating conditions or disorders where
vasodilation and/or inhibition of platelet aggregation or other
disorders where prostacyclin has shown benefit, such as in treating
pulmonary hypertension. Accordingly, the present invention provides
methods for inducing prostacyclin-like effects in a subject
comprising administering a pharmaceutically effective amount of one
or more of the compounds described herein, such as those of
structure I above, preferably orally, to a patient in need of such
treatment. As an example, the vasodilating effects of the present
compounds can be used to treat pulmonary hypertension, which result
from various forms of connective tissue disease, such as lupus,
scleroderma or mixed connective tissue disease. These compounds are
thus useful for the treatment of pulmonary hypertension.
[0072] In another embodiment, the present invention also provides
methods of promoting prostacyclin-like effect in a subject by
administering a pharmaceutically effective amount of a compound of
structure II:
##STR00006##
wherein,
[0073] R.sup.1 is independently selected from the group consisting
of H, substituted and unsubstituted alkyl groups, arylalkyl groups
and groups wherein OR.sup.1 form a substituted or unsubstituted
glycolamide ester;
[0074] R.sup.2 and R.sup.3 may be the same or different and are
independently selected from the group consisting of H, phosphate
and groups wherein OR.sup.2 and OR.sup.3 form esters of amino acids
or proteins, with the proviso that all of R.sup.1, R.sup.2 and
R.sup.3 are not H;
[0075] an enantiomer of the compound; and
[0076] a pharmaceutically acceptable salt of the compound.
[0077] In groups wherein OR.sup.1 form a substituted or
unsubstituted glycolamide ester, R.sup.1 can be
--CH.sub.2CONR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5 may be the
same or different and are independently selected from the group
consisting of H, OH, substituted and unsubstituted alkyl groups,
--(CH.sub.2).sub.mCH.sub.3, --CH.sub.2OH, and
--CH.sub.2(CH.sub.2).sub.nOH, with the proviso that m is 0, 1, 2, 3
or 4, and n is 0, 1, 2, 3 or 4.
[0078] In other methods of inducing vasodilation or treating
hypertension, R.sup.1 can be a C.sub.1-C.sub.4 alkyl group, such as
methyl, ethyl, propyl or butyl. In other methods R.sup.1 is a
substituted or unsubstituted benzyl groups, such as
--CH.sub.2C.sub.6H.sub.5, --CH.sub.2C.sub.6H.sub.4NO.sub.2,
--CH.sub.2C.sub.6H.sub.4OCH.sub.3, --CH.sub.2C.sub.6H.sub.4Cl,
--CH.sub.2C.sub.6H.sub.4(NO.sub.2).sub.2, or
--CH.sub.2C.sub.6H.sub.4F. The benzyl group can be ortho, meta,
para, ortho/para substituted and combinations thereof. Suitable
substituents on the aromatic ring include halogens (fluorine,
chlorine, bromine, iodine), --NO.sub.2 groups, --OR.sup.16 groups
wherein R.sup.16 is H or a C.sub.1-C.sub.4 alkyl group, and
combinations thereof.
[0079] Alternatively, when R.sup.1 is --CH.sub.2CONR.sup.4R.sup.5
then R.sup.4 and R.sup.5 may be the same or different and are
independently selected from the group consisting of H, OH,
--CH.sub.3, and --CH.sub.2CH.sub.2OH. In these methods, where
R.sup.1 is not H, generally one or both of R.sup.2 and R.sup.3 are
H.
[0080] In some methods, one or both of R.sup.2 and R.sup.3 are H
and R.sup.1 is --CH.sub.3, --CH.sub.2C.sub.6H.sub.5. In other
methods where one or both of R.sup.2 and R.sup.3 are H, then
R.sup.1 is --CH.sub.2CONR.sup.4R.sup.5, and one or both of R.sup.4
and R.sup.5 are H, --OH, --CH.sub.3, --CH.sub.2CH.sub.2OH.
[0081] In methods where one or both of R.sup.2 and R.sup.3 are not
H, R.sup.2 and R.sup.3 can be independently selected from phosphate
and groups wherein OR.sup.2 and OR.sup.3 are esters of amino acids,
dipeptides, esters of tripeptides and esters of tetrapeptides. In
some embodiments, only one of R.sup.2 or R.sup.3 is a phosphate
group. In methods where at least one of R.sup.2 and R.sup.3 is not
H, generally R.sup.1 is H. In other methods, one of R.sup.2 or
R.sup.3 is H and the other R.sup.2 or R.sup.3 is as defined
elsewhere herein. In some methods, R.sup.2 is H and R.sup.3 is not
H. In additional embodiments, R.sup.1 and R.sup.3 are H and R.sup.2
is a group wherein OR.sup.2 is an ester of an amino acid or a
dipeptide. In further embodiments, R.sup.1 and R.sup.2 are H and
R.sup.3 is a group wherein OR.sup.3 is an ester of an amino acid or
a dipeptide.
[0082] In the methods, where one or both of the OR.sup.2 and
OR.sup.3 groups form esters of amino acids or peptides, i.e.,
dipeptides, tripeptides or tetrapeptides, these can be depicted
generically as --COCHR.sup.6NR.sup.7R.sup.8 wherein R.sup.6 is
selected from the group consisting of amino acid side chains,
R.sup.7 and R.sup.8 may be the same or different and are
independently selected from the group consisting of H, and
--COCHR.sup.9NR.sup.10R.sup.11. In the embodiments wherein the
amino acid is proline, R.sup.7 together with R.sup.6 forms a
pyrrolidine ring structure. R.sup.6 can be any of the naturally
occurring amino acid side chains, for example --CH.sub.3 (alanine),
--(CH.sub.2).sub.3NHCNH.sub.2NH (arginine), --CH.sub.2CONH.sub.2
(asparagine), --CH.sub.2COOH (aspartic acid), --CH.sub.2SH
(cysteine), --(CH.sub.2).sub.2CONH.sub.2 (glutamine),
--(CH.sub.2).sub.2COOH (glutamic acid), --H (glycine),
--CHCH.sub.3CH.sub.2CH.sub.3 (isoleucine),
--CH.sub.2CH(CH.sub.3).sub.2 (leucine), --(CH.sub.2).sub.4NH.sub.2
(lysine), --(CH.sub.2).sub.2SCH.sub.3 (methionine), --CH2Ph
(phenylalanine), --CH.sub.2OH (serine), --CHOHCH.sub.3 (threonine),
--CH(CH.sub.3).sub.2 (valine),
##STR00007##
--(CH.sub.2).sub.3NHCONH.sub.2 (citrulline) or
--(CH.sub.2).sub.3NH.sub.2 (ornithine). Ph designates a phenyl
group.
[0083] In the above methods, R.sup.7 and R.sup.8 may be the same or
different and are selected from the group consisting of H, and
--COCHR.sup.9NR.sup.10R.sup.11, wherein R.sup.9 is a side chain of
amino acid, R.sup.10 and R.sup.11 may be the same or different and
are selected from the group consisting of H, and
--COCHR.sup.12NR.sup.13R.sup.14, wherein R.sup.12 is an amino acid
side chain, R.sup.13 and R.sup.14 may be the same or different and
are independently selected from the group consisting of H, and
--COCHR.sup.15NH.sub.2. One skilled in the art will realize that
the peptide chains can be extended on the following scheme to the
desired length and include the desired amino acid residues.
[0084] In the embodiments where either or both of OR.sup.2 and
OR.sup.3 groups form an ester of a peptide, such as dipeptide,
tripeptide, tetrapeptide, etc. the peptides can be either
homopeptides, i.e., repeats of the same amino residue, or
heteropeptides, i.e., made up of different combinations of amino
acids.
[0085] As will be understood by the skilled artisan when only one
of R.sup.7 and R.sup.8 includes a peptide bond to further amino
acid, such as in the di, tri and tetrapeptides, the resulting
peptide chain will be linear. When both R.sup.7 and R.sup.8 include
a peptide bond, then the peptide can be branched.
[0086] In still other methods R.sup.1 is H and one of R.sup.2 or
R.sup.3 is a phosphate group or H while the other R.sup.2 or
R.sup.3 is a group such the OR.sup.2 or OR.sup.3 is an ester of an
amino acid, such as an ester of glycine or alanine.
[0087] In some methods, the administered compound can have an oral
bioavailability that is at least 25%, 50% 100%, 200%, 400% of the
oral bioavailability of treprostinil. It is generally preferred to
administer compounds that have higher absolute oral
bioavailabilities, such as 15%, 20%, 25%, 30% and 40%, 45%, 50%,
55%, 60% or more when administered orally.
[0088] Treprostinil has also been discovered to inhibit metastasis
of cancer cells as disclosed in U.S. patent application Ser. No.
10/006,197 filed Dec. 10, 2001 and Ser. No. 10/047,802 filed Jan.
16, 2002, both of which are hereby incorporated into this
application. Accordingly, the compounds described above, and in
particular those of structure I and II, can also be used in the
treatment of cancer and cancer related disorders, and as such the
present invention provides pharmaceutical compositions and methods
for treating cancer. Suitable formulations and methods of using the
present compounds can be achieved by substituting the compounds of
the present invention, such as those of structure I and II and in
particular prodrugs of treprostinil, for the active compounds
disclosed in U.S. patent application Ser. Nos. 10/006,197 and
10/047,802 filed Jan. 16, 2002.
[0089] Synthesis of the following compounds of structure I and
structure II can be achieved as follows:
Synthesis of Methyl Ester of Treprostinil (2) and Biphosphate Ester
of Treprostinil
##STR00008##
[0090] Synthesis of Methyl Ester of Treprostinil (2)
[0091] Methyl ester of treprostinil (2) was prepared by treating
1.087 g (2.8 mmoles) of treprostinil (1) with 50 ml of a saturated
solution of dry hydrochloric acid in methanol. After 24 hours at
room temperature, the methanol was evaporated to dryness and the
residue was taken in 200 ml dichloromethane. The dichloromethane
solution was washed with a 10% aqueous potassium carbonate
solution, and then with water to a neutral pH, it was dried over
sodium sulfate, filtered and the solvent was removed in vacuo
affording treprostinil methyl ester (2) in 98% yield as a yellow
oil. The crude methyl ester was used as such in subsequent
reactions.
Synthesis of Biphosphate Ester of Treprostinil (4)
[0092] The procedure was adapted after Steroids, 2(6),
567-603(1963). The methyl ester of treprostinil (2) (60 mg, 0.15
mmoles) was dissolved in 2 ml dry pyridine and a pyridinium
solution of the previously prepared pyridinium solution of
2-cyanoethylphosphate 1M (0.3 ml, 0.3 mmoles) (cf. Methods in
Enzymology, 1971, 18(c), 54-57) were concentrated to dryness in
vacuo at 40.degree. C. Anhydrous pyridine was added and the
reaction mixture was again concentrated; the operation was repeated
twice in order to remove water completely. Finally the residue was
dissolved in 2 ml anhydrous pyridine and 190 mg (0.9 mmoles)
dicyclohexylcarbodiimide were added as a solution in 2 ml anhydrous
pyridine. The reaction mixture in a closed flask was stirred
magnetically for 48 hours at room temperature. 1 ml water was added
and after one hour, the mixture was concentrated to a thick paste
in vacuo. The reaction mixture was treated overnight at room
temperature with 3 ml of a 1/9 water/methanol solution containing
35 mg sodium hydroxide. The white solid (dicyclohexylurea) formed
was removed by filtration and it was washed well with water. The
aqueous-methanolic solution was concentrated almost to dryness in
vacuo, water was added and the solution was extracted with
n-butanol (3.times.2 ml), then with methylene chloride (1.times.2
ml). The pH of the solution was adjusted to 9.0 by treatment with a
sulfonic acid ion exchange resin (H+ cycle-Dowex), treatment with
Dowex resin for a longer time (.about.12 hours) lead to both the
cleavage of the TBDMS group and the recovery of the free carboxyl
group. The resin was filtered and the solution was concentrated to
dryness affording the corresponding bisphosphate 4 (43 mg, yield
52%).
Synthesis of 3'-Monophosphate Ester of Treprostinil (8) and
2-Monophosphate Ester of Treprostinil (10)
##STR00009##
[0093] Synthesis of Monoprotected TBDMS Methyl Ester of
Treprostinil (5 and 6)
[0094] The procedure was adapted from Org. Synth., 1998, 75,
139-145. The treprostinil methyl ester (2) (305.8 mg, 0.75 mmoles)
was dissolved in 15 ml anhydrous dichloromethane and the solution
was cooled on an ice bath to 0.degree. C. Imidazole (102 mg, 1.5
mmoles) and tert-butyldimethyl silyl chloride (226.2 mg, 1.5
mmoles) were added and the mixture was maintained under stirring at
0.degree. C. for 30 minutes, then stirred overnight at room
temperature. Water (25 ml) was added and the organic layer was
separated. The aqueous layer was then extracted with
dichloromethane (3.times.50 ml). The organic layers were dried over
Na.sub.2SO.sub.4, the solution was filtered and the solvent was
removed in vacuo affording 447 mg crude reaction product. The crude
reaction product was separated by column chromatography (silica
gel, 35% ethyl acetate/hexanes) affording 140 mg bis-TBDMS
protected Treprostinil methyl ester, 160 mg 2-TBDMS protected
treprostinil methyl ester (6) and 60 mg 3'-TBDMS protected
Treprostinil methyl ester (5).
Synthesis of Monophosphate Ester of Treprostinil 8/10
[0095] The procedure was adapted after Steroids, 1963, 2(6),
567-603 and is the same for (8) and (10) starting from (6) and (5),
respectively. The TBDMS protected methyl ester of treprostinil (6)
(46 mg, 0.09 mmoles) was dissolved in 2 ml dry pyridine and a
pyridinium solution of the previously prepared pyridinium solution
of 2-cyanoethyiphosphate 1M (0.2 ml, 0.2 mmoles) (cf. Methods in
Enzymology, 1971, 18(c), 54-57) were concentrated to dryness in
vacuo at 40.degree. C. Anhydrous pyridine was added and the
reaction mixture was again concentrated; the operation was repeated
twice in order to remove water completely. Finally the residue was
dissolved in 2 ml anhydrous pyridine and 116 mg (0.56 mmoles)
dicyclohexylcarbodiimide were added as a solution in 2 ml anhydrous
pyridine. The reaction mixture in a closed flask was stirred
magnetically for 48 hours at room temperature in the dark. 5 ml
water were added and after one hour, the mixture was concentrated
to a thick paste in vacuo. The reaction mixture was treated
overnight at room temperature with 10 ml of a 1/9 water/methanol
solution containing 100 mg sodium hydroxide. The white solid
(dicyclohexylurea) formed was removed by filtration and it was
washed well with water. The aqueous-methanolic solution was
concentrated almost to dryness in vacuo, water was added and the
solution was extracted with n-butanol (3.times.10 ml), then with
methylene chloride (1.times.10 ml). The pH of the solution was
adjusted to 9.0 by treatment with a sulfonic acid ion exchange
resin (H+ cycle-Dowex); treatment with Dowex resin for a longer
time (-.about.12 hours) lead to both the cleavage of the TBDMS
group and the recovery of the free carboxyl group. The resin was
filtered and the solution was concentrated to dryness affording the
corresponding monophosphate 8 (33 mg, yield 68%).
Synthesis of Methyl Ester of Treprostinil (2)
##STR00010##
[0097] (2) (1 g; 2.56 mmol) was added to methanol (50 ml) prior
saturated with gaseous hydrochloric acid and the mixture swirled to
give a clear solution that was left to stand overnight at room
temperature. Solvent was removed in vacuo and the residue was
neutralized with a 20% potassium carbonate solution and extracted
in dichloromethane. The organic layer was washed with water, dried
over anhydrous magnesium sulfate and evaporated to yield the crude
product (0.96 g). Purification by preparative tlc (silica gel
plate; eluent: 7:3 (v/v) hexane-ethyl acetate) afforded 2 (0.803;
77.5%), colorless oil.
Synthesis of Treprostinil Diethanolamine (UT-15C)
[0098] Treprostinil acid is dissolved in a 1:1 molar ratio mixture
of ethanol:water and diethanolamine is added and dissolved. The
solution is heated and acetone is added as an antisolvent during
cooling.
Synthesis of Diglycil Ester of Treprostinil Methyl Ester (12)
##STR00011##
[0100] To a magnetically stirred solution of (2) methyl ester 2
(0.268 g; 0.66 mmol) in dichloromethane (30 ml)
N-carbobenzyloxyglycine p-nitrophenyl ester (0.766 g; 2.32 mmol)
and 4-(dimethyamino)pyridine (250 mg; 2.05 mmol) were successively
added. The resulted yellow solution was stirred at 20.degree. C.
for 24 hrs., then treated with 5% sodium hydroxide solution (20 ml)
and stirring continued for 15 mm. Dichloromethane (50 ml) was
added, layers separated and the organic phase washed with a 5%
sodium hydroxide solution (6.times.20 ml), water (30 ml), 10%
hydrochloric acid (2.times.40 ml), 5% sodium bicarbonate solution
(40 ml) and dried over anhydrous sodium sulfate. Removal of the
solvent afforded crude (11) (0.61 g), pale-yellow viscous oil.
Purification by flash column chromatography on silica gel eluting
with gradient 9/1 to 1/2 (v/v) hexane-ethyl ether afforded 0.445 g
(85.3%) of 11, white crystals, m.p. 70-72.degree. C. 'F1-NMR
[CDCl.sub.3; .delta.(ppm)]: 3.786 (s)(3H, COOCH.sub.3), 3.875
(d)(2H) and 3.940 (d)(2H)(NH--CH.sub.2--COO), 4.631 (s) (2H,
OCH.sub.2COOCH3), 4.789 (m)(1H, adjacent to OOC--CH.sub.2NHcbz) and
4.903 (m) (1H, adjacent to OOCCH.sub.2NHcbz), 5.09 (s)(4H,
C.sub.6H.sub.5CH.sub.2O), 5.378 (m)(1H) and 5.392 (m)(1H)(NH),
7.295-7.329 (m)(10H, C.sub.6H.sub.5). LR ESI-MS (m/z): 787.1
[M+H].sup.+, 804.1 [M+NH4].sup.+, 809.3 [M+Na].sup.+, 825.2
[M+K].sup.+, 1590.5 [2M+NH.sub.4]+, 1595.6 [2M+Na]+.
Methyl Ester, Diglycyl Ester (12)
[0101] A solution of ester (11) (0.4 g; 0.51 mmol) in methanol (30
ml) was introduced in the pressure bottle of a Parr hydrogenation
apparatus, 10% palladium on charcoal (0.2 g; 0.197 mmol Pd) was
added, apparatus closed, purged thrice with hydrogen and loaded
with hydrogen at 50 p.s.i. Stirring was started and hydrogenation
carried out for 5 hrs. at room temperature. Hydrogen was removed
from the installation by vacuum suction and replaced with argon.
The catalyst was filtered off through celite deposited on a fit and
the filtrate concentrated in vacuo to give 0.240 g (91%) of 4,
white solid m.p. 98-100.degree. C.
Synthesis of Benzyl Ester of Treprostinil (13)
##STR00012##
[0103] To a stirred solution of (2) (2 g; 5.12 mmol) in anhydrous
tetrahydrofuran (20 ml) benzyl bromide (0.95 ml; 7.98 mmol) and
freshly distilled triethylamine (1.6 ml; 11.48 mmol) were
consecutively added at room temperature and the obtained solution
was refluxed with stirring for 12 hrs. A white precipitate was
gradually formed. Solvent was distilled off in vacuo and the
residue treated with water (30 ml). Upon extraction with methylene
chloride emulsion formation occurs. The organic and aqueous layers
could be separated only after treatment with 5% hydrochloric acid
solution (20 ml). The organic layer was washed with water, dried on
anhydrous sodium sulfate, and evaporated, the residue was further
dried under reduced pressure over phosphorus pentoxide to give a
yellow viscous oil (2.32 g) that was purified by preparative thin
layer chromatography (silica gel plate; eluent: 1:2, v/v,
hexane/ethyl ether). Yield: 81.2%.
Synthesis of Bis-Glycyl Ester of Treprostinil (15)
##STR00013##
[0104] Benzy Ester, Di-cbzGly Ester (14)
[0105] To a magnetically stirred solution of benzyl ester 13 (1 g;
2.08 mmol) in dichloromethane (50 ml) N-carbobenzyloxyglycine
p-nitrophenyl ester (2.41 g; 7.28 mmol) and 4-(dimethyamino)
pyridine (788 mg; 6.45 mmol) were added. The resulted yellow
solution was stirred at 20.degree. C. for 21 hrs., then
successively washed with a 5% sodium hydroxide solution (6.times.45
ml), 10% hydrochloric acid (2.times.40 ml), 5% sodium bicarbonate
solution (40 ml) and dried over anhydrous sodium sulfate. Removal
of the solvent, followed by drying over phosphorus pentoxide under
reduced pressure, afforded crude 14 (2.61 g), pale-yellow oil.
Purification by flash column chromatography on silica gel eluting
with gradient 9:1 to 1:2 (v/v) hexane-ethyl ether gave (14_(1.51 g;
84.1%) as a colorless, very viscous oil.
Diglycyl Ester (15)
[0106] A solution of ester (14) (0.4 g; 0.46 mmol) in methanol (30
ml) was hydrogenated over 10% Pd/C as described for ester (12).
Work-up and drying over phosphorus pentoxide in vacuo yielded 0.170
g (72.7%) of ester 15, white solid m.p. 155-158.degree. C.
Synthesis of 3'-Glycyl Ester of Treprostinil 19
##STR00014##
[0107] Benzyl Ester, t-Butyldimethysilyl Monoester (16)
[0108] A solution of tert-butyldimethylsilyl chloride (0.45 g; 2.98
mmol) in dichloromethane (8 ml) was added dropwise over 10 min., at
room temperature, into a stirred solution of benzyl ester 13 (0.83
g; 1.73 mmol) and imidazole (0.33 g; 4.85 mmol) in dichloromethane
(20 ml). Stirring was continued overnight then water (20 ml) was
added, the mixture stirred for one hour, layers separated, organic
layer dried over anhydrous sodium sulfate and concentrated in vacuo
to give a slightly yellow oil (1.15 g). The crude product is a
mixture of the mono-TBDMS (16) and di-TBDMS esters (.sup.1H-NMR).
Column chromatography on silica gel, eluting with a 9:1 (v/v)
hexane-ethyl acetate mixture, readily afforded the di-ester (0.618
g) in a first fraction, and ester 16 (0.353 g; yield relative to
13:34.4%) in subsequent fractions. Analytical tlc on silica gel of
the ester 16 showed only one spot (eluent: 3:2 (v/v) hexane-ethyl
ether). Consequently, under the above reaction conditions, the
other possible isomer (mono-TBDMS ester at the side-chain hydroxyl)
was not observed.
[0109] Another experiment in which the molar ratio
tert-butyldimethylsilyl chloride:ester 13 was lowered to 1.49
(followed by flash column chromatography of the product on silica
gel, eluting with gradient 9.5/0.5 to 3/1 (v/v) hexane-ethyl ether)
lead to a decreased content (36.5%, as pure isolated material) of
the undesired di-OTBDMS by-product. The mono-OTBDMS ester fractions
(45.1%; isolated material) consisted of ester 16 (98%) and its
side-chain isomer (2%) that could be distinctly separated; the
latter was evidenced (tlc, NMR) only in the last of the monoester
fractions.
Benzyl Ester, Cbz-Glycyl Monoester (18)
[0110] To a magnetically stirred solution of ester 16 (0.340 g;
0.57 mmol) in dichloromethane (15 ml) N-carbobenzyloxyglycine
p-nitrophenyl ester (0.445 g; 1.35 mmol) and 4-(dimethyamino)
pyridine (150 mg; 1.23 mmol) were successively added. The solution
was stirred at 20.degree. C. for 40 hrs. Work-up as described for
esters 11 and 14 yielded a crude product (0.63 g) containing 90% 17
and 10% 18 (.sup.1H-NMR). To completely remove the protective TBDMS
group, this mixture was dissolved in ethanol (30 ml) and subjected
to acid hydrolysis (5% HCl, 7 ml) by stirring overnight at room
temperature. Solvent was then removed under reduced pressure and
the residue extracted in dichloromethane (3.times.50 ml); the
organic layer was separated, washed once with water (50 ml), dried
over sodium sulfate and concentrated in vacuo to give crude ester
18 (0.51 g). Purification by flash column chromatography as for
esters 11 and 14 afforded ester 18 (0.150 g; overall yield: 39.1%)
as a colorless, viscous oil.
Glycyl Monoester (19)
[0111] A solution of ester 18 (0.15 g; 0.22 mmol) in methanol (30
ml) was hydrogenated over 10% Pd/C as described for ester 12 and
15. Work-up and drying over phosphorus pentoxide in vacuo yielded
ester 10 (0.98 g; 98.0%), white, shiny crystals m.p. 74-76.degree.
C. LR ESI-MS (m/z): 448.2 [M+H].sup.+, 446.4 [M-H].sup.-.
Synthesis of 3'-L-Leucyl Ester of Treprostinil 22
##STR00015##
[0112] Benzyl Ester, t-Butyldimethysilyl Monoester, Cbz-L-Leucyl
Monoester (20)
[0113] To a stirred solution of ester 16 (0.38 g: 0.64 mmol) and
N-carbobenzyloxy-L-leucine N-hydroxysuccinimide ester (0.37 g; 1.02
mmol) in 10 ml dichloromethane 4-(dimethyamino)pyridine (0.17 g;
1.39 mmol) was added, then stirring continued at room temperature
for 2 days. The solvent was removed in vacuo and the crude product
(0.9 g) subjected to flash column chromatography on silica gel
eluting with 9:1 hexane-ethyl acetate; the firstly collected
fraction yielded an oil (0.51 g) which, based on the its NMR
spectrum and tlc, was proved to be a 2:1 mixture of ester 20 and
the starting ester 16. Preparative tlc on silica gel (eluent: ethyl
acetate-hexane 1:4) gave pure 20, colorless oil (overall yield
based on 7:62.6%).
Benzyl Ester, Cbz-L-Leucyl Monoester (21)
[0114] De-protection of the cyclopentenyl hydroxyl in the
t-butyldimethysilyl monoester 20 succeeded by treatment with
diluted hydrochloric acid solution as described for 18, with the
exception that a 1:5 (v/v) chloroform-ethanol mixture, instead of
ethanol alone, was used to ensure homogeneity. Work-up afforded 20,
colorless oil, in 87.6% yield.
L-Leucyl Monoester (22)
[0115] Hydrogenolysis of the benzyl and N-carbobenzyloxy groups in
21 was carried out as for 18. Work-up afforded 22 (95.3%), white
solid, m.p. 118-120.degree. C.
Synthesis of 2-L-Leucyl Ester of Treprostinil 25
##STR00016##
[0116] Benzyl Ester, Cbz-L-Ieucyl Monoesters (21, 23) and -Diester
(24)
[0117] To a stirred solution of ester 13 (0.53 g: 1.10 mmol) and
N-carbobenzyloxy-L-leucine N-hydroxysuccinimide ester (0.76 g; 2.05
mmol) in dichloromethane (30 ml) 4-(dimethyamino) pyridine (0.29 g;
2.37 mmol) was added, then stirring continued at room temperature
for 1 day. The solution was diluted with dichioromethane (40 mnl),
successively washed with a 5% sodium hydroxide solution (4.times.25
ml), 10% hydrochloric acid (2.times.30 ml), 5% sodium bicarbonate
solution (50 ml), dried over anhydrous sodium sulfate and
concentrated under reduced pressure to give the crude product (0.85
g), as a viscous, yellow oil. Thin layer chromatography revealed a
complex mixture in which esters 13 and 21 as well as cbz-L-leucine
could be identified through the corresponding r.sub.F values, only
as minor products. The crude product was flash-chromatographed
through a silica gel column eluting with gradient hexane-ethyl
ether. At 7:3 (v/v) hexane-ethyl ether, the first fraction gave the
cbz-L-leucyl diester 24 (6% of the product subjected to
chromatography) while the two subsequent fractions afforded the
cbz-L-leucyl monoester 23 (54% of the crude product, as pure
isolated 23; 57.6% yield, relative to 2). Purity of both compounds
was verified by analytical tlc and NMR. The other isomer,
cbz-L-leucyl monoester 21 constituted only about 5% of the crude
product and was isolated by preparative tlc of the latter only a
3.1 23/21 mixture
##STR00017##
L-Leucyl Monoester (25)
[0118] Hydrogenolysis of 23 to the ester 25 was performed as
described for compound 12 but reaction was carried out at 35
p.s.i., overnight. Work-up and drying over phosphorus pentoxide in
vacuo afforded 25, white solid m. p. 153-155.degree. C., in
quantitative yield.
Synthesis of 3'-L-Alanyl Ester of Treprostinil 30
##STR00018##
[0119] N-Cbz-L-Alanyl p-Nitro Phenyl Ester (27)
[0120] To a stirred solution containing N-carbobenzyloxy-L-alanine
(1 g; 4.48 mmol) and p-nitrophenol (1 g; 7.19 mmol) in anhydrous
tetrahydrofuran (7 ml) a fine suspension of
1,3-dicyclohexylcarbodiimide (1.11 g; 5.38 mmol) in tetrahydrofuran
(5 ml) was added over 30 min. Stirring was continued at room
temperature for 18 hrs., glacial acetic acid (0.3 ml) added,
1,3-dicyclohexylurea filtered off and solvent removed in vacuo, at
40.degree. C., to give a viscous, yellow-reddish oil (2.5 g). The
.sup.1H-NMR spectrum showed a mixture consisting of
N-carbobenzyloxy-Lalanine p-nitrophenyl ester (27), unreacted
p-nitrophenol and a small amount of DCU, which was used as such in
the next reaction step.
##STR00019##
Benzyl Ester, Cbz-L-Alanyl Monoester (29)
[0121] A solution of 4-(dimethylamino)pyridine (0.30 g; 2.49 mmol)
in dichloromethane (3 ml) was quickly dropped (over 5 min.) into a
magnetically stirred solution of ester 16 (0.37 g; 0.62 mmol) and
crude N-carbobenzyloxy-L-alanine p-nitrophenyl ester (0.98 g) in
dichloromethane (12 ml). The mixture was stirred overnight at room
temperature, then diluted with dichloromethanc (50 ml), and
thoroughly washed with a 5% sodium hydroxide solution (7.times.35
ml), 10% hydrochloric acid (3.times.35 ml), 5.degree./a sodium
bicarbonate solution (50 ml), dried over anhydrous sodium sulfate
and concentrated under reduced pressure to give the crude ester 28
(1.1 g). The latter was dissolved in ethanol (30 ml), 5%
hydrochloric acid (8 ml) and chloroform (5 ml) were added and the
solution stirred overnight. Solvents were removed in vacuo, the
residue taken-up in dichloromethane, washed to pH 7 with a 5%
sodium hydrogencarbonate solution, dried over anhydrous sodium
sulfate and the solvent evaporated affording crude 29 (1.04 g).
Purification by column chromatography on silica gel, eluting with
gradient hexane-ethyl ether, enabled separation of a fraction (at
hexane:ethyl ether=1:1 v/v) of pure 29 as a colorless very viscous
oil (0.11 g; 25.8% overall yield, based on 16).
L-Alanyl Monoester (30)
[0122] Removal of the benzyl and N-carbobenzyloxy groups in 29 was
achieved through catalytic hydrogenation as described for 12. Ester
30 was obtained (yield: 97.2%) as a pale-yellow, partially
crystallized, oil.
Synthesis of the 3'-L-Valine Ester of Treprostinil Benzyl Ester
33
##STR00020##
[0123] Synthesis of the Benzyl Ester of Treprostinil 13
[0124] The benzyl ester 11 was synthesized by adapting the method
described by J. C. Lee et al. in Organic Prep. and Proc. Intl.,
1996, 28(4), 480-483. To a solution of 1 (620 mg, 1.6 mmoles) and
cesium carbonate (782.4 mg, 2.4 mmoles) in acetonitrile (30 ml) was
added benzyl bromide (0.48 ml, 4 mmoles) and the mixture was
stirred at reflux for 1 hour. After cooling at room temperature,
the precipitate was filtered off and the filtrate was concentrated
in vacuo. The residue was dissolved in chloroform (150 ml) and
washed with a 2% aqueous solution of NaHCO.sub.3 (3.times.30 ml).
The organic layer was washed with brine, dried on Na.sub.2SO.sub.4,
filtered and the solvent was removed in vacuo to afford 750 mg of
the crude benzyl ester 13 (yield 98%) as a yellow viscous oil. The
crude benzyl ester 13 can be purified by column chromatography
(100-0% dichioromethane(methanol) but it can also be used crude in
subsequent reactions.
Synthesis of the TBDMS Protected Treprostinil Benzyl Ester 16
[0125] The procedure for the synthesis of the TBDMS protected
benzyl ester was adapted from Organic Synth., 1998, 75, 139-145.
The benzyl ester 13 (679 mg, 1.4 mmoles) was dissolved in anhydrous
dichloromethanc (20 ml) and the solution was cooled to 0.degree. C.
on an ice bath. Imidazole (192 mg, 2.8 mmoles) and
t-butyldimethylsilyl chloride (TBDMSCl) (420 mg, 2.8 mmoles) were
added and the mixture was maintained under stirring for another
half hour on the ice bath and then it was left overnight at room
temperature. 40 ml water was added to the reaction mixture and the
organic layer was separated. The aqueous layer was extracted with
3.times.50 ml dichloromethane. The combined organic layers were
dried over Na.sub.2SO.sub.4, filtered and the solvent was removed
in vacuo. This afforded 795 mg of material which proved to be a
mixture of the desired mono TBDMS protected 5 benzyl ester with the
bis-TBDMS protected benzyl ester. Pure 16 (249 mg) was obtained by
column chromatography on silica gel (eluent 35% ethyl
acetate/hexane).
Synthesis of N-Cbz-L-Valine Ester of the TBDMS Protected
Treprostinil Benzyl Ester 31
[0126] The procedure used was adapted from Tetrahedron Lett., 1978,
46, 4475-4478. A solution of NCbz-L-valine (127 mg, 0.5 mmoles),
N,N-dicyclohexylcarbodiimide (DCC) (111 mg, 0.5 mmoles), compound
16 (249 mg, 0.4 mmoles) and 4-(dimethylamino)pyridine (DMAP) (6 mg,
0.05 mmoles) in anhydrous dichloromethane (15 ml) was stirred at
room temperature until esterification was complete. The solution
was filtered and the formed N,N-dicyclohexylurea was filtered. The
filtrate was diluted with dichloromethane (80 ml) and washed with
water (3.times.30 ml), a 5% aqueous acetic acid solution
(2.times.30 ml) and then again with water (3.times.30 ml). The
organic layer was dried over Na.sub.2SO.sub.4 and the solvent was
evaporated in vacuo affording 369 mg crude 31. Pure 31 was obtained
by chromatography (silica gel, 35% ethyl acetate/hexane).
Synthesis of the 3'-N-Cbz-L-Valine Ester of Treprostinil Benzyl
Ester 32
[0127] Cleavage of the TBDMS group in compound 31 was achieved
using an adaptation of the procedure described in Org. Letters,
2000, 2(26), 4177-4180. The N-Cbz-L-valine ester of the TBDMS
protected benzyl ester 31 (33 mg, 0.04 mmoles) was dissolved in
methanol (5 ml) and tetrabutylammonium tribromide (TBATB) (2 mg,
0.004 mmoles) was added. The reaction mixture was stirred at room
temperature for 24 hrs until the TBDMS deprotection was complete.
The methanol was evaporated and the residue was taken in
dichloromethane. The dichloromethane solution was washed with brine
and then dried over Na.sub.2SO.sub.4. After filtering the drying
agent the solvent was evaporated to dryness affording 30.2 mg of
crude compound 32.
Synthesis of the 3'-L-Valine Ester of Treprostinil 33
[0128] The benzyl and benzyl carboxy groups were removed by
catalytic hydrogenation at atmospheric pressure in the presence of
palladium 10% wt on activated carbon. The 3'-N-Cbz-L-valine ester
of benzyl ester 32 (30.2 mg, 0.04 mmoles) was dissolved in methanol
(10 ml) and a catalytic amount of Pd/C was added. Under magnetic
stirring the air was removed from the flask and then hydrogen was
admitted. The reaction mixture was maintained under hydrogen and
stirring at room temperature for 24 hrs, then the hydrogen was
removed with vacuum. The reaction mixture was then filtered through
a layer of celite and the solvent was removed in vacuo to afford
the pure 3'-L-valine ester of Treprostinil 33 (15 mg, 0.03
mmoles).
Synthesis of 2-L-Valine Ester of Treprostinil 36/Bis-L-Valine Ester
of Trenrostinil 37
Synthesis of 2-L-Alanine Ester of Treprostinil 36'/Bis-L-Alanine
Ester of Treprostinil 37'
##STR00021##
[0129] Synthesis of 2-N-Cbz-L-Valine Ester of Treprostinil Benzyl
Ester 34 and Bis-N-Cbz-L-Valine Ester of Treprostinil Benzyl Ester
35
[0130] The procedure used was adapted from Tetrahedron Lett., 1978,
46, 4475-4478. A solution of NCbz-L-valine (186 mg, 0.7 mmoles),
N,N-dicyclohexylcarbodiimide (DCC) (167 mg, 0.8 mmoles), compound
13 (367 mg, 0.8 mmoles) and 4-(dimethylamino)pyridine (DMAP) (12
mg, 0.09 mmoles) in anhydrous dichloromethane (15 ml) was stirred
at room temperature until esterification was complete. The solution
was filtered and the formed N,N-dicyclohexylurea was filtered. The
filtrate was diluted with dichloromethane (100 ml) and washed with
water (3.times.50 ml), a 5% aqueous acetic acid solution
(2.times.50 ml) and then again with water (3.times.50 ml). The
organic layer was dried over Na.sub.2SO.sub.4 and the solvent was
evaporated in vacuo affording 556 mg crude product. The product was
separated by chromatography (silica gel, 35% ethyl acetate/hexane)
yielding 369.4 mg 2-valine ester 34 and 98 mg bis-valine ester
35.
Synthesis of 2 N-Cbz-L-Alanine Ester of Treprostinil Benzyl Ester
34' and Bis-N-Cbz-L-Alanine Ester of Treprostinil Benzyl Ester
35'
[0131] The procedure used was adapted from Tetrahedron Lett., 1978,
46, 4475-4478. A solution of NCbz-L-alanine (187 mg, 0.84 mmoles),
N,N-dicyclohexylcarbodiimide (DCC) (175 mg, 0.85 mmoles), compound
13 (401 mg, 0.84 mmoles) and 4-(dimethylamino)pyridine (UMAP) (11.8
mg, 0.1 mmoles) in anhydrous dichloromethane (15 ml) was stirred at
room temperature until esterification was complete. The solution
was filtered and the formed N,N-dicyclohexylurea was filtered. The
filtrate was diluted with dichloromethane (100 ml) and washed with
water (3.times.50 ml), a 5% aqueous acetic acid solution
(2.times.50 ml) and then again with water (3.times.50 ml). The
organic layer was dried over Na.sub.2SO.sub.4 and the solvent was
evaporated in vacuo affording 516 mg crude product. The product was
separated by chromatography (silica gel, 35% ethyl acetate/hexane)
yielding 93.4 mg 2-alanine ester 34' and 227 mg bis-alanine ester
35'.
Synthesis of 2-L-Valine Ester of Treprostinil 36/Bis-L-Valine Ester
of Treprostinil 37
[0132] The benzyl and benzyl carboxy groups were removed by
catalytic hydrogenation at atmospheric pressure in the presence of
palladium 10% wt on activated carbon. The 2-N-Cbz-L-valine ester of
Treprostinil benzyl ester 34 (58.2 mg, 0.08
mmoles)/bis-N-Cbz-L-valine ester of Treprostinil benzyl ester 35
(55.1 mg, 0.06 mmoles) was dissolved in methanol (10 ml) and a
catalytic amount of Pd/C was added. Under magnetic stirring the air
was removed from the flask and hydrogen was admitted. The reaction
mixture was maintained under hydrogen and stirring at room
temperature for 20 hrs, then hydrogen was removed with vacuum. The
reaction mixture was then filtered through a layer of celite and
the solvent was removed in vacuo to afford the pure 2-L-valine
ester of Treprostinil 36 (40 mg, 0.078 mmoles)/bis-L-valine ester
of Treprostinil 37 (23 mg, 0.04 mmoles).
Synthesis of 2-L-Alanine Ester of Treprostinil 36'/Bis-L-Alanine
Ester of Treprostinil 37'
[0133] The benzyl and benzyl carboxy groups were removed by
catalytic hydrogenation at atmospheric pressure in the presence of
palladium 10% wt on activated carbon. The 2-N-Cbz-L-alanine ester
of Treprostinil benzyl ester 34' (87.4 mg, 0.13
mmoles)/bis-N-Cbz-L-alanine ester of Treprostinil benzyl ester 35'
(135 mg, 0.15 mmoles) was dissolved in methanol (15 ml) and a
catalytic amount of Pd/C was added. Under magnetic stirring the air
was removed from the flask and hydrogen was admitted. The reaction
mixture was maintained under hydrogen and stirring at room
temperature for 20 hrs, then hydrogen was removed with vacuum. The
reaction mixture was then filtered through a layer of celite and
the solvent was removed in vacuo to afford the pure 2-L-valine
ester of Treprostinil 36' (57 mg, 0.12 mmoles)/bis-L-alanine ester
of Treprostinil 37' (82 mg, 0.15 mmoles).
Synthesis of Benzyl Esters of Treprostinil 38 a-e
##STR00022##
[0134] a 4-NO.sub.2C.sub.6H.sub.4CH.sub.2; b
4-(CH.sub.3O)C.sub.6H.sub.4CH.sub.2; c 2-ClC.sub.6H.sub.4CH.sub.2;
d 2,4-(NO.sub.2).sub.2C.sub.6H.sub.3CH.sub.2; e
4-FC.sub.6H.sub.4CH.sub.2 Synthesis of the benzyl esters of
treprostinil 38 a-e was performed using the procedure for the
benzyl ester 13.
[0135] Enantiomers of these compounds, shown below, can be
synthesized using reagents and synthons of enantiomeric chirality
of the above reagents.
##STR00023##
[0136] (-)-treprostinil can be synthesized as follows:
##STR00024##
[0137] (a) (S)-2-methyl-CBS-oxazaborolidine, BH.sub.3.SMe.sub.2,
THF, -30.degree. C., 85%. (b) TBDMSCl, imidazole, CH.sub.2Cl.sub.2,
95%. (c) Co.sub.2(CO).sub.8, CH.sub.2Cl.sub.2, 2 hr. r.t., then
CH.sub.3CN, 2 hr. reflux. 98%. (d) K.sub.2CO.sub.3, Pd/C (10%),
EtOH, 50 psi/24 hr. 78% (e) NaOH, EtOH, NaBH.sub.4. 95%. (f) BnBr,
NaH, THF, 98%. (g). CH.sub.3OH, TsOH. 96%. (h) i. p-nitrobenzoic
acid, DEAD, TPP, benzene. (i) CH.sub.3OH, KOH. 94%. (j) Pd/C (10%),
EtOH, 50 psi/2 hr. quant. (k). Ph.sub.2PLi, THF. (1) i.
ClCH.sub.2CN, K.sub.2CO.sub.3. ii, KOH, CH.sub.3OH, reflux. 83% (2
steps).
[0138] Briefly, the enantiomer of the commercial drug
(+)-Treprostinil was synthesized using the stereoselective
intramolecular Pauson Khand reaction as a key step and Mitsunobu
inversion of the side-chain hydroxyl group. The absolute
configuration of (-)-Treprostinil was confirmed by an X-ray
structure of the L-valine amide derivative.
[0139] The following procedure was used to make
(-)-treprostinil-methyl-L-valine amide: To a stirred solution of
(-)-Treprostinil (391 mg, 1 mmol) and L-valine methyl ester
hydrochloride (184 mg, 1.1 mmol) in DMF (10 ml) under Ar was
sequentially added pyBOP reagent (1.04 g, 2 mmol), diisopropylethyl
amine (0.52 ml, 3 mmol). The reaction mixture was stirred at room
temperature overnight (15 hrs). Removal of the solvent in vacuo and
purification by chromatography yielded white solid 12 (481 mg,
86%), which was recrystallized (10% ethyl acetate in hexane) to
give suitable crystals for X-ray.
[0140] Various modifications of these synthetic schemes capable of
producing additional compounds discussed herein will be readily
apparent to one skilled in the art.
[0141] There are two major barriers to deliver treprostinil in the
circulatory system. One of these barriers is that treprostinil
undergoes a large first pass effect. Upon first circulating through
the liver, about 60% of treprostinil plasma levels are metabolized,
which leaves only about 40% of the absorbed dose. Also, a major
barrier to oral delivery for treprostinil is that the compound is
susceptible to an efflux mechanism in the gastrointestinal tract.
The permeability of treprostinil has been measured across Caco-2
cell monolayers. The apical to basal transport rate was measured to
be 1.39.times.10.sup.6 cm/sec, which is indicative of a highly
permeable compound. However, the basal to apical transport rate was
12.3.times.10.sup.6 cm/sec, which suggests that treprostinil is
efficiently effluxed from the serosal to lumenal side of the
epithelial cell. These data suggest that treprostinil is
susceptible to p-glycoprotein, a membrane bound multidrug
transporter. It is believed that the p-glycoprotein efflux pump
prevents certain pharmaceutical compounds from traversing the
mucosal cells of the small intestine and, therefore, from being
absorbed into systemic circulation.
[0142] Accordingly, the present invention provides pharmaceutical
compositions comprising treprostinil, the compound of structure I
or the compound of structure II, or their pharmaceutically
acceptable salts and combinations thereof in combination with one
or more inhibitors of p-glycoprotein. A number of known
non-cytotoxic pharmacological agents have been shown to inhibit
p-glycoprotein are disclosed in U.S. Pat. Nos. 6,451,815,
6,469,022, and 6,171,786.
[0143] P-glycoprotein inhibitors include water soluble forms of
vitamin E, polyethylene glycol, poloxamers including Pluronic F-68,
polyethylene oxide, polyoxyethylene castor oil derivatives
including Cremophor EL and Cremophor RH 40, Chrysin, (+)-Taxifolin,
Naringenin, Diosmin, Quercetin, cyclosporin A (also known as
cyclosporine), verapamil, tamoxifen, quinidine, phenothiazines, and
9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4-tetrahydro-6,7,-dimethoxy-2-
-isoquinolinyl)ethyl]phenyl]-4-acridinecarboxamide or a salt
thereof.
[0144] Polyethylene glycols (PEGs) are liquid and solid polymers of
the general formula H(OCH.sub.2CH.sub.2).sub.nOH, where n is
greater than or equal to 4, having various average molecular
weights ranging from about 200 to about 20,000. PEGs are also known
as alpha-hydro-omega-hydroxypoly-(oxy-1,2-ethanediyl)polyethylene
glycols. For example, PEG 200 is a polyethylene glycol wherein the
average value of n is 4 and the average molecular weight is from
about 190 to about 210. PEG 400 is a polyethylene glycol wherein
the average value of n is between 8.2 and 9.1 and the average
molecular weight is from about 380 to about 420. Likewise, PEG 600,
PEG 1500 and PEG 4000 have average values of n of 12.5-13.9, 29-36
and 68-84, respectively, and average molecular weights of 570-630,
1300-1600 and 3000-3700, respectively, and PEG 1000, PEG 6000 and
PEG 8000 have average molecular weights of 950-1050, 5400-6600, and
7000-9000, respectively. Polyethylene glycols of varying average
molecular weight of from 200 to 20000 are well known and
appreciated in the art of pharmaceutical science and are readily
available.
[0145] The preferred polyethylene glycols for use in the instant
invention are polyethylene glycols having an average molecular
weight of from about 200 to about 20,000. The more preferred
polyethylene glycols have an average molecular weight of from about
200 to about 8000. More specifically, the more preferred
polyethylene glycols for use in the present invention are PEG 200,
PEG 400, PEG 600, PEG 1000, PEG 1450, PEG 1500, PEG 4000, PEG 4600,
and PEG 8000. The most preferred polyethylene glycols for use in
the instant invention is PEG 400, PEG 1000, PEG 1450, PEG 4600 and
PEG 8000.
[0146] Polysorbate 80 is an oleate ester of sorbitol and its
anhydrides copolymerized with approximately 20 moles of ethylene
oxide for each mole of sorbitol and sorbitol anhydrides.
Polysorbate 80 is made up of sorbitan mono-9-octadecanoate
poly(oxy-1,2-ethandiyl) derivatives. Polysorbate 80, also known as
Tween 80, is well known and appreciated in the pharmaceutical arts
and is readily available.
[0147] Water-soluble vitamin E, also known as d-alpha-tocopheryl
polyethylene glycol 1000 succinate [TPGS], is a water-soluble
derivative of natural-source vitamin E. TPGS may be prepared by the
esterification of the acid group of crystalline d-alpha-tocopheryl
acid succinate by polyethylene glycol 1000. This product is well
known and appreciated in the pharmaceutical arts and is readily
available. For example, a water-soluble vitamin E product is
available commercially from Eastman Corporation as Vitamin E
TPGS.
[0148] Naringenin is the bioflavonoid compound
2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one
and is also known as 4',5,7-trihydroxyflavanone. Naringenin is the
aglucon of naringen which is a natural product found in the fruit
and rind of grapefruit. Naringenin is readily available to the
public from commercial sources.
[0149] Quercetin is the bioflavonoid compound
2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one and
is also known as 3,3',4',5,7-pentahydroxyflavone. Quercetin is the
aglucon of quercitrin, of rutin and of other glycosides. Quercetin
is readily available to the public from commercial sources.
[0150] Diosmin is the naturally occurring flavonic glycoside
compound
7-[[6-O-6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranosyl]oxy]-5-hydr-
oxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one. Diosmin
can be isolated from various plant sources including citrus fruits.
Diosmin is readily available to the public from commercial
sources.
[0151] Chrysin is the naturally occurring compound
5,7-dihydroxy-2-phenyl-4H-1-benzopyran-4-one which can be isolated
from various plant sources. Chrysin is readily available to the
public from commercial sources.
[0152] Poloxamers are
alpha-hydro-omega-hydroxypoly(oxyethylene)poly
(oxypropylene)poly(oxyethylene) block copolymers. Poloxamers are a
series of closely related block copolymers of ethylene oxide and
propylene oxide conforming to the general formula
HO(C.sub.2H.sub.4O).sub.a(C.sub.3H.sub.6O).sub.b(C.sub.2H.sub.4O).sub.aH.
For example, poloxamer 124 is a liquid with "a" being 12, "b" being
20, and having an average molecular weight of from about 2090 to
about 2360; poloxamer 188 is a solid with "a" being 80, "b" being
27, and having an average molecular weight of from about 7680 to
about 9510; poloxamer 237 is a solid with "a" being 64, "b" being
37, and having an average molecular weight of from about 6840 to
about 8830; poloxamer 338 is a solid with "a" being 141, "b" being
44, and having an average molecular weight of from about 12700 to
about 17400; and poloxamer 407 is a solid with "a" being 101, "b"
being 56, and having an average molecular weight of from about 9840
to about 14600. Poloxamers are well known and appreciated in the
pharmaceutical arts and are readily available commercially. For
example, Pluronic F-68 is a commercially available poloxamer from
BASF Corp. The preferred poloxamers for use in the present
invention are those such as poloxamer 188, Pluronic F-68, and the
like.
[0153] Polyoxyethylene castor oil derivatives are a series of
materials obtained by reacting varying amounts of ethylene oxide
with either castor oil or hydrogenated castor oil. These
polyoxyethylene castor oil derivatives are well known and
appreciated in the pharmaceutical arts and several different types
of material are commercially available, including the Cremophors
available from BASF Corporation. Polyoxyethylene castor oil
derivatives are complex mixtures of various hydrophobic and
hydrophilic components. For example, in polyoxyl 35 castor oil
(also known as Cremophor EL), the hydrophobic constituents comprise
about 83% of the total mixture, the main component being glycerol
polyethylene glycol ricinoleate. Other hydrophobic constituents
include fatty acid esters of polyethylene glycol along with some
unchanged castor oil. The hydrophilic part of polyoxyl 35 castor
oil (17%) consists of polyethylene glycols and glyceryl
ethoxylates.
[0154] In polyoxyl 40 hydrogenated castor oil (Cremophor RH 40)
approximately 75% of the components of the mixture are hydrophobic.
These comprise mainly fatty acid esters of glycerol polyethylene
glycol and fatty acid esters of polyethylene glycol. The
hydrophilic portion consists of polyethylene glycols and glycerol
ethoxylates. The preferred polyoxyethylene castor oil derivatives
for use in the present invention are polyoxyl 35 castor oil, such
as Cremophor EL, and polyoxyl 40 hydrogenated castor oil, such as
Cremophor RH 40. Cremophor EL and Cremophor RH 40 are commercially
available from BASF Corporation.
[0155] Polyethylene oxide is a nonionic homopolymer of ethylene
oxide conforming to the general formula (OCH.sub.2CH.sub.2).sub.n
in which n represents the average number of oxyethylene groups.
Polyethylene oxides are available in various grades which are well
known and appreciated by those in the pharmaceutical arts and
several different types of material are commercially available. The
preferred grade of polyethylene oxide is NF and the like which are
commercially available.
[0156] (+)-Taxifolin is
(2R-trans)-2-(3,4-dihydroxyphenyl)-2,3-dihydro-3,5,7-trihydroxy-4H-1-benz-
o pyran-4-one. Other common names for (+)-taxifolin are
(+)-dihydroquercetin; 3,3',4',5,7-pentahydroxy-flavanone;
diquertin; taxifoliol; and distylin. (+)-Taxifolin is well know and
appreciated in the art of pharmaceutical arts and is readily
available commercially.
[0157] The preferred p-glycoprotein inhibitor for use in the
present invention are water soluble vitamin E, such as vitamin E
TPGS, and the polyethylene glycols. Of the polyethylene glycols,
the most preferred p-glycoprotein inhibitors are PEG 400, PEG 1000,
PEG 1450, PEG 4600 and PEG 8000.
[0158] Administration of a p-glycoprotein inhibitor may be by any
route by which the p-glycoprotein inhibitor will be bioavailable in
effective amounts including oral and parenteral routes. Although
oral administration is preferred, the p-glycoprotein inhibitors may
also be administered intravenously, topically, subcutaneously,
intranasally, rectally, intramuscularly, or by other parenteral
routes. When administered orally, the p-glycoprotein inhibitor may
be administered in any convenient dosage form including, for
example, capsule, tablet, liquid, suspension, and the like.
[0159] Generally, an effective p-glycoprotein inhibiting amount of
a p-glycoprotein inhibitor is that amount which is effective in
providing inhibition of the activity of the p-glycoprotein mediated
active transport system present in the gut. An effective
p-glycoprotein inhibiting amount can vary between about 5 mg to
about 1000 mg of p-glycoprotein inhibitor as a daily dose depending
upon the particular p-glycoprotein inhibitor selected, the species
of patient to be treated, the dosage regimen, and other factors
which are all well within the abilities of one of ordinary skill in
the medical arts to evaluate and assess. A preferred amount however
will typically be from about 50 mg to about 500 mg, and a more
preferred amount will typically be from about 100 mg to about 500
mg. The above amounts of a p-glycoprotein inhibitor can be
administered from once to multiple times per day. Typically for
oral dosing, doses will be administered on a regimen requiring one,
two or three doses per day.
[0160] Where water soluble vitamin E or a polyethylene glycol is
selected as the p-glycoprotein inhibitor, a preferred amount will
typically be from about 5 mg to about 1000 mg, a more preferred
amount will typically be from about 50 mg to about 500 mg, and a
further preferred amount will typically be from about 100 mg to
about 500 mg. The most preferred amount of water soluble vitamin E
or a polyethylene glycol will be from about 200 mg to about 500 mg.
The above amounts of water soluble vitamin E or polyethylene glycol
can be administered from once to multiple times per day. Typically,
doses will be administered on a regimen requiring one, two or three
doses per day with one and two being preferred.
[0161] As used herein, the term "co-administration" refers to
administration to a patient of both a compound that has
vasodilating and/or platelet aggregation inhibiting properties,
including the compounds described in U.S. Pat. Nos. 4,306,075 and
5,153,222 which include treprostinil and structures I and II
described herein, and a p-glycoprotein inhibitor so that the
pharmacologic effect of the p-glycoprotein inhibitor in inhibiting
p-glycoprotein mediated transport in the gut is manifest at the
time at which the compound is being absorbed from the gut. Of
course, the compound and the p-glycoprotein inhibitor may be
administered at different times or concurrently. For example, the
p-glycoprotein inhibitor may be administered to the patient at a
time prior to administration of the therapeutic compound so as to
pre-treat the patient in preparation for dosing with the
vasodilating compound. Furthermore, it may be convenient for a
patient to be pre-treated with the p-glycoprotein inhibitor so as
to achieve steady state levels of p-glycoprotein inhibitor prior to
administration of the first dose of the therapeutic compound. It is
also contemplated that the vasodilating and/or platelet aggregation
inhibiting compounds and the p-glycoprotein inhibitor may be
administered essentially concurrently either in separate dosage
forms or in the same oral dosage form.
[0162] The present invention further provides that the vasodilating
and/or platelet aggregation inhibiting compound and the
p-glycoprotein inhibitor may be administered in separate dosage
forms or in the same combination oral dosage form.
Co-administration of the compound and the p-glycoprotein inhibitor
may conveniently be accomplished by oral administration of a
combination dosage form containing both the compound and the
p-glycoprotein inhibitor.
[0163] Thus, an additional embodiment of the present invention is a
combination pharmaceutical composition for oral administration
comprising an effective vasodilating and/or platelet aggregation
inhibiting amount of a compound described herein and an effective
p-glycoprotein inhibiting amount of a p-glycoprotein inhibitor.
This combination oral dosage form may provide for immediate release
of both the vasodilating and/or platelet aggregation inhibiting
compound and the p-glycoprotein inhibitor or may provide for
sustained release of one or both of the vasodilating and/or
platelet aggregation inhibiting compound and the p-glycoprotein
inhibitor. One skilled in the art would readily be able to
determine the appropriate properties of the combination dosage form
so as to achieve the desired effect of co-administration of the
vasodilating and/or platelet aggregation inhibiting compound and
the p-glycoprotein inhibitor.
[0164] Accordingly, the present invention provides for an
enhancement of the bioavailability of treprostinil, a drug of
structure I or II, and pharmaceutically acceptable salts thereof by
co-administration of a p-glycoprotein inhibitor. By
co-administration of these compounds and a p-glycoprotein
inhibitor, the total amount of the compound can be increased over
that which would otherwise circulate in the blood in the absence of
the p-glycoprotein inhibitor. Thus, co-administration in accordance
with the present invention can cause an increase in the AUC of the
present compounds over that seen with administration of the
compounds alone.
[0165] Typically, bioavailability is assessed by measuring the drug
concentration in the blood at various points of time after
administration of the drug and then integrating the values obtained
over time to yield the total amount of drug circulating in the
blood. This measurement, called the Area Under the Curve (AUC), is
a direct measurement of the bioavailability of the drug.
[0166] Without limiting the scope of the invention, it is believed
that in some embodiments derivatizing treprostinil at the R.sup.2
and R.sup.3 hydroxyl groups can help overcome the barriers to oral
treprostinil delivery by blocking these sites, and thus the
metabolism rate may be reduced to permit the compound to bypass
some of the first pass effect. Also, with an exposed amino acid,
the prodrug may be actively absorbed from the dipeptide transporter
system that exists in the gastrointestinal tract. Accordingly, the
present invention provides compounds, such as those found in
structures I and II, that reduce the first pass effect of
treprostinil and/or reduce the efflux mechanism of the
gastrointestinal tract.
[0167] In some embodiments of the method of treating hypertension
in a subject, the subject is a mammal, and in some embodiments is a
human.
[0168] Pharmaceutical formulations may include any of the compounds
of any of the embodiments described above, either alone or in
combination, in combination with a pharmaceutically acceptable
carrier such as those described herein.
[0169] The instant invention also provides for compositions which
may be prepared by mixing one or more compounds of the instant
invention, or pharmaceutically acceptable salts thereof, with
pharmaceutically acceptable carriers, excipients, binders, diluents
or the like, to treat or ameliorate a variety of disorders related
vasoconstriction and/or platelet aggregation. A therapeutically
effective dose further refers to that amount of one or more
compounds of the instant invention sufficient to result in
amelioration of symptoms of the disorder. The pharmaceutical
compositions of the instant invention can be manufactured by
methods well known in the art such as conventional granulating,
mixing, dissolving, encapsulating, lyophilizing, emulsifying or
levigating processes, among others. The compositions can be in the
form of, for example, granules, powders, tablets, capsules, syrup,
suppositories, injections, emulsions, elixirs, suspensions or
solutions. The instant compositions can be formulated for various
routes of administration, for example, by oral administration, by
transmucosal administration, by rectal administration, transdermal
or subcutaneous administration as well as intrathecal, intravenous,
intramuscular, intraperitoneal, intranasal, intraocular or
intraventricular injection. The compound or compounds of the
instant invention can also be administered by any of the above
routes, for example in a local rather than a systemic fashion, such
as injection as a sustained release formulation. The following
dosage forms are given by way of example and should not be
construed as limiting the instant invention.
[0170] For oral, buccal, and sublingual administration, powders,
suspensions, granules, tablets, pills, capsules, gelcaps, and
caplets are acceptable as solid dosage forms. These can be
prepared, for example, by mixing one or more compounds of the
instant invention, or pharmaceutically acceptable salts thereof,
with at least one additive or excipient such as a starch or other
additive. Suitable additives or excipients are sucrose, lactose,
cellulose sugar, mannitol, maltitol, dextran, sorbitol, starch,
agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum
arabic, gelatins, collagens, casein, albumin, synthetic or
semi-synthetic polymers or glycerides, methyl cellulose,
hydroxypropylmethyl-cellulose, and/or polyvinylpyrrolidone.
Optionally, oral dosage forms can contain other ingredients to aid
in administration, such as an inactive diluent, or lubricants such
as magnesium stearate, or preservatives such as paraben or sorbic
acid, or anti-oxidants such as ascorbic acid, tocopherol or
cysteine, a disintegrating agent, binders, thickeners, buffers,
sweeteners, flavoring agents or perfuming agents. Additionally,
dyestuffs or pigments may be added for identification. Tablets may
be further treated with suitable coating materials known in the
art.
[0171] Additionally, tests have shown that the present compounds,
including treprostinil, and in particular the compounds of
structure I and II have increased bioavailability when delivered to
the duodenum. Accordingly, one embodiment of the present invention
involves preferential delivery of the desired compound to the
duodenum as well as pharmaceutical formulations that achieve
duodenal delivery. Duodenal administration can be achieved by any
means known in the art. In one of these embodiments, the present
compounds can be formulated in an enteric-coated dosage form.
Generally, enteric-coated dosage forms are usually coated with a
polymer that is not soluble at low pH, but dissolves quickly when
exposed to pH conditions of 3 or above. This delivery form takes
advantage of the difference in pH between the stomach, which is
about 1 to 2, and the duodenum, where the pH tends to be greater
than 4.
[0172] Liquid dosage forms for oral administration may be in the
form of pharmaceutically acceptable emulsions, syrups, elixirs,
suspensions, slurries and solutions, which may contain an inactive
diluent, such as water. Pharmaceutical formulations may be prepared
as liquid suspensions or solutions using a sterile liquid, such as,
but not limited to, an oil, water, an alcohol, and combinations of
these. Pharmaceutically suitable surfactants, suspending agents,
emulsifying agents, may be added for oral or parenteral
administration.
[0173] As noted above, suspensions may include oils. Such oil
include, but are not limited to, peanut oil, sesame oil, cottonseed
oil, corn oil and olive oil. Suspension preparation may also
contain esters of fatty acids such as ethyl oleate, isopropyl
myristate, fatty acid glycerides and acetylated fatty acid
glycerides. Suspension formulations may include alcohols, such as,
but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol,
glycerol and propylene glycol. Ethers, such as but not limited to,
poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil
and petrolatum; and water may also be used in suspension
formulations.
[0174] Injectable dosage forms generally include aqueous
suspensions or oil suspensions which may be prepared using a
suitable dispersant or wetting agent and a suspending agent.
Injectable forms may be in solution phase or in the form of a
suspension, which is prepared with a solvent or diluent. Acceptable
solvents or vehicles include sterilized water, Ringer's solution,
or an isotonic aqueous saline solution. Alternatively, sterile oils
may be employed as solvents or suspending agents. Preferably, the
oil or fatty acid is non-volatile, including natural or synthetic
oils, fatty acids, mono-, di- or tri-glycerides.
[0175] For injection, the pharmaceutical formulation may be a
powder suitable for reconstitution with an appropriate solution as
described above. Examples of these include, but are not limited to,
freeze dried, rotary dried or spray dried powders, amorphous
powders, granules, precipitates, or particulates. For injection,
the formulations may optionally contain stabilizers, pH modifiers,
surfactants, bioavailability modifiers and combinations of these.
The compounds may be formulated for parenteral administration by
injection such as by bolus injection or continuous infusion. A unit
dosage form for injection may be in ampoules or in multi-dose
containers.
[0176] Besides those representative dosage forms described above,
pharmaceutically acceptable excipients and carries are generally
known to those skilled in the art and are thus included in the
instant invention. Such excipients and carriers are described, for
example, in "Remingtons Pharmaceutical Sciences" Mack Pub. Co., New
Jersey (1991), which is incorporated herein by reference.
[0177] The formulations of the invention may be designed for to be
short-acting, fast-releasing, long-acting, and sustained-releasing
as described below. Thus, the pharmaceutical formulations may also
be formulated for controlled release or for slow release.
[0178] The instant compositions may also comprise, for example,
micelles or liposomes, or some other encapsulated form, or may be
administered in an extended release form to provide a prolonged
storage and/or delivery effect. Therefore, the pharmaceutical
formulations may be compressed into pellets or cylinders and
implanted intramuscularly or subcutaneously as depot injections or
as implants such as stents. Such implants may employ known inert
materials such as silicones and biodegradable polymers.
[0179] Specific dosages may be adjusted depending on conditions of
disease, the age, body weight, general health conditions, sex, and
diet of the subject, dose intervals, administration routes,
excretion rate, and combinations of drugs. Any of the above dosage
forms containing effective amounts are well within the bounds of
routine experimentation and therefore, well within the scope of the
instant invention.
[0180] A therapeutically effective dose may vary depending upon the
route of administration and dosage form. The preferred compound or
compounds of the instant invention is a formulation that exhibits a
high therapeutic index. The therapeutic index is the dose ratio
between toxic and therapeutic effects which can be expressed as the
ratio between LD.sub.50 and ED.sub.50. The LD.sub.50 is the dose
lethal to 50% of the population and the ED.sub.50 is the dose
therapeutically effective in 50% of the population. The LD.sub.50
and ED.sub.50 are determined by standard pharmaceutical procedures
in animal cell cultures or experimental animals.
[0181] A method of preparing pharmaceutical formulations includes
mixing any of the above-described compounds with a pharmaceutically
acceptable carrier and water or an aqueous solution.
[0182] Pharmaceutical formulations and medicaments according to the
invention include any of the compounds of any of the embodiments of
compound of structure I, II or pharmaceutically acceptable salts
thereof described above in combination with a pharmaceutically
acceptable carrier. Thus, the compounds of the invention may be
used to prepare medicaments and pharmaceutical formulations. In
some such embodiments, the medicaments and pharmaceutical
formulations comprise any of the compounds of any of the
embodiments of the compounds of structure I or pharmaceutically
acceptable salts thereof. The invention also provides for the use
of any of the compounds of any of the embodiments of the compounds
of structure I, II or pharmaceutically acceptable salts thereof for
prostacyclin-like effects. The invention also provides for the use
of any of the compounds of any of the embodiments of the compounds
of structure I, II or pharmaceutically acceptable salts thereof or
for the treatment of pulmonary hypertension.
[0183] The invention also pertains to kits comprising one or more
of the compounds of structure I or II along with instructions for
use of the compounds. In another embodiment, kits having compounds
with prostacyclin-like effects described herein in combination with
one or more p-glycoprotein inhibitors is provided along with
instructions for using the kit.
[0184] By way of illustration, a kit of the invention may include
one or more tablets, capsules, caplets, gelcaps or liquid
formulations containing the bioenhancer of the present invention,
and one or more tablets, capsules, caplets, gelcaps or liquid
formulations containing a prostacyclin-like effect compound
described herein in dosage amounts within the ranges described
above. Such kits may be used in hospitals, clinics, physician's
offices or in patients' homes to facilitate the co-administration
of the enhancing and target agents. The kits should also include as
an insert printed dosing information for the co-administration of
the enhancing and target agents.
[0185] The following abbreviations and definitions are used
throughout this application:
[0186] Generally, reference to a certain element such as hydrogen
or H is meant to include all isotopes of that element. For example,
if an R group is defined to include hydrogen or H, it also includes
deuterium and tritium.
[0187] As used herein, the term "p-glycoprotein inhibitor" refers
to organic compounds which inhibit the activity of the
p-glycoprotein mediated active transport system present in the gut.
This transport system actively transports drugs which have been
absorbed from the intestinal lumen and into the gut epithelium back
out into the lumen. Inhibition of this p-glycoprotein mediated
active transport system will cause less drug to be transported back
into the lumen and will thus increase the net drug transport across
the gut epithelium and will increase the amount of drug ultimately
available in the blood.
[0188] The phrases "oral bioavailability" and "bioavailability upon
oral administration" as used herein refer to the systemic
availability (i.e., blood/plasma levels) of a given amount of drug
administered orally to a patient.
[0189] The phrase "unsubstituted alkyl" refers to alkyl groups that
do not contain heteroatoms. Thus the phrase includes straight chain
alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl,
heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The
phrase also includes branched chain isomers of straight chain alkyl
groups, including but not limited to, the following which are
provided by way of example: --CH(CH.sub.3).sub.2,
--CH(CH.sub.3)(CH.sub.2CH.sub.3), --CH(CH.sub.2CH.sub.3).sub.2,
--C(CH.sub.3).sub.3, --C(CH.sub.2CH.sub.3).sub.3,
--CH.sub.2CH(CH.sub.3).sub.2,
--CH.sub.2CH(CH.sub.3)(CH.sub.2CH.sub.3),
--CH.sub.2CH(CH.sub.2CH.sub.3).sub.2, --CH.sub.2C(CH.sub.3).sub.3,
--CH.sub.2C(CH.sub.2CH.sub.3).sub.3,
--CH(CH.sub.3)CH(CH.sub.3)(CH.sub.2CH.sub.3),
--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2,
--CH.sub.2CH.sub.2CH(CH.sub.3)(CH.sub.2CH.sub.3),
--CH.sub.2CH.sub.2CH(CH.sub.2CH.sub.3).sub.2,
--CH.sub.2CH.sub.2C(CH.sub.3).sub.3,
--CH.sub.2CH.sub.2C(CH.sub.2CH.sub.3).sub.3,
--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2,
--CH(CH.sub.3)CH(CH.sub.3)CH(CH.sub.3).sub.2,
--CH(CH.sub.2CH.sub.3)CH(CH.sub.3)CH(CH.sub.3)(CH.sub.2CH.sub.3),
and others. The phrase also includes cyclic alkyl groups such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and
cyclooctyl and such rings substituted with straight and branched
chain alkyl groups as defined above. The phrase also includes
polycyclic alkyl groups such as, but not limited to, adamantyl
norbornyl, and bicyclo[2.2.2]octyl and such rings substituted with
straight and branched chain alkyl groups as defined above. Thus,
the phrase unsubstituted alkyl groups includes primary alkyl
groups, secondary alkyl groups, and tertiary alkyl groups.
Unsubstituted alkyl groups may be bonded to one or more carbon
atom(s), oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in
the parent compound. Preferred unsubstituted alkyl groups include
straight and branched chain alkyl groups and cyclic alkyl groups
having 1 to 20 carbon atoms. More preferred such unsubstituted
alkyl groups have from 1 to 10 carbon atoms while even more
preferred such groups have from 1 to 5 carbon atoms. Most preferred
unsubstituted alkyl groups include straight and branched chain
alkyl groups having from 1 to 3 carbon atoms and include methyl,
ethyl, propyl, and --CH(CH.sub.3).sub.2.
[0190] The phrase "substituted alkyl" refers to an unsubstituted
alkyl group as defined above in which one or more bonds to a
carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen and
non-carbon atoms such as, but not limited to, a halogen atom in
halides such as F, Cl, Br, and I; and oxygen atom in groups such as
hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; a
sulfur atom in groups such as thiol groups, alkyl and aryl sulfide
groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a
nitrogen atom in groups such as amines, amides, alkylamines,
dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides,
imides, and enamines; a silicon atom in groups such as in
trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl
groups, and triarylsilyl groups; and other heteroatoms in various
other groups. Substituted alkyl groups also include groups in which
one or more bonds to a carbon(s) or hydrogen(s) atom is replaced by
a bond to a heteroatom such as oxygen in carbonyl, carboxyl, and
ester groups; nitrogen in groups such as imines, oximes,
hydrazones, and nitriles. Preferred substituted alkyl groups
include, among others, alkyl groups in which one or more bonds to a
carbon or hydrogen atom is/are replaced by one or more bonds to
fluorine atoms. One example of a substituted alkyl group is the
trifluoromethyl group and other alkyl groups that contain the
trifluoromethyl group. Other alkyl groups include those in which
one or more bonds to a carbon or hydrogen atom is replaced by a
bond to an oxygen atom such that the substituted alkyl group
contains a hydroxyl, alkoxy, aryloxy group, or heterocyclyloxy
group. Still other alkyl groups include alkyl groups that have an
amine, alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine,
diarylamine, heterocyclylamine, (alkyl)(heterocyclyl)amine,
(aryl)(heterocyclyl)amine, or diheterocyclylamine group.
[0191] The phrase "unsubstituted arylalkyl" refers to unsubstituted
alkyl groups as defined above in which a hydrogen or carbon bond of
the unsubstituted alkyl group is replaced with a bond to an aryl
group as defined above. For example, methyl (--CH.sub.3) is an
unsubstituted alkyl group. If a hydrogen atom of the methyl group
is replaced by a bond to a phenyl group, such as if the carbon of
the methyl were bonded to a carbon of benzene, then the compound is
an unsubstituted arylalkyl group (i.e., a benzyl group). Thus the
phrase includes, but is not limited to, groups such as benzyl,
diphenylmethyl, and 1-phenylethyl (--CH(C.sub.6H.sub.5)(CH.sub.3))
among others.
[0192] The phrase "substituted arylalkyl" has the same meaning with
respect to unsubstituted arylalkyl groups that substituted aryl
groups had with respect to unsubstituted aryl groups. However, a
substituted arylalkyl group also includes groups in which a carbon
or hydrogen bond of the alkyl part of the group is replaced by a
bond to a non-carbon or a non-hydrogen atom. Examples of
substituted arylalkyl groups include, but are not limited to,
--CH.sub.2C(.dbd.O)(C.sub.6H.sub.5), and --CH.sub.2(2-methylphenyl)
among others.
[0193] A "pharmaceutically acceptable salt" includes a salt with an
inorganic base, organic base, inorganic acid, organic acid, or
basic or acidic amino acid. As salts of inorganic bases, the
invention includes, for example, alkali metals such as sodium or
potassium; alkaline earth metals such as calcium and magnesium or
aluminum; and ammonia. As salts of organic bases, the invention
includes, for example, trimethylamine, triethylamine, pyridine,
picoline, ethanolamine, diethanolamine, and triethanolamine. As
salts of inorganic acids, the instant invention includes, for
example, hydrochloric acid, hydroboric acid, nitric acid, sulfuric
acid, and phosphoric acid. As salts of organic acids, the instant
invention includes, for example, formic acid, acetic acid,
trifluoroacetic acid, fumaric acid, oxalic acid, lactic acid,
tartaric acid, maleic acid, citric acid, succinic acid, malic acid,
methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic
acid. As salts of basic amino acids, the instant invention
includes, for example, arginine, lysine and ornithine. Acidic amino
acids include, for example, aspartic acid and glutamic acid.
[0194] "Treating" within the context of the instant invention,
means an alleviation of symptoms associated with a biological
condition, disorder, or disease, or halt of further progression or
worsening of those symptoms, or prevention or prophylaxis of the
disease or disorder. For example, within the context of treating
patients having pulmonary hypertension, successful treatment may
include a reduction direct vasodilation of pulmonary and/or
systemic arterial vascular beds and inhibition of platelet
aggregation. The result of this vasodilation will generally reduce
right and left ventricular afterload and increased cardiac output
and stroke volume. Dose-related negative inotropic and lusitropic
effects can also result. The outward manifestation of these
physical effects can include a decrease in the symptoms of
hypertension, such as shortness of breath, and an increase in
exercise capacity.
[0195] The present invention, thus generally described, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the present invention.
EXAMPLES
Example 1
[0196] In this Example, the bioavailability of treprostinil in rats
after dosing orally, intraduodenally, intracolonically and via the
portal vein was compared to determine possible barriers to
bioavailability. In addition to bioavailability, a number of
pharmacokinetic parameters were determined.
Animal Dosing
[0197] The bioavailability of treprostinil was evaluated in
Sprague-Dawley, male rats. Fifteen surgically modified rats were
purchased from Hilltop Lab Animals (Scottdale, Pa.). The animals
were shipped from Hilltop to Absorption Systems' West Chester
University facility (West Chester, Pa.), where they were housed for
at least twenty-four hours prior to being used in the study. The
animals were fasted for approximately 16 hours prior to dosing. The
fifteen rats used in this study were divided into five groups (I,
II, III, IV and V).
[0198] The weight of the animals and the dosing regimen are
presented in Table 1.
TABLE-US-00001 TABLE 1 Dose Weight Route of Study Volume Dose Group
Rat # (g) Administration Day (mL/kg) (mg/kg) I 118 327 Intravenous
0 2 1 119 329 Intravenous 0 2 1 120 320 Intravenous 0 2 1 II 121
337 Intraportal Vein 0 2 1 122 319 Intraportal Vein 0 2 1 123 330
Intraportal Vein 0 2 1 III 124 329 Intraduodenal 0 2 1 125 331
Intraduodenal 0 2 1 126 324 Intraduodenal 0 2 1 IV 127 339
Intracolonic 0 2 1 128 333 Intracolonic 0 2 1 129 320 Intracolonic
0 2 1 V 130 293 Oral 0 2 1 131 323 Oral 0 2 1 132 332 Oral 0 2
1
[0199] Samples were withdrawn at the following time points.
[0200] IV and IPV: 0 (pre-dose) 2, 5, 15, 30, 60, 120, 240, 360,
480 minutes
[0201] ID, IC and Oral: 0 (pre-dose), 5, 15, 30, 60, 120, 240, 360,
480 minutes
[0202] Approximately 0.50 to 0.75 mL of whole blood was collected
from the jugular vein of a cannulated rat. The blood was
transferred to heparinized tubes and placed on ice until
centrifuged. Following centrifugation the plasma was placed on ice
until frozen at -70.quadrature.C prior to shipment to Absorption
Systems
Analysis of Plasma Samples
[0203] Samples were analyzed using the following methodology:
Dosing Solution Preparation
[0204] The dosing solution was prepared by combining 15.2 mg of
treprostinil diethanolamine (12.0 mg of the free acid form) with 24
mL of 5% dextrose. The solution was then sonicated until dissolved
for a final concentration of 0.5 mg/mL. The final pH of the dosing
solution was 4.6. At the time of dosing, the dosing solution was
clear and homogenous.
Standards and Sample Preparation
[0205] To determine the concentration of treprostinil in rat plasma
samples, standards were prepared with rat plasma collected in
heparin obtained from Lampire Biological Laboratories (Lot
#021335263) to contain 1000, 300, 100, 30, 10, 3, 1 and 0.3 ng/mL
of treprostinil. Plasma standards were treated identically to the
plasma samples.
[0206] Plasma samples were prepared by solid phase extraction.
After an extraction plate was equilibrated, 150 .mu.L of a plasma
sample was placed into the well and vacuumed through. The
extraction bed was then washed with 600 .mu.L of
acetonitrile:deionized water (25:75) with 0.2% formic acid. The
compound was eluted with 600 .mu.L of 90% acetonitrile and 10%
ammonium acetate. The eluates were collected and evaporated to
dryness. The residue was reconstituted with 150 .mu.L of
acetonitrile:deionized water (50:50) with 0.5 .mu.g/mL of
tolbutamide (used as an internal standard).
HPLC Conditions
Column: Keystone Hypersil BDS C18 30.times.2 mm i.d., 3 .mu.m.
[0207] Mobile Phase Buffer: 25 mM NH.sub.4OH to pH 3.5 w/85% formic
acid. Reservoir A: 10% buffer and 90% water. Reservoir B: 10%
buffer and 90% acetonitrile.
Mobile Phase Composition:
Gradient Program:
TABLE-US-00002 [0208] Time Duration Grad. Curve % A % B -0.1 0.10 0
80 20 0 3.00 1.0 10 90 3.00 1.00 1.0 0 100 4.00 2.00 0 80 20
[0209] Flow Rate: 300 .mu.L/min. [0210] Inj. Vol.: 10 .mu.L [0211]
Run Time: 6.0 min. [0212] Retention Time: 2.6 min. [0213] Mass
Spectrometer Instrument: PE SCIEX API 2000 [0214] Interface:
Electrospray ("Turbo Ion Spray") [0215] Mode: Multiple Reaction
Monitoring (MRM)
TABLE-US-00003 [0215] Precursor Ion Product Ion Treprostinil 389.2
331.2 IS 269.0 170.0
TABLE-US-00004 Nebulizing Drying Gas: Curtain Gas: 25 Ion Spray:
Gas: 25 60, 350.degree. C. -5000 V Orifice: -80 V Ring: -350 V Q0:
10 V IQ1: 11 V ST: 15 V R01: 11 V IQ2: 35 V R02: 40 V IQ3: 55 V
R03: 45 V CAD Gas: 4
Method Validation
[0216] Table 2 lists the average recoveries (n=6) and coefficient
of variation (c.v.) for rat plasma spiked with treprostinil. All
samples were compared to a standard curve prepared in 50:50
dH.sub.2O:acetonitrile with 0.5 .mu.g/mL of tolbutamide to
determine the percent of treprostinil recovered from the
plasma.
TABLE-US-00005 TABLE 2 Accuracy and Precision of Method Spiked
Coefficient of Concentration Percent Recovered Variation 1000 ng/mL
85.6 5.2 100 ng/mL 89.6 11.6 10 ng/mL 98.8 7.0
Pharmacokinetic Analysis
[0217] Pharmacokinetic analysis was performed on the average plasma
concentration for each time point.
[0218] The data were subjected to non-compartmental analysis using
the pharmacokinetic program WinNonlin v. 3.1 (2).
Results
Clinical Observations
[0219] Prior to beginning the experiments it was noted that
supra-pharmacological doses of treprostinil would be needed to
achieve plasma concentrations that could be analyzed with adequate
sensitivity. Using the dose of 1 mg/kg some adverse effects were
noted in animals dosed intravenously and via the intraportal
vein.
[0220] All rats dosed intravenously displayed signs of extreme
lethargy five minutes after dosing but fully recovered to normal
activity thirty minutes post-dosing. In addition, fifteen minutes
after dosing all three animals dosed via the portal vein exhibited
signs of lethargy. One rat (#123) expired before the thirty-minute
sample was drawn. The other rats fully recovered. The remaining
animals did not display any adverse reactions after administration
of the compound.
Sample Analysis
[0221] Average plasma concentrations for each route of
administration are shown in Table 3.
TABLE-US-00006 TABLE 3 Average (n = 3) plasma concentrations
(ng/mL) Pre- Time (min) dose 2 5 15 30 60 120 240 360 480
Intravenous 0 1047.96 364.28 130.91 55.56 14.45 4.45 1.09 0.50 0.30
Intraportal Vein* 0 302.28 97.39 47.98 21.94 11.06 3.87 2.51 4.95
5.14 Intraduodenal 0 -- 61.76 31.67 18.57 13.55 5.91 1.11 0.89 0.90
Intracolonic 0 -- 7.46 3.43 3.52 1.48 0.64 0.36 0.062.sup..lamda.
0.202.sup..lamda. Oral 0 -- 4.52 2.90 3.67 2.06 4.52 1.82 0.90 0.96
*n = 2, .sup..lamda.concentration falls below the limit of
quantitation (LOQ) of the analytical method
[0222] The plasma concentration versus time curves for intravenous,
intraportal, intraduodenal, intracolonic and oral dosing are shown
in FIGS. 1 and 2. FIG. 3 shows the average plasma concentration
versus time curves for all five routes of administration. In the
experiments shown in these figures, the diethanolamine salt was
used. Table 4 shows the pharmacokinetic parameters determined for
treprostinil. The individual bioavailabilities of each rat are
found in Table 5.
TABLE-US-00007 TABLE 4 Average Bioavailability and Pharmacokinetic
Parameters of Treprostinil in Rats Average Average Volume of CLs
Route of AUC.sub.480 min C.sub.max T.sub.max T.sub.1/2
Bioavailability Distribution* (mL min.sup.-1 Administration (min
ng/mL) (ng/mL) (min) (min) (%) .+-. SD (L kg.sup.-1) kg.sup.-1)*
Intravenous 11253.49 2120.sup..PSI. 0 94 NA 1.98 88.54 Intraportal
Vein 4531.74 302 2 ND 40.3 .+-. 5.5 ND ND Intraduodenal 2712.55 62
5 ND 24.1 .+-. 0.5 ND ND Intracolonic 364.63 8 5 ND 3.2 .+-. 2.5 ND
ND Oral 1036.23 5 5 ND 9.2 .+-. 1.4 ND ND *Normalized to the
average weight of the rats ND:Not determined .sup..PSI.Extrapolated
Value
TABLE-US-00008 TABLE 5 Individual Bioavailabilities of Treprostinil
in Rats Route of Individual AUC.sub.480 min Individual
Administration Rat # (min ng/mL) Bioavailability (%) Intravenous
118 10302.85 NA 119 9981.52 NA 120 13510.65 NA Intraportal Vein 121
4970.67 44.2 122 4093.21 36.4 123 ND ND Intraduodenal 124 2725.68
24.2 125 2763.60 24.6 126 2646.05 23.5 Intracolonic 127 72.63 0.7
128 395.08 3.5 129 625.20 5.6 Oral 130 998.70 8.9 131 907.60 8.1
132 1203.73 10.7 NA: Not applicable ND: Not determined
Conclusions
[0223] Treprostinil has a terminal plasma half-life of 94 minutes.
The distribution phase of treprostinil has a half-life of 10.3
minutes and over 90% of the distribution and elimination of the
compound occurs by 60 minutes post-dosing. The volume of
distribution (Vd=1.98 L/kg) is greater than the total body water of
the rat (0.67 L/kg) indicating extensive partitioning into tissues.
The systemic clearance of treprostinil (88.54 mL/min/kg) is greater
than the hepatic blood flow signifying that extra-hepatic clearance
mechanisms are involved in the elimination of the compound.
[0224] First pass hepatic elimination of treprostinil results in an
average intraportal vein bioavailability of 40.3%. Fast but
incomplete absorption is observed after intraduodenal, intracolonic
and oral dosing (T.sub.max.ltoreq.5 min). By comparing the
intraportal vein (40.3%) and intraduodenal bioavailability (24.1%)
it appears that approximately 60% of the compound is absorbed in
the intestine. The average intraduodenal bioavailibility is almost
three times greater than the oral bioavailibility suggesting that
degradation of treprostinil in the stomach or gastric emptying may
influence the extent of systemic absorption.
Example 2
[0225] In this Example, Treprostinil concentrations were determined
in male Sprague-Dawley rats following a single oral dose of the
following compounds:
##STR00025##
Treprostinil Benzyl Ester
##STR00026##
[0226] Treprostinil Diglycine
##STR00027##
[0227] Treprostinil Methyl Ester
Experimental
Dosing Solution Preparation
[0228] All dosing vehicles were prepared less than 2 hours prior to
dosing.
1. Treprostinil Methyl Ester
[0229] A solution of treprostinil methyl ester was prepared by
dissolving 2.21 mg of treprostinil methyl ester with 0.85 mL of
dimethylacetamide (DMA). This solution was then diluted with 7.65
mL of PEG 400:Polysorbate 80:Water, 40:1:49. The final
concentration of the dosing vehicle was 0.26 mg/mL of treprostinil
methyl ester equivalent to 0.25 mg/mL of Treprostinil. The dosing
vehicle was a clear solution at the time of dosing.
2. Treprostinil Benzyl Ester
[0230] A solution of treprostinil benzyl ester was prepared by
dissolving 2.58 mg of treprostinil benzyl ester with 0.84 mL of
dimethylacetamide (DMA). This solution was then diluted with 7.54
mL of PEG 400:Polysorbate 80:Water, 40:1:49. The final
concentration of the dosing vehicle was 0.268 mg/mL of treprostinil
benzyl ester equivalent to 0.25 mg/mL of Treprostinil. The dosing
vehicle was a clear solution at the time of dosing.
3. Treprostinil Diglycine
[0231] A solution of treprostinil diglycine was prepared by
dissolving 1.86 mg of compound with 0.58 mL of dimethylacetamide
(DMA). This solution was then diluted with 5.18 mL of PEG
400:Polysorbate 80:Water, 40:1:49. The final concentration of the
dosing vehicle was 0.323 mg/mL of treprostinil diglycine equivalent
to 0.25 mg/mL of Treprostinil. The dosing vehicle was a clear
solution at the time of dosing.
Animal Dosing
[0232] The plasma concentrations of Treprostinil following
administration of each prodrug were evaluated in male
Sprague-Dawley rats. Rats were purchased from Hilltop Lab Animals
(Scottdale, Pa.). The animals were shipped from Hilltop to
Absorption Systems' West Chester University facility (West Chester,
Pa.). They were housed for at least twenty-four hours prior to
being used in the study. The animals were fasted for approximately
16 hours prior to dosing. The rats used in this study were divided
into three groups (I, II and III). Groups I-III were dosed on the
same day.
[0233] The weight of the animals and the dosing regimen are
presented in Table 6.
TABLE-US-00009 TABLE 6 Study Design Route of Dose Weight Ad-
Compound Volume Dose* Group Rat # (kg) ministration Dosed (mL/kg)
(mg/kg) I 638 306 Oral Treprostinil 2 0.520 639 310 Oral methyl
ester 640 319 Oral II 641 319 Oral Treprostinil 2 0.616 642 309
Oral benzyl ester 643 320 Oral III 644 318 Oral Treprostinil 2
0.646 645 313 Oral diglycine 646 322 Oral *This dose of prodrug =
0.500 mg/kg of the active, Treprostinil
[0234] Animals were dosed via oral gavage. Blood samples were taken
from a jugular vein cannula at the following time points:
[0235] 0 (pre-dose) 5, 15, 30, 60, 120, 240, 360 and 480
minutes
[0236] The blood samples were withdrawn and placed into tubes
containing 30 .mu.L of a solution of 500 units per mL of heparin in
saline, and centrifuged at 13,000 rpm for 10 minutes. Approximately
200 .mu.L of plasma was then removed and dispensed into
appropriately labeled polypropylene tubes containing 4 .mu.L of
acetic acid in order to stabilize any prodrug remaining in the
samples. The plasma samples were frozen at -20.degree. C. and were
transported on ice to Absorption Systems Exton Facility. There they
were stored in a -80.degree. C. freezer pending analysis.
Analysis of Plasma Samples
[0237] Plasma samples were analyzed as described in Example 1. In
brief, Treprostinil was extracted from the plasma via liquid-liquid
extraction then analyzed by LC/MS/MS. The analytical validation
results were reported previously in Example 1. The lower limit of
quantification (LLOQ) of the analytical method was 0.01 ng/mL.
Samples were not assayed for unchanged prodrug.
Acceptance Criteria for Analytical Runs
[0238] Two standard curves, with a minimum of five points per
curve, and a minimum of two quality control samples (QCs) were
dispersed throughout each run. Each route of administration was
bracketed by a standard curve used for back-calculation. The
standards and QCs must be within .+-.15% (20% for the LLOQ)
accuracy and precision for the run to be accepted. At least 75% of
all standards and QCs must pass the acceptance criteria.
Pharmacokinetic Analysis
[0239] Pharmacokinetic analysis was performed on the plasma
concentration of Treprostinil for each individual rat at each time
point and on the average plasma concentration for all three rats in
the group for each time point. The data were subjected to
non-compartmental analysis using the pharmacokinetic program
WinNonLin v. 3.1 (2).
Results
Study Observations
[0240] No adverse reactions were observed following oral
administration of treprostinil methyl ester, treprostinil benzyl
ester or treprostinil diglycine.
Plasma Stability of Prodrugs in Acidified Rat Plasma
[0241] In order to terminate any conversion of prodrug to active
after samples were withdrawn the plasma was acidified. Acetic acid
(v/v) was added to each plasma sample immediately after
centrifugation of the red blood cells to a concentration of 2%.
In-vitro plasma stability of each prodrug was performed to insure
that the compound was stable in acidified plasma. To perform this
assay 2% acetic acid was added to blank rat plasma obtained from
Lampire Biological. The acidified rat plasma was equilibrated at
37.degree. C. for three minutes prior to addition of prodrug. The
initial concentration of each prodrug was 1000 ng/mL. A 100 .mu.L
aliquot of plasma (n=3 per time point) was taken at 0, 60 and 120
minutes. Each aliquot was combined with 20 .mu.L of HCl and
vortexed. Liquid-liquid extraction was then performed and the
concentration of Treprostinil in each sample determined. The
concentration of Treprostinil at each time point in acidified rat
plasma is given in Table 7. Small amounts of Treprostinil appear to
be present in the neat compound sample of treprostinil methyl ester
and treprostinil diglycine. The concentration of Treprostinil
remained constant throughout the course of the experiment,
indicating that there was no conversion of prodrug into active
compound occurring in acidified plasma.
TABLE-US-00010 TABLE 7 Plasma Stability of Prodrugs in Acidified
Dog Plasma Treprostinil Concentration (ng/mL) .+-. SD (n = 3)
Treprostinil Treprostinil Treprostinil Time (min) methyl ester
benzyl ester diglycine 0 56.8 .+-. 9.3 <0.01 54.9 .+-. 4.3 60
55.1 .+-. 5.0 <0.01 51.8 .+-. 5.9 120 53.8 .+-. 1.3 <0.01
54.5 .+-. 0.8 Total % Treprostinil 5.7 <0.01 5.5
Average Treprostinil plasma concentrations following administration
of treprostinil methyl ester, treprostinil benzyl ester or
treprostinil diglycine are shown in Table 8.
TABLE-US-00011 TABLE 8 Treprostinil Concentrations (Average .+-. SD
(n = 3) Plasma Concentrations (ng/mL) Oral Dosing Pre- 5 15 30 60
120 240 360 480 Solution Dose (min) (min) (min) (min) (min) (min)
(min) (min) Treprostinil 0 <0.01 0.2 .+-. 0.0 0.3 .+-. 0.1 0.5
.+-. 0.1 1.5 .+-. 0.8 0.2 .+-. 0.7 <0.01 0.1 .+-. 0.1 methyl
ester Treprostinil 0 3.1 .+-. 2.8 1.9 .+-. 0.8 2.5 .+-. 1.5 3.2
.+-. 1.9 7.3 .+-. 4.9 1.6 .+-. 1.2 0.4 .+-. 0.40 0.6 .+-. 0.9
benzyl ester Treprostinil 0 <0.01 1.1 .+-. 1.9 6.6 .+-. 10.7 0.5
.+-. 0.3* 40. .+-. 5.8 9.0 .+-. 13.5 2.1 .+-. 2.9 1.3 .+-. 0.8
diglycine *Due to insufficient amount of sample collected this time
point is the average of n = 2 rats.
[0242] FIGS. 4-7 contain graphical representations of the plasma
concentration versus time curves for Treprostinil in rat following
administration of each prodrug. Table 9 lists each figure and the
information displayed.
TABLE-US-00012 TABLE 9 List of Figures FIG. Description 4 Oral Dose
of Treprostinil methyl ester 5 Oral Dose of Treprostinil benzyl
ester 6 Oral Dose of Treprostinil diglycine 7 Oral Dose of
Treprostinil benzyl ester and Treprostinil diglycine Compared to
Treprostinil Alone from Example 1
Pharmacokinetic Analysis
[0243] Bioavailability of the prodrug was determined relative to
that of the active compound based on Example 1 in which
Treprostinil was dosed to rats. The following formula was used to
determine relative bioavailability (F):
Relative F=(AUC.sub.(Prodrug Dose)/Dose)/(AUC.sub.(Treprostinil
Dose)/Dose)*100
[0244] Bioavailability was also determined relative to an
intravenous dose of Treprostinil in rats determined in Example 1.
Results are listed in Table 10.
TABLE-US-00013 TABLE 10 Average Relative Bioavailability and
Pharmacokinetic Parameters of Treprostinil in Rats Test Average
Relative Compound Dose AUC.sub.0-t C.sub.max T.sub.max
Bioavailability Bioavailability Administered (mg/kg) (min ng/mL)
(ng/mL) (min) (%) .+-. SD (n = 3) (%) .+-. SD (n = 3) Treprostinil
0.5 212 1.50 120 41.0 .+-. 16 3.8 .+-. 2 methyl ester Treprostinil
0.5 1171 7.20 120 226 .+-. 155 20.8 .+-. 14 benzyl ester
Treprostinil 0.5 2242 9.04 240 433 .+-. 631 39.9 .+-. 58
diglycine
Conclusions
[0245] In this study the relative oral bioavailabilities of
prodrugs of Treprostinil were determined in rats. Treprostinil
methyl ester resulted in Treprostinil area under the plasma
concentration versus time curves (AUCs) less than that after dosing
the active compound. Prodrugs treprostinil benzyl ester and
treprostinil diglycine both had Treprostinil average AUCs greater
than that after dosing of the active compound. Treprostinil
diglycine had the highest relative bioavailability of 433% with
over 4 times more Treprostinil reaching the systemic circulation.
The Cmax of 9 ng/mL of Treprostinil following administration of
treprostinil diglycine occurred at 240 minutes post-dosing. The
Cmax following dosing of Treprostinil is 5 ng/mL and occurs only 5
minutes post-dosing. Treprostinil benzyl ester had a relative
bioavailability of 226.+-.155% with a Cmax of 7.2 ng/mL occurring
120 minutes post-dosing. It should also be noted that the AUCs are
not extrapolated to infinity.
REFERENCES
[0246] 1. WinNonlin User's Guide, version 3.1, 1998-1999, Pharsight
Co., Mountain View, Calif. 94040.
Example 3
[0247] This example illustrates a pharmacokinetic study of
treprostinil following administration of a single duodenal dose of
treprostinil and various prodrugs of the present invention.
[0248] In this study, the area under the curve of Treprostinil in
male Sprague-Dawley rats following a single intraduodenal dose of
treprostinil monophosphate (ring), treprostinil monovaline (ring),
treprostinil monoalanine (ring) or treprostinil monoalanine
(chain), prodrugs of treprostinil was compared. The compounds were
as follows:
##STR00028##
having the following substituents:
TABLE-US-00014 Compound R.sup.1 R.sup.2 R.sup.3 treprostinil H
--PO.sub.3H.sub.3 H monophosphate (ring) treprostinil H
--COCH(CH(CH.sub.3).sub.2)NH.sub.2 H monovaline (ring) treprostinil
H --COCH(CH.sub.3)NH.sub.2 H monoalanine (ring) treprostinil H H
--COCH(CH.sub.3)NH.sub.2 monoalanine (chain)
Experimental
Dosing Solution Preparation
[0249] All dosing vehicles were prepared less than 2 hours prior to
dosing.
1. Treprostinil Monophosphate (Ring)
[0250] A dosing solution of treprostinil monophosphate (ring) was
prepared by dissolving 1.01 mg of treprostinil monophosphate (ring)
in 0.167 mL of dimethylacetamide (DMA) until dissolved. This
solution was further diluted with 1.50 mL of PEG 400:Polysorbate
80:Water, 40:1:49. The final concentration of the dosing vehicle
was 0.603 mg/mL of prodrug equivalent to 0.5 mg/mL of Treprostinil.
The dosing vehicle was a clear solution at the time of dosing.
2. Treprostinil Monovaline (Ring)
[0251] A 50 mg/mL solution of treprostinil monovaline (ring) was
prepared in dimethylacetamide (DMA). A 25 .mu.L aliquot of the 50
mg/mL stock solution was then diluted with 175 .mu.L of DMA and 1.8
mL of PEG 400:Polysorbate 80:Water, 40:1:49. The final
concentration of the dosing vehicle was 0.625 mg/mL of prodrug
equivalent to 0.5 mg/mL of Treprostinil. The dosing vehicle was a
clear solution at the time of dosing.
3. Treprostinil Monoalanine (Ring)
[0252] A solution of treprostinil monoalanine (ring) was prepared
by dissolving 1.05 mg of treprostinil monoalanine (ring) in 0.178
mL of dimethylacetamide (DMA) until dissolved. This solution was
further diluted with 1.60 mL of PEG 400:Polysorbate 80:Water,
40:1:49. The final concentration of the dosing vehicle was 0.590
mg/mL of treprostinil monoalanine (ring) equivalent to 0.5 mg/mL of
Treprostinil. The dosing vehicle was a clear solution at the time
of dosing.
4. Treprostinil Monoalanine (Chain)
[0253] A solution of treprostinil monoalanine (chain) was prepared
by dissolving 0.83 mg of treprostinil monoalanine (chain) in 0.14
mL of dimethylacetamide (DMA) until dissolved. This solution was
further diluted with 1.26 mL of PEG 400:Polysorbate 80:Water,
40:1:49. The final concentration of the dosing vehicle was 0.591
mg/mL of treprostinil monoalanine (chain) equivalent to 0.5 mg/mL
of Treprostinil. The dosing vehicle was a clear solution at the
time of dosing.
Animal Dosing
[0254] The plasma concentrations of Treprostinil following oral
administration of each prodrug were evaluated in male
Sprague-Dawley rats. Twelve rats were purchased from Hilltop Lab
Animals (Scottdale, Pa.). The animals were shipped from Hilltop to
Absorption Systems' West Chester University facility (West Chester,
Pa.). They were housed for at least twenty-four hours prior to
being used in the study. The animals were fasted for approximately
16 hours prior to dosing. The twelve rats used in this study were
divided into four groups. All groups were dosed on day 1 of the
study. The weight of the animals and the dosing regimen are
presented in Table 11.
TABLE-US-00015 TABLE 11 Dose Volume Dose* Rat # Weight (g) Compound
(mL/kg) (mg/kg) 130 327 treprostinil monophosphate (ring) 1 0.603
131 321 treprostinil monophosphate (ring) 1 0.603 132 310
treprostinil monophosphate (ring) 1 0.603 133 328 treprostinil
monovaline (ring) 1 0.625 134 326 treprostinil monovaline (ring) 1
0.625 135 346 treprostinil monovaline (ring) 1 0.625 136 321
treprostinil monoalanine (chain) 1 0.591 137 319 treprostinil
monoalanine (chain) 1 0.591 138 330 treprostinil monoalanine
(chain) 1 0.591 139 316 treprostinil monoalanine (ring) 1 0.590 140
330 treprostinil monoalanine (ring) 1 0.590 141 339 treprostinil
monoalanine (ring) 1 0.590 *This dose of prodrug = 0.500 mg/kg of
treprostinil
[0255] Animals were dosed via an indwelling duodenal cannula. Blood
samples were taken from a jugular vein cannula at the following
time points: 0 (pre-dose) 5, 15, 30, 60, 120, 240, 360 and 480
minutes.
[0256] The blood samples were withdrawn and placed into tubes
containing 30 .mu.L of a solution of 500 units per mL of heparin in
saline, and centrifuged at 13,000 rpm for 10 minutes. Approximately
200 .mu.L of plasma was then removed and dispensed into
appropriately labeled polypropylene tubes containing 4 .mu.L of
acetic acid in order to stabilize any prodrug remaining in the
samples. The plasma samples were frozen at -20.degree. C. and were
transported on ice to Absorption Systems Exton Facility. There they
were stored in a -80.degree. C. freezer pending analysis.
Analysis of Plasma Samples
[0257] Plasma samples were analyzed using the methods described
above. In brief, Treprostinil was extracted from the plasma via
solid phase extraction then analyzed by LC/MS/MS. The lower limit
of quantification (LLOQ) of the analytical method was 0.03
ng/mL.
Acceptance Criteria for Analytical Runs
[0258] Four standard curves, with a minimum of five points per
curve, and a minimum of two quality control samples (QCs) at 3
concentrations were dispersed throughout each run. Each prodrug set
was bracketed by a standard curve used for back-calculation. The
standards and QCs must be within .+-.15% (20% for the LLOQ)
accuracy and precision for the run to be accepted. At least 75% of
all standards and QCs must pass the acceptance criteria.
Pharmacokinetic Analysis
[0259] Pharmacokinetic analysis was performed on the plasma
concentration of Treprostinil for each individual rat at each time
point and on the average plasma concentration for all three rats in
the group for each time point.
[0260] The data were subjected to non-compartmental analysis using
the pharmacokinetic program WinNonLin v. 3.1 (2).
Results
Study Observations
[0261] No adverse reactions were observed following intraduodenal
administration of treprostinil monophosphate (ring), treprostinil
monovaline (ring), treprostinil monoalanine (ring) or treprostinil
monoalanine (chain).
Ex-Vivo Plasma Stability of Prodrugs in Acidified Rat Plasma
[0262] In order to terminate any conversion of prodrug to active
after samples were withdrawn, the plasma was acidified. Acetic acid
(v/v) was added to each plasma sample immediately after separation
of the red blood cells to a concentration of 2%. In-vitro plasma
stability of each prodrug was performed to insure that the compound
was stable in acidified plasma. To perform this assay 2% acetic
acid was added to blank rat plasma obtained from Lampire
Biological. The acidified rat plasma was brought to room
temperature for three minutes prior to addition of prodrug. The
initial concentration of each prodrug was 1000 ng/mL. A 100 .mu.L
aliquot of plasma (n=3 per time point) was taken at 0, 60 and 120
minutes. Sample preparation of each plasma sample was performed as
described above and the concentration of Treprostinil
monitored.
[0263] Treprostinil concentrations did not increase in any of the
acidified plasma samples spiked with prodrug over the two-hour
period of the experiment.
Sample Analysis
[0264] Average Treprostinil plasma concentrations following
administration of treprostinil monophosphate (ring), treprostinil
monovaline (ring), treprostinil monoalanine (ring) or treprostinil
monoalanine (chain) are shown in Table 12.
TABLE-US-00016 TABLE 12 AVERAGE .+-. SD (N = 3) PLASMA TREPROSTINIL
CONCENTRATIONS (NG/ML) Oral Dosing Pre- 5 15 30 60 120 240 360 480
Solution dose (min) (min) (min) (min) (min) (min) (min) (min)
treprostinil 0 8.62 .+-. 3.0 6.57 .+-. 1.7 3.31 .+-. 1.2 4.31 .+-.
0.8 2.07 .+-. 0.4 0.91 .+-. 0.5 0.26 .+-. 0.08 0.3 .+-. 0.08
monophosphate (ring) treprostinil 0 0.76 .+-. 0.2 0.91 .+-. 0.7
1.52 .+-. 0.6 1.53 .+-. 0.6 1.65 .+-. 0.7 0.66 .+-. 0.1 0.15 .+-.
0.03 0.05 .+-. 0.02 monovaline (ring) treprostinil 0 2.42 .+-. 0.6
2.52 .+-. 0.4 2.91 .+-. 0.6 3.25 .+-. 1.5 1.69 .+-. 0.4 0.55 .+-.
0.2 0.20 .+-. 0.1 0.22 .+-. 0.2 monoalanine (ring) treprostinil 0
9.53 .+-. 2.6 3.92 .+-. 0.6 3.83 .+-. 0.7 2.74 .+-. 0.9 0.86 .+-.
0.4 0.29 .+-. 0.2 0.08 .+-. 0.04 0.19 .+-. 0.3 monoalanine
(chain)
[0265] FIGS. 8-12 contain graphical representations of the plasma
concentration versus time curves for Treprostinil in rat following
administration of each prodrug. Table 13 lists each figure and the
information displayed.
TABLE-US-00017 TABLE 13 FIG. Description 8 Intraduodenal dose of
treprostinil monophosphate (ring) 9 Intraduodenal dose of
treprostinil monovaline (ring) 10 Intraduodenal dose of
treprostinil monoalanine (ring) 11 Intraduodenal dose of
treprostinil monoalanine (chain) 12 Intraduodenal dose of each
prodrug compared to treprostinil alone from Example 1
Pharmacokinetic Analysis
[0266] Bioavailability of the prodrug was determined relative to
that of the active compound based on a previous study in which
Treprostinil was dosed to rats. The following formula was used to
determine relative bioavailability (F):
Relative F=(AUC.sub.(ProdrugDose)/Dose)/(AUC.sub.(Treprostinil
Dose)/Dose)*100
[0267] Absolute bioavailability was also estimated using data from
an intravenous dose of Treprostinil in rats determined in Example
1. Results are listed in Table 14.
TABLE-US-00018 TABLE 14 List of Figures FIG. Description 8
Intraduodenal Dose of treprostinil monophosphate (ring) 9
Intraduodenal Dose of treprostinil monovaline (ring) 10
Intraduodenal Dose of treprostinil monoalanine (ring) 11
Intraduodenal Dose of treprostinil monoalanine (chain) 12
Intraduodenal Dose of Each Prodrug Compared to Treprostinil Alone
from Example 1
Conclusions
[0268] The relative intraduodenal bioavailabilities of four
prodrugs of Treprostinil were determined in rats. All the compounds
had relative intraduodenal bioavailabilities less than that of the
active compound. treprostinil monophosphate (ring) and treprostinil
monoalanine (ring) had the highest relative intraduodenal
bioavailability at 56% and 38% respectively. The T.sub.max for
treprostinil monophosphate (ring) and treprostinil monoalanine
(chain) occurred 5 minutes post-dosing. treprostinil monovaline
(ring) and treprostinil monoalanine (ring) had longer absorption
times with T.sub.max values of 120 and 60 minutes respectively.
Maximum Treprostinil concentrations were highest following
treprostinil monophosphate (ring) and treprostinil monoalanine
(chain) dosing. They reached approximately 9 ng/mL 5 minutes
post-dosing. The bioavailabilities are much greater when dosed
intraduodenally than when dosed orally as measured by treprostinil
plasma levels.
REFERENCES
[0269] 1. WinNonlin User's Guide, version 3.1, 1998-1999, Pharsight
Co., Mountain View, Calif. 94040.
Example 4
[0270] In this Example, Treprostinil concentrations will be
determined in male Sprague-Dawley rats following a single oral or
intraduodenal dose of the following compounds of structure II:
##STR00029##
having the following substituents:
TABLE-US-00019 Cpd. R.sup.1 R.sup.2 R.sup.3 A --CH.sub.2CONH.sub.2
H H B --CH.sub.2CON(CH.sub.2).sub.2OH H H C
--CH.sub.2CON(CH.sub.3).sub.2 H H D --CH.sub.2CONHOH H H E
--CH.sub.2C.sub.6H.sub.4NO.sub.2 (p)* H H F
--CH.sub.2C.sub.6H.sub.4OCH.sub.3 (p)* H H G
--CH.sub.2C.sub.6H.sub.4Cl (o)* H H H
--CH.sub.2C.sub.6H.sub.4(NO.sub.2).sub.2* H H (o,p) I
--CH.sub.2C.sub.6H.sub.4F (p)* H H J H --PO.sub.3H.sub.3 H K H H
--PO.sub.3H.sub.3 L H --COCH.sub.2NH.sub.2 H M H H
--COCH.sub.2NH.sub.2 N H --COCH(CH.sub.3)NH.sub.2 H O H H
--COCH(CH.sub.3)NH.sub.2 P H --COCH(CH.sub.3)NH.sub.2
--COCH(CH.sub.3)NH.sub.2 *o denotes ortho substitution, m denotes
meta substitution and p denotes para substitution.
[0271] Examples of these compounds include:
##STR00030## ##STR00031## ##STR00032##
[0272] Prodrug preparation and analysis will take place as
described in Examples 1 and 2 above. Additionally, the oral
bioavailability of treprostinil, treprostinil sodium and the
compounds shown in Example 2 and this Example will be administered
in close proximity to or simultaneously with various different
p-glycoprotein inhibiting compounds at varying concentrations and
tested to determine the effect of the p-glycoprotein inhibitors on
the oral bioavailability of the compounds. The p-glycoprotein
inhibitors will be administered both intravenously and orally.
Example 5
[0273] Clinical Studies with Treprostinil Diethanolamine
[0274] Introduction
[0275] Prior to proceeding directly into clinical studies with a
sustained release (SR) solid dosage form of UT-15C (treprostinil
diethanolamine), a determination of the pharmacokinetics of an oral
"immediate release" solution was performed. The first clinical
study (01-101) evaluated the ability of escalating doses of an oral
solution of UT-15C to reach detectable levels in plasma, potential
dose-plasma concentration relationship, bioavailability and the
overall safety of UT-15C. Volunteers were dosed with the solutions
in a manner that simulated a sustained release formulation
releasing drug over approximately 8 hours.
[0276] The second clinical study (01-102) assessed the ability of
two SR solid dosage form prototypes (i.e., 1. microparticulate
beads in a capsule and, 2. tablet) to reach detectable levels in
plasma and the potential influence of food on these plasma drug
concentrations. The SR prototypes were designed to release UT-15C
over approximately an 8 hour time period.
[0277] Details of the two clinical studies are described below.
Clinical Study 01-101
[0278] A Safety, Tolerability, and Pharmacokinetic Study of
Multiple Escalating Doses of UT-15C (Treprostinil Diethanolamine)
Administered as an Oral Solution in Healthy Adult Volunteers
(Including Study of Bioavailability).
[0279] The oral solution of UT-15C was administered to 24 healthy
volunteers to assess the safety and pharmacokinetic profile of
UT-15C as well as its bioavailability. To mimic a SR release
profile, doses were administered every two hours for four doses at
either 0.05 mg per dose (total=0.2 mg), 0.125 mg per dose
(total=0.5 mg), 0.25 mg per dose (total=1.0 mg), or 0.5 mg per dose
(total=2.0 mg). Study endpoints included standard safety
assessments (adverse events, vital signs, laboratory parameters,
physical examinations, and electrocardiograms) as well as
pharmacokinetic parameters.
[0280] All subjects received all four scheduled doses and completed
the study in its entirety. Treprostinil plasma concentrations were
detectable in all subjects following administration of an oral
solution dose of UT-15C. Both AUC.sub.inf and C.sub.max increased
in a linear fashion with dose for each of the four dose aliquots.
The highest concentration observed in this study was 5.51 ng/mL
after the third 0.25 mg solution dose aliquot of the 2.0 mg UT-15C
total dose. Based on historical intravenous treprostinil sodium
data, the mean absolute bioavailability values for the 0.2 mg, 0.5
mg, 1.0 mg and 2.0 mg doses of UT-15C were estimated to be 21%,
23%, 24% and 25%, respectively. The results of this study are
respectively shown in FIGS. 13A-13D.
[0281] UT-15C was well tolerated by the majority of subjects at all
doses given. There were no clinically significant, treatment
emergent changes in hematology, clinical chemistry, urinalysis,
vital signs, physical exams, and ECGs. The most frequently reported
adverse events were flushing, headache, and dizziness. This safety
profile with UT-15C (treprostinil diethanolamine) is consistent
with the reported safety profile and product labeling of Remodulin
(treprostinil sodium) and other prostacyclin analogs. Thus,
changing the salt form of treprostinil did not result in any
unexpected safety issues following the protocol specified dosing
regimen (i.e. single dose every 2 hours for four total doses on a
single day).
Clinical Study 01-102
[0282] A Safety, Tolerability, and Pharmacokinetic Study Comparing
a Single Dose of a Sustained Release Capsule and Tablet Formulation
of UT-15C (Treprostinil Diethanolamine) Administered to Healthy
Adult Volunteers in the Fasted and Fed State
[0283] The 01-102 study was designed to evaluate and compare the
safety and pharmacokinetic profiles of a (1) UT-15C SR tablet
prototype and, (2) UT-15C SR capsule prototype (microparticulate
beads in a capsule) in both the fasted and fed state. Each of the
SR dosage forms were designed to release UT-15C (1 mg) over an
approximate 8-hour time period. Fourteen healthy adult volunteers
were assigned to receive the SR tablet formulation while an
additional fourteen volunteers were assigned to receive the SR
capsule formulation. Subjects were randomized to receive a single
dose (1 mg) of their assigned SR prototype in both the fasted and
fed state. A crossover design was employed with a seven day
wash-out period separating the fed/fasted states. For the fed
portion of the study, subjects received a high calorie, high fat
meal. Study endpoints included standard safety assessments (adverse
events, vital signs, laboratory parameters, physical examinations,
and electrocardiograms) as well as pharmacokinetic parameters.
[0284] All subjects administered UT-15C SR tablets and capsules had
detectable treprostinil plasma concentrations. Calculations of area
under the curve from zero to twenty-four hours (AUC.sub.0-24)
indicate that total exposure to UT-15C SR occurred in the following
order: Tablet Fed>Capsule Fasted>Tablet Fasted>Capsule
Fed. FIG. 14 displays the pharmacokinetic profiles of the two
formulations in the fasted and fed states.
[0285] UT-15C SR tablets and capsules were tolerated by the
majority of subjects. All adverse events were mild to moderate in
severity and were similar to those described in Study 01-101 and in
Remodulin's product labeling. Additionally, there were no
treatment-emergent changes in vital signs, laboratory parameters,
physical examinations, or electrocardiograms throughout the
study.
[0286] These results demonstrate that detectable and potentially
therapeutic drug concentrations can be obtained from a solid dosage
form of UT-15C and that these concentrations can be maintained over
an extended period of time through sustained release formulation
technology.
Polymorphs of Treprostinil Diethanolamine
[0287] Two crystalline forms of UT-15C were identified as well as
an amorphous form. The first, which is metastable, is termed Form
A. The second, which is thermodynamically more stable, is Form B.
Each form was characterized and interconversion studies were
conducted to demonstrate which form was thermodynamically stable.
Form A is made according to the methods in Table 15. Form B is made
from Form A, in accordance with the procedures of Table 16.
TABLE-US-00020 TABLE 15 XRPD Sample Solvent Conditions.sup.a
Habit/Description Result.sup.b ID tetrahydrofuran FE opaque white
solids; morphology A 1440- unknown, birefringent 72-02 SE glassy
transparent solids A (PO) 1440- 72-03 SC (60.degree. C.)
translucent, colorless glassy sheets A 1440- of material,
birefringent 72-16 Toluene slurry (RT), 6 d white solids; opaque
masses of A + B 1440- smaller particles 72-01 toluene:IPA
SC(60.degree. C.) white solids; spherical clusters of A 1480-
(11.4:1) fibers, birefringent 21-03 Water FE opaque white solids;
morphology A 1440- unknowm, birefringent 72-07 SE opaque ring of
solids, birefringent A + B 1440- 72-08 freeze dry white, glassy
transparent solids A + B 1480- 58-02 water:ethanol FE opaque white
solids; morphology A + 1440- (1:1) unknown, birefringent 11.5 pk
72-09 FE clear and oily substance with some B 1480- opaque solids
79-02 SE glassy opaque ring of solid A 1440- 72-10 .sup.aFE = fast
evaporation; SE = slow evaporation; SC = slow cool .sup.bIS =
insufficient sample; PO = preferred orientation; LC = low
crystallinity; pk = peak .sup.cXRPD = X-ray powder diffraction
TABLE-US-00021 TABLE 16 XRPD Solvent Conditions Habit/Description
Result Sample ID ethanol/water FE glassy appearing solids of
--.sup.b 1519-68-01 (1:1) unknown morphology; birefringent
1,4-dioxane slurry(50.degree. C.), 6 d white solids; opaque masses
of B 1519-73-02.sup.a material; morphology unknown
slurry(50.degree. C.), 2 d small grainy solids; with B 1557-12-01
birefringence subsample of -- B 1557-15-01 1557-12-01 subsample of
white solids B 1557-15-02 1557-12-01 slurry(50.degree. C.), 2 d --
B 1557-17-01 isopropanol slurry(RT), 1 d white solids --.sup.b
1519-96-03 tetrahydrofuran slurry(RT), 1 d -- --.sup.b 1519-96-02
toluene slurry(50.degree. C.), 6 d white solids B 1519-73-01
.sup.aSeeds of sample #1480-58-01 (A + B) added .sup.bSamples not
analyzed
Characterization of Crystal Forms:
Form A
[0288] The initial material synthesized (termed Form A) was
characterized using X-ray powder diffraction (XRPD), differential
scanning calorimetry (DSC), thermogravimetry (TG), hot stage
microscopy, infrared (IR) and Raman spectroscopy, and moisture
sorption. Representative XRPD of Form A is shown in FIG. 15. The IR
and Raman spectra for Form A are shown in FIGS. 16 and 17,
respectively. The thermal data for Form A are shown in FIG. 18. The
DSC thermogram shows an endotherm at 103.degree. C. that is
consistent with melting (from hot stage microscopy). The sample was
observed to recrystallize to needles on cooling from the melt. The
TG data shows no measurable weight loss up to 100.degree. C.,
indicating that the material is not solvated. The moisture sorption
data are shown graphically in FIG. 19. Form A material shows
significant weight gain (>33%) during the course of the
experiment (beginning between 65 to 75% RH), indicating that the
material is hygroscopic. In addition, hygroscopicity of
treprostinil diethanolamine was evaluated in humidity chambers at
approximately 52% RH and 68% RH. The materials were observed to
gain 4.9% and 28% weight after 23 days in the .about.52% RH and
.about.68% RH chambers, respectively.
[0289] Based on the above characterization data, Form A is a
crystalline, anhydrous material which is hygroscopic and melts at
103.degree. C.
Form B
[0290] Treprostinil diethanolamine Form B was made from heated
slurries (50.degree. C.) of Form A in 1,4 dioxane and toluene, as
shown in Table 16. Material isolated from 1,4-dioxane was used to
fully characterize Form B. A representative XRPD pattern of Form B
is shown in FIG. 20. Form A and Form B XRPD patterns are similar,
however, significant differences are observed in the range of
approximately 12-17.degree.2.theta. (FIG. 20).
[0291] The thermal data for Form B are shown in FIG. 21. The DSC
thermogram (Sample ID 1557-17-01) shows a single endotherm at
107.degree. C. that is consistent with a melting event (as
determined by hotstage microscopy). The TG shows minimal weight
loss up to 100.degree. C.
[0292] The moisture sorption/desorption data for Form B are shown
in FIG. 22. There is minimal weight loss at 5% RH and the material
absorbs approximately 49% water at 95% RH. Upon desorption from 95%
down to 5% RH, the sample loses approximately 47%.
[0293] Form A and Form B can easily be detected in the DSC curve.
Based on the above characterization data, Form B appears to be a
crystalline material which melts at 107.degree. C.
Thermodynamic Properties:
[0294] Inter-conversion experiments were carried out in order to
determine the thermodynamically most stable form at various
temperatures. These studies were performed in two different
solvents, using Forms A and B material, and the data are summarized
in Table 17. Experiments in isopropanol exhibit full conversion to
Form B at ambient, 15.degree. C., and 30.degree. C. after 7 days,
11 days, and 1 day, respectively. Experiments in tetrahydrofuran
also exhibit conversion to Form B at ambient, 15.degree. C., and
30.degree. C. conditions. Full conversion was obtained after 11
days at 15.degree. C., and 1 day at 30.degree. C. At ambient
conditions, however, a minor amount of Form A remained after 7 days
based on XRPD data obtained. Full conversion would likely occur
upon extended slurry time. Based on these slurry inter-conversion
experiments, Form B appears to be the most thermodynamically stable
form. Form A and Form B appear to be related monotropically with
Form B being more thermodynamically stable.
TABLE-US-00022 TABLE 17 Interconversion Studies of Treprostinil
Diethanolamine Experiment/ Sample No. Forms Solvent Starting
Materials Temperature Time 1557-22- A vs. B isopropanol solid
mixture ambient 7 days 01 # 1557-20-01.sup.a 1557-47- A vs. B solid
mixture 15.degree. C. 11 days 02 # 1557-35-01.sup.d 1557-33- A vs.
B solid mixture 30.degree. C. 1 day 02 # 1557-35-01.sup.d 1557-21-
A vs. B solid mixture 50.degree. C. -- 02.sup.e # 1557-20-01.sup.b
1557-20- A vs. B tetrahydrofuran solid mixture ambient 7 days 03 #
1557-20-01.sup.c 1557-47- A vs. B solid mixture 15.degree. C. 11
days 01 # 1557-35-01.sup.d 1557-33- A vs. B solid mixture
30.degree. C. 1 day 01 # 1557-35-01.sup.d 1557-21- A vs. B solid
mixture 50.degree. C. -- 01.sup.e # 1557-20-01.sup.c
.sup.asaturated solution Sample ID 1557-21-03 .sup.bsaturated
solution Sample ID 1519-96-03 .sup.csaturated solution Sample ID
1519-96-02 .sup.dsaturated solution prepared just prior to addition
of solids .sup.esamples not analyzed as solubility (at 50.degree.
C.) of treprostinil diethanolamine was very high and solutions
became discolored.
[0295] All references disclosed herein are specifically
incorporated by reference thereto.
[0296] While preferred embodiments have been illustrated and
described, it should be understood that changes and modifications
can be made therein in accordance with ordinary skill in the art
without departing from the invention in its broader aspects as
defined herein.
* * * * *