U.S. patent application number 15/716007 was filed with the patent office on 2018-06-07 for treprostinil prodrugs.
This patent application is currently assigned to United Therapeutics Corporation. The applicant listed for this patent is United Therapeutics Corporation. Invention is credited to Hitesh BATRA, Liang GUO, Ken PHARES, Adam Marc SILVERSTEIN.
Application Number | 20180153847 15/716007 |
Document ID | / |
Family ID | 60120132 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180153847 |
Kind Code |
A1 |
PHARES; Ken ; et
al. |
June 7, 2018 |
TREPROSTINIL PRODRUGS
Abstract
Provided are novel prodrugs of treprostinil, as well as methods
of making and methods of using these prodrugs.
Inventors: |
PHARES; Ken; (Hillsborough,
NC) ; BATRA; Hitesh; (Herndon, NC) ; GUO;
Liang; (Vienna, VA) ; SILVERSTEIN; Adam Marc;
(Cary, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
United Therapeutics Corporation |
Silver Spring |
MD |
US |
|
|
Assignee: |
United Therapeutics
Corporation
Silver Spring
MD
|
Family ID: |
60120132 |
Appl. No.: |
15/716007 |
Filed: |
September 26, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62399737 |
Sep 26, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/222 20130101;
A61K 9/0019 20130101; A61K 31/216 20130101; A61K 31/335 20130101;
A61K 31/265 20130101; A61K 31/192 20130101; A61P 9/12 20180101;
A61K 31/27 20130101 |
International
Class: |
A61K 31/27 20060101
A61K031/27; A61K 31/216 20060101 A61K031/216; A61K 31/192 20060101
A61K031/192; A61K 31/265 20060101 A61K031/265; A61K 9/00 20060101
A61K009/00; A61P 9/12 20060101 A61P009/12 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. A method of treating pulmonary hypertension comprising
administering subcutaneously to a patient suffering from pulmonary
hypertension an effective amount of a prodrug of treprostinil,
wherein the prodrug converts in whole or in part to treprostinil in
vivo following administration and has reduced affinity for one or
more of the IP, DP or EP receptors locally at the site of injection
as compared to treprostinil.
5. The method of claim 4, wherein said administering is continuous
subcutaneous administering.
6. The method of claim 4, wherein said administering results in no
or less pain at a site of the injection compared to administering
treprostinil.
7. A method of treating pulmonary hypertension comprising
administering subcutaneously to a patient suffering from pulmonary
hypertension an effective amount of a prodrug of treprostinil,
wherein the prodrug is a compound having the following formula:
##STR00107## wherein X is OR.sub.9 or NR.sub.1R.sub.6; wherein
R.sub.9 is H or C.sub.1-C.sub.4 alkyl, which may be optionally
substituted with a terminal hydroxyl or carboxy group; wherein
R.sub.1 is H or C.sub.1-C.sub.4 alkyl and R.sub.6 is ##STR00108##
or wherein R.sub.1 and R.sub.6 are such that NR.sub.1R.sub.6 is an
amide of an amino acid; R.sub.7 is H or C.sub.1-C.sub.4 alkyl,
which may be substituted with a terminal hydroxy or carboxy group;
R.sub.8 is H or C.sub.1-C.sub.4 alkyl; each of R.sub.2 and R.sub.3
is independently selected from H, C.sub.1-4 alkyl, ##STR00109##
phosphate and a group, in which OR.sub.2 or OR.sub.3 forms an ester
of an amino acid; Y is OR.sub.4 or NR.sub.4R.sub.5, each of R.sub.4
and R.sub.5 is independently selected from H and C.sub.1-4 alkyl;
with a proviso that all of R.sub.9, R.sub.2 and R.sub.3 are not H;
or a pharmaceutically acceptable salt of the compound.
8. The method of claim 4, wherein the prodrug is a compound having
the following formula: ##STR00110## wherein X is OH or
NR.sub.1R.sub.6, wherein R.sub.1 is H or C.sub.1-C.sub.4 alkyl and
R.sub.6 is ##STR00111## or wherein R.sub.1 and R.sub.6 are such
that NR.sub.1R.sub.6 is an amide of an amino acid; R.sub.7 is H or
C.sub.1-C.sub.4 alkyl, which may be substituted with a terminal
hydroxy or carboxy group, R.sub.8 is H or C.sub.1-C.sub.4 alkyl and
each of R.sub.2 and R.sub.3 is independently selected from H,
C.sub.1-4 alkyl, or ##STR00112## wherein Y is OR.sub.4 or
NR.sub.4R.sub.5, wherein each of R.sub.4 and R.sub.5 is
independently selected from H and C.sub.1-4 alkyl; with a proviso
that when X is OH, both of R.sub.2 and R.sub.3 are not H; or a
pharmaceutically acceptable salt of the compound
9. The method of claim 8 wherein: X is OH or ##STR00113##
10. The method of claim 7, wherein X is OH.
11. The method of claim 10, wherein each of R.sub.2 and R.sub.3 is
independently selected from C.sub.1-4 alkyl.
12. (canceled)
13. The method of claim 10, wherein each of R.sub.2 and R.sub.3 is
independently selected from H, and ##STR00114##
14. The method of claim 13, wherein one of R.sub.2 and R.sub.3 is
##STR00115## and the other of R.sub.2 and R.sub.3 is H.
15. The method of claim 14, wherein Y is OR.sub.4.
16. (canceled)
17. The method of claim 14, wherein Y is NR.sub.4R.sub.5.
18. (canceled)
19. (canceled)
20. (canceled)
21. The method of claim 10, wherein at least one of R.sub.2 and
R.sub.3 is phosphate.
22. (canceled)
23. (canceled)
24. The method of claim 10, wherein at least R.sub.2 and R.sub.3 is
a group, in which OR.sub.2 or OR.sub.3 forms an ester of an amino
acid.
25. (canceled)
26. The method of claim 7, wherein X is NR.sub.1R.sub.6 and each of
R.sub.2 and R.sub.3 are H.
27. The method of claim 26, wherein R.sub.1 is H.
28. The method of claim 27, wherein R.sub.6 is ##STR00116##
29. (canceled)
30. (canceled)
31. The method of claim 27, wherein R.sub.6 is ##STR00117##
32. (canceled)
33. The method of claim 26, wherein NR.sub.1R.sub.6 is an amide of
an amino acid.
34. The method of claim 7, wherein X is OR.sub.9, R.sub.9 is
C.sub.1-C.sub.4 alkyl, which may be optionally substituted with a
terminal hydroxyl or carboxy group, R.sub.2 and R.sub.3 are each
H.
35. The method of claim 34, wherein R.sub.9 is C.sub.1-C.sub.4
substituted with a terminal hydroxyl group.
36. The method of claim 4, wherein the prodrug has one of the
following formulas: ##STR00118## ##STR00119## ##STR00120##
##STR00121##
37. A compound or a pharmaceutically acceptable salt thereof,
wherein the compound having one of the following formulas:
##STR00122## ##STR00123## ##STR00124##
38. (canceled)
39. (canceled)
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. The method of claim 4, wherein the patient is a human, wherein
said prodrug has a half-life of less than 120 minutes.
49. (canceled)
50. (canceled)
51. The method of claim 48, wherein said prodrug has the half-life
in plasma of less than 15 minutes.
Description
RELATED APPLICATIONS
[0001] The present application claims priority to U.S. provisional
application No. 62/399,737 filed Sep. 26, 2016, which is
incorporated herein by reference in its entirety.
FIELD
[0002] The present application generally relates to prostacyclins
and more particularly, to prodrugs of treprostinil and to methods
of making and using such prodrugs.
BACKGROUND
[0003] Pulmonary hypertension is a progressive and life-threatening
disease characterized by increased pressure in the pulmonary
vasculature that can lead to, inter alia, heart failure.
[0004] Pulmonary hypertension (PH) has been previously classified
as primary (idiopathic) or secondary. The World Health Organization
(WHO) has classified pulmonary hypertension into five groups:
[0005] Group 1: pulmonary arterial hypertension (PAH); [0006] Group
1': Pulmonary veno-occlusive disease (PVOD) and/or pulmonary
capillary haemangiomatosis (PCH) [0007] Group 2: PH with left heart
disease; [0008] Group 3: PH with lung disease and/or hypoxemia;
[0009] Group 4: PH due to chronic thrombotic and/or embolic
disease; and [0010] Group 5: miscellaneous conditions; unclear
multifactorial mechanisms (e.g., sarcoidosis, histiocytosis X,
lymphangiomatosis and compression of pulmonary vessels).
[0011] There are currently a number of approved products for
certain types of pulmonary hypertension, including Group 1 (PAH).
Those products include products containing treprostinil as the
active ingredient, such as Remodulin.RTM. treprostinil injection.
Treprostinil, however, is sometimes associated with site pain when
administered subcutaneously. Thus, a need exists for administering
treprostinil without causing site pain.
SUMMARY
[0012] One embodiment is a method of treating a disease or
condition, comprising selecting a patient, who is suffering from
the disease or condition and who has experienced site pain upon
subcutaneous administration of treprostinil or a pharmaceutically
salt thereof and administering subcutaneously to the patient an
effective amount of a prodrug of treprostinil, wherein the disease
or condition is one or more disease or condition selected from the
group consisting of pulmonary hypertension, congestive heart
failure, peripheral vascular disease, Raynaud's phenomenon,
Scleroderma, renal insufficiency, peripheral neuropathy, digital
ulcers, intermittent claudication, ischemic limb disease,
peripheral ischemic lesions, pulmonary fibrosis and asthma.
[0013] Another embodiment is a method of treating pulmonary
hypertension comprising administering subcutaneously to a patient
suffering from pulmonary hypertension an effective amount of a
prodrug of treprostinil.
[0014] Yet another embodiment is a compound or a pharmaceutically
acceptable salt thereof, wherein the compound having one of the
following formulas:
##STR00001## ##STR00002## ##STR00003##
FIGURES
[0015] FIG. 1 is a scheme illustrating synthesis of cyclopentyl
ring carbamate prodrug I.
[0016] FIG. 2 is a scheme illustrating synthesis of side chain
carbamate prodrug II.
[0017] FIG. 3 is a scheme illustrating synthesis of cyclopentyl
ring carbonate prodrug III.
[0018] FIG. 4 is a scheme illustrating synthesis of side chain
carbonate prodrug IV.
[0019] FIG. 5 is a scheme illustrating synthesis of acetate amide
prodrug VII.
[0020] FIG. 6 is a scheme illustrating synthesis of a key starting
material.
[0021] FIG. 7 presents chemical formula of selected prodrugs.
[0022] FIG. 8 is a plot presenting plasma concentrations of
Remodulin and a prodrug of treprostinil as a function of time. The
data points shown for intravenous and subcutaneous Remodulin (shown
as "IV" and "SQ" respectively in the figure) were obtained from
clinical trials involving patients suffering from pulmonary
hypertension to show that subcutaneously administered treprostinil
was bioequivalent to intravenously administered treprostinil. The
plots marked "Upper Limit" and "Lower Limit" reflect a range of
bioequivalence for subcutaneous Remodulin. The plot for the prodrug
of treprostinil (shown as "PRODRUG") represents one possible set of
data points that would approximate the plot of subcutaneously
administered Remodulin, where the prodrug has been converted in
vivo to treprostinil free acid, and the amount of free acid
treprostinil in plasma is plotted.
[0023] FIG. 9 reports withdrawal time due to site pain in tested
rats, which were administered one of the following: a) saline, b) a
(Remodulin) placebo formulation, which contained citrate buffer,
sodium chloride and m-cresol, while not containing treprostinil; c)
a first Remodulin formulation with a treprostinil concentration of
1 .mu.g/mL, where the formulation contained treprostinil, citrate
buffer, sodium chloride and m-cresol; d) a second Remodulin
formulation with a treprostinil concentration of 100 .mu.g/mL,
where the formulation contained treprostinil, citrate buffer,
sodium chloride and m-cresol, with the vertical bars showing how
quickly the tested rat withdrew its paw in response to a heat
stimulus following administration of the formulations at t=0, 15
min and 90 min.
[0024] FIG. 10A-D show selected prodrugs.
[0025] FIG. 11 shows chromatographic overlay of treprostinil and
eight selected prodrugs using ACE Excel 2 C18 column. The results
of the specificity study indicate that treprostinil is well
separated from all prodrugs except Prodrug XIV which co-elutes.
[0026] FIG. 12 shows chromatographic overlay of treprostinil and
eight selected prodrugs using Waters BEH C18 column.
[0027] FIG. 13 shows chromatographic overlay of treprostinil and
eight selected prodrugs using ACE Excel 2 C18-AR column (C18-Phenyl
phase).
[0028] FIG. 14 shows chromatographic overlay of treprostinil and
eight selected prodrugs using Waters CSH Phenyl Hexyl column.
[0029] FIG. 15 summarizes half-live values for selected
prodrugs.
[0030] FIG. 16 is a schematic depiction of intraplantar model
induction and treatment used in Examples 9 and 11. Thermal test was
performed immediately post Von Frey test (up to 10 minutes
difference).
[0031] FIG. 17 presents results for Von Frey Response test of cycle
1 (g). Lower force indicates greater sensitivity. For each of
baseline, 15 min and 90 min test points, columns represent the
following left to right: Saline (Group 1), PBS (Group 2),
treprostinil 100 .mu.g/ml (Group 3), Prodrug I 100 .mu.g/ml (Group
4), Prodrug II 100 .mu.g/ml (Group 5), Prodrug VII 100 .mu.g/ml
(Group 6), Prodrug VIII 100 .mu.g/ml (Group 7).
[0032] FIG. 18 presents results for Von Frey Response test of cycle
2 (g). Lower force indicates greater sensitivity. For each of
baseline, 15 min and 90 min test time-points, columns represent the
following left to right: Saline (Group 8), PBS (Group 9),
treprostinil 1 .mu.g/ml (Group 10), Prodrug I 1 .mu.g/ml (Group
11), Prodrug II 1 .mu.g/ml (Group 12), Prodrug VII 1 .mu.g/ml
(Group 13), Prodrug VIII 1 .mu.g/ml (Group 14).
[0033] FIG. 19 presents results for Thermal Response test of cycle
1 (sec). Lower/faster time indicates greater sensitivity. For each
of baseline, 15 min and 90 min test time-points, columns represent
the following left to right: Saline (Group 1), PBS (Group 2),
treprostinil 100 .mu.g/ml (Group 3), Prodrug I 100 .mu.g/ml (Group
4), Prodrug II 100 .mu.g/ml (Group 5), Prodrug VII 100 .mu.g/ml
(Group 6), Prodrug VIII 100 .mu.g/ml (Group 7).
[0034] FIG. 20 presents results for Thermal Response test of cycle
2 (sec). Lower/faster time indicates greater sensitivity. For each
of Baseline, 15 min and 90 min test time-points, columns represent
the following left to right: Saline (Group 8), PBS (Group 9),
treprostinil 1 .mu.g/ml (Group 10), Prodrug I 1 .mu.g/ml (Group
11), Prodrug II 1 .mu.g/ml (Group 12), Prodrug VII 1 .mu.g/ml
(Group 13), Prodrug VIII 1 .mu.g/ml (Group 14).
[0035] FIG. 21 presents mean clinical score of cycle 1 (points).
Increased/Higher score indicates more observations of adverse
events. For each of 15 min and 90 min test time-points, data
presented the following left to right: Saline (Group 1), PBS (Group
2), treprostinil 100 .mu.g/ml (Group 3), Prodrug I 100 .mu.g/ml
(Group 4), Prodrug II 100 .mu.g/ml (Group 5), Prodrug VII 100
.mu.g/ml (Group 6), Prodrug VIII 100 .mu.g/ml (Group 7). For the 15
min time point, non-zero observations are as follows left to right:
treprostinil 100 .mu.g/ml (Group 3), Prodrug I 100 .mu.g/ml (Group
4), Prodrug II 100 .mu.g/ml (Group 5), Prodrug VII 100 .mu.g/ml
(Group 6), Prodrug VIII 100 .mu.g/ml (Group 7). For the 90 min time
point, non-zero observations are as follows left to right:
treprostinil 100 .mu.g/ml (Group 3), Prodrug I 100 .mu.g/ml (Group
4), Prodrug II 100 .mu.g/ml (Group 5), Prodrug VII 100 .mu.g/ml
(Group 6).
[0036] FIG. 22 presents mean clinical score of cycle 2 (points).
Increased/Higher score indicates more observations of adverse
events. For each of 15 min and 90 min test time-points, data
presented the following left to right: Saline (Group 8), PBS (Group
9), treprostinil 1 .mu.g/ml (Group 10), Prodrug I 1 .mu.g/ml (Group
11), Prodrug II 1 .mu.g/ml (Group 12), Prodrug VII 1 .mu.g/ml
(Group 13), Prodrug VIII 1 .mu.g/ml (Group 14). For each of 15 min
and 90 min test time-points, the only non-zero observations
correspond to Treprostinil 1 .mu.g/ml (Group 10).
[0037] FIG. 23 is a schematic depiction of intraplantar model
induction and treatment used in Example 10.
[0038] FIG. 24 presents results for Von Frey Response test (g).
Lower force indicated greater sensitivity. For each of Baseline, 15
min and 90 min test points, columns represent the following left to
right: Phosphate Buffer (Group 1), treprostinil 100 .mu.g/ml (Group
2), treprostinil 1 .mu.g/ml (Group 3), PRODRUG VII 100 .mu.g/ml
(Group 4), PRODRUG VII 1 .mu.g/ml (Group 5), Prodrug XV 100
.mu.g/ml (Group 6), Prodrug XV 1 .mu.g/ml (Group 7).
[0039] FIG. 25 presents results for Thermal Response test (sec).
Lower/faster time indicates greater sensitivity. For each of
Baseline, 15 min and 90 min test points, columns represent the
following left to right: Phosphate Buffer (Group 1), treprostinil
100 .mu.g/ml (Group 2), treprostinil 1 .mu.g/ml (Group 3), Prodrug
VII 100 .mu.g/ml (Group 4), Prodrug VII 1 .mu.g/ml (Group 5),
Prodrug XV 100 .mu.g/ml (Group 6), Prodrug XV 1 .mu.g/ml (Group
7).
[0040] FIG. 26 presents mean clinical score of cycle 1 (points).
Increased/Higher score indicates more observations of adverse
events. For each of 15 min and 90 min test time-points, data
presented the following left to right: Phosphate Buffer (Group 1),
treprostinil 100 .mu.g/ml (Group 2), treprostinil 1 .mu.g/ml (Group
3), Prodrug VII 100 .mu.g/ml (Group 4), Prodrug VII 1 .mu.g/ml
(Group 5), Prodrug XV 100 .mu.g/ml (Group 6), Prodrug XV 1 .mu.g/ml
(Group 7). Zero score was observed for phosphate buffer for each of
15 min and 90 min points. Thus, the first from the left non-zero
column represents treprostinil 100 .mu.g/ml (Group 2).
[0041] FIG. 27 presents results for Von Frey Response test of cycle
1 (g). Lower force indicates greater sensitivity. For each of
baseline, 15 min and 90 min test points, columns represent the
following left to right: Phosphate Buffer (Group 1), treprostinil
100 .mu.g/ml (Group 2), PRODRUG VII 100 .mu.g/ml (Group 3), Prodrug
III 100 .mu.g/ml (Group 4), Prodrug IV 100 .mu.g/ml (Group 5),
Prodrug XIV 100 .mu.g/ml (Group 6).
[0042] FIG. 28 presents results for Von Frey Response test of cycle
2 (g). Lower force indicates greater sensitivity. For each of
baseline, 15 min and 90 min test time-points, columns represent the
following left to right: Phosphate Buffer (Group 7), treprostinil 1
.mu.g/ml (Group 8), PRODRUG VII 1 .mu.g/ml (Group 9), Prodrug III 1
.mu.g/ml (Group 10), Prodrug IV 1 .mu.g/ml (Group 11), Prodrug XIV
1 .mu.g/ml (Group 12).
[0043] FIG. 29 presents results for Thermal Response test of cycle
1 (sec). Lower/faster time indicates greater sensitivity. For each
of baseline, 15 min and 90 min test time-points, columns represent
the following left to right: Phosphate Buffer (Group 1),
treprostinil 100 .mu.g/ml (Group 2), PRODRUG VII 100 .mu.g/ml
(Group 3), Prodrug III 100 .mu.g/ml (Group 4), Prodrug IV 100
.mu.g/ml (Group 5), Prodrug XIV 100 .mu.g/ml (Group 6).
[0044] FIG. 30 presents results for Thermal Response test of cycle
2 (sec). Lower/faster time indicates greater sensitivity. For each
of baseline, 15 min and 90 min test time-points, columns represent
the following left to right: Phosphate Buffer (Group 7),
treprostinil 1 .mu.g/ml (Group 8), PRODRUG VII 1 .mu.g/ml (Group
9), Prodrug III 1 .mu.g/ml (Group 10), Prodrug IV 1 .mu.g/ml (Group
11), Prodrug XIV 1 .mu.g/ml (Group 12).
[0045] FIG. 31 presents mean clinical score of cycle 1 (points).
Increased/Higher score indicates more observations of adverse
events. For each of 15 min and 90 min test time-points, data
presented the following left to right: Phosphate Buffer (Group 1),
treprostinil 100 .mu.g/ml (Group 2), PRODRUG VII 100 .mu.g/ml
(Group 3), Prodrug III 100 .mu.g/ml (Group 4), Prodrug IV 100
.mu.g/ml (Group 5), Prodrug XIV 100 .mu.g/ml (Group 6). Zero score
was observed for phosphate buffer for each of 15 min and 90 min
points. Thus, the first from the left non-zero column represents
Treprostinil 100 .mu.g/ml (Group 2).
[0046] FIG. 32 presents mean clinical score of cycle 2 (points).
Increased/Higher score indicates more observations of adverse
events. For each of 15 min and 90 min test time-points, data
presented the following left to right: Phosphate Buffer (Group 7),
treprostinil 1 .mu.g/ml (Group 8), PRODRUG VII 1 .mu.g/ml (Group
9), Prodrug III 1 .mu.g/ml (Group 10), Prodrug IV 1 .mu.g/ml (Group
11), Prodrug XIV 1 .mu.g/ml (Group 12). Zero score was observed for
phosphate buffer for each of 15 min and 90 min points. Thus, the
first from the left non-zero column represents Treprostinil 1
.mu.g/ml (Group 8).
[0047] FIG. 33 presents a summary of radiotelemetry data for heart
rate. Data presented as Means.+-.SEM.
[0048] FIG. 34 presents a summary of radiotelemetry data for
systolic blood pressure. Data presented as Means.+-.SEM.
[0049] FIG. 35 presents a summary of radiotelemetry data for
diastolic blood pressure. Data presented as Means.+-.SEM.
[0050] FIG. 36 presents a summary of radiotelemetry data for mean
arterial pressure. Data presented as Means.+-.SEM.
[0051] FIG. 37 presents a summary of radiotelemetry data for pulse
pressure. Data presented as Means.+-.SEM.
[0052] FIG. 38 presents a summary of radiotelemetry data for body
temperature. Data presented as Means.+-.SEM.
[0053] FIG. 39 schematically depicts a scheme for synthesis of
Prodrug VIII.
DETAILED DESCRIPTION
[0054] Unless otherwise specified, "a" or "an" refers to one or
more.
[0055] Treprostinil, the active ingredient in Remodulin (injected
or intravenous treprostinil), Tyvaso.RTM. (inhaled treprostinil),
and Orenitram.RTM. (oral solid dosage form of treprostinil), was
described in U.S. Pat. No. 4,306,075. Methods of making
treprostinil and other prostacyclin derivatives are described, for
example, in Moriarty, et al., J. Org. Chem. 2004, 69, 1890-1902,
Drug of the Future, 2001, 26(4), 364-374, U.S. Pat. Nos. 6,441,245,
6,528,688, 6,700,025, 6,809,223, 6,756,117, 8,461,393, 8,481,782;
8,242,305, 8,497,393, 8,940,930, 9,029,607, 9,156,786 and 9,388,154
9,346,738; U.S. Published Patent Application Nos. 2012-0197041,
2013-0331593, 2014-0024856, 2015-0299091, 2015-0376106,
2016-0107973, 2015-0315114, 2016-0152548, and 2016-0175319; PCT
Publication No. WO2016/0055819 and WO2016/081658.
[0056] Various uses and/or various forms of treprostinil are
disclosed, for examples, in U.S. Pat. Nos. 5,153,222, 5,234,953,
6,521,212, 6,756,033, 6,803,386, 7,199,157, 6,054,486, 7,417,070,
7,384,978, 7,879,909, 8,563,614, 8,252,839, 8,536,363, 8,410,169,
8,232,316, 8,609,728, 8,350,079, 8,349,892, 7,999,007, 8,658,694,
8,653,137, 9,029,607, 8,765,813, 9,050,311, 9,199,908, 9,278,901,
8,747,897, 9,358,240, 9,339,507, 9,255,064, 9,278,902, and
9,278,903, U.S. Published Patent Application Nos. 2009-0036465,
2008-0200449, 2008-0280986, 2009-0124697, 2014-0275616,
2014-0275262, 2013-0184295, 2014-0323567, 2016-0030371,
2016-0051505, 2016-0030355, 2016-0143868, 2015-0328232,
2015-0148414, 2016-0045470, and 2016-0129087, and PCT Publications
Nos. WO00/57701, WO20160105538, and WO2016038532.
[0057] Treprostinil has the following chemical formula:
##STR00004##
[0058] The present inventors discovered that administering certain
prodrugs of treprostinil by injection, such as subcutaneous
administration, may lead to less or no pain compared to
administering treprostinil or a salt of treprostinil, such as
treprostinil sodium, via the same administration route in the same
concentration.
[0059] One embodiment may be a method of treating a disease or
condition that can be treated by administering to a patient an
effective amount of a prodrug of treprostinil. The condition can be
pulmonary hypertension, and the patient can be a human. The
administration can occur by injection, such as subcutaneous
injection. In some embodiments, the prodrug can be administered
substantially continuously to the patient, such as by using an
appropriate pump.
[0060] Another embodiment may be a method of a method of treating a
disease or condition that can be treated by treprostinil comprising
selection of a patient, who suffers from the disease or condition
and who has experienced site pain upon administering treprostinil
or a salt of treprostinil, such as treprostinil sodium, and
administering to the patient an effective amount of a prodrug of
treprostinil. The condition can be pulmonary hypertension, and the
patient can be a human. The administration can occur by injection,
such as subcutaneous injection. In some embodiments, the prodrug
can be administered substantially continuously to the patient, such
as by using an appropriate pump.
[0061] Diseases/conditions that may be treated by treprostinil
include, but are not limited to, pulmonary hypertension, including
pulmonary arterial hypertension (PAH) and chronic thromboembolic
pulmonary hypertension; heart failure, such as congestive heart
failure; ischemic diseases, such as peripheral vascular disease,
Raynaud's phenomenon, Raynaud's disease, Buerger's disease,
Scleroderma, renal insufficiency, intermittent claudication,
ischemic limb disease, peripheral ischemic lesions; peripheral
neuropathy, including diabetic neuropathy; extremity lesions and/or
ulcers, such as foot ulcers and/or digital, ulcers (both finger
and/or toe), which may or may not be caused by an ischemic disease,
such as peripheral vascular disease, Raynaud's phenomenon,
Raynaud's disease, Buerger's disease, Scleroderma, intermittent
claudication, ischemic limb disease, and/or by peripheral
neuropathy, such as diabetic neuropathy; pulmonary fibrosis, cystic
fibrosis; asthma; cancer, which may be a cancer selected from the
group consisting of lung, liver, brain, pancreatic, kidney,
prostate, breast, colon and head-neck cancer.
[0062] In some embodiments, less or no pain associated with
administering a treprostinil prodrug as compared to administering
treprostinil or a salt of treprostinil has a number of benefits.
For example, patients that could not tolerate pain associated with
treprostinil may be able obtain the benefits of treprostinil
treatment by receiving the prodrug. A visual analogue score (VAS
score) may be collected continuously from a patient throughout the
duration of an infusion, such as subcutaneous infusion. The VAS
score may be then plotted as a function of time to calculate a pain
area-under-curve (AUC). Less or no pain compared to administering
treprostinil or a salt of treprostinil, such as treprostinil
sodium, which may be achieved by administering treprostinil prodrug
may mean a lower pain AUC for the treprostinil prodrug compared to
treprostinil or a salt of treprostinil, such as treprostinil
sodium. The VAS score method may allow quantification of more than
just pain intensity as it may also allow integration of the
intensity as well as monitoring change in the intensity with time.
The VAS score method is disclosed, for example, in Lydick E, et al.
Quality of Life Research. 1995; 4:41-45; and Van Wijk A J et al.
Eur J Pain. 2013; 17:394-401.
[0063] The term "effective amount" may mean an amount of a
treprostinil prodrug, which may be necessary to treat the disease
or condition. In some embodiments, an effective amount of
treprostinil prodrug may be the same or similar to an effective
amount of treprostinil for treating the same disease or condition.
In some embodiments, an effective amount of treprostinil prodrug
may be different from an effective amount of treprostinil for
treating the same disease or condition. A person of ordinary skill
in the art would be able to determine and "effective amount" of the
treprostinil prodrug based, for example, on the relevant disease or
condition, the amount of treprostinil known to treat, ameliorate,
or prevent the disease or condition, and the rate at which the
prodrug converts to treprostinil in vivo.
[0064] In some embodiments, the prodrug may be a prodrug may be a
prodrug disclosed in U.S. Pat. Nos. 7,384,978, 7,417,070,
7,544,713, 8,252,839, 8,410,169, 8,536,363, 9,050,311, 9,199,908,
9,278,901, 9,422,223 and 9,624,156, which are incorporated herein
by reference in their entirety.
[0065] In some embodiments, the prodrug may be a prodrug disclosed
in U.S. Pat. Nos. 9,371,264, 9,394,227, 9,505,737, and 9,643,911,
which are incorporated herein by reference in their entirety.
[0066] In some embodiments, the prodrug may be one of prodrugs
discussed below.
[0067] For example, in some embodiments, the prodrug may be a
compound having the following formula:
##STR00005##
wherein X is OR.sub.9 or NR.sub.1R.sub.6; wherein R.sub.9 is H or
C.sub.1-C.sub.4 alkyl, which may be optionally substituted with a
terminal hydroxyl or carboxy group; wherein R.sub.1 is H or
C.sub.1-C.sub.4 alkyl and R.sub.6 is
##STR00006##
or wherein R.sub.1 and R.sub.6 are such that NR.sub.1R.sub.6 is an
amide of an amino acid; R.sub.7 is H or C.sub.1-C.sub.4 alkyl,
which may be substituted with a terminal hydroxy or carboxy group;
R.sub.8 is H or C.sub.1-C.sub.4 alkyl; each of R.sub.2 and R.sub.3
is independently selected from H, C.sub.1-4 alkyl,
##STR00007##
phosphate and a group, in which OR.sub.2 or OR.sub.3 forms an ester
of an amino acid; Y is OR.sub.4 or NR.sub.4R.sub.5, each of R.sub.4
and R.sub.5 is independently selected from H and C.sub.1-4 alkyl;
with a proviso that all of R.sub.9, R.sub.2 and R.sub.3 are not H;
or
[0068] a pharmaceutically acceptable salt of the compound.
[0069] In some embodiments, the prodrug may be a compound having
the following formula:
##STR00008##
wherein X is OH or NR.sub.1R.sub.6, wherein R.sub.1 is H or
C.sub.1-C.sub.4 alkyl and R.sub.6 is
##STR00009##
or wherein R.sub.1 and R.sub.6 are such that NR.sub.1R.sub.6 is an
amide of an amino acid; R.sub.7 is H or C.sub.1-C.sub.4 alkyl,
which may be substituted with a terminal hydroxy or carboxy group,
R.sub.8 is H or C.sub.1-C.sub.4 alkyl and each of R.sub.2 and
R.sub.3 is independently selected from H, C.sub.1-4 alkyl, or
##STR00010##
wherein Y is OR.sub.4 or NR.sub.4R.sub.5, wherein each of R.sub.4
and R.sub.5 is independently selected from H and C.sub.1-4 alkyl;
with a proviso that when X is OH, both of R.sub.2 and R.sub.3 are
not H; or a pharmaceutically acceptable salt of the compound.
[0070] In some embodiments, the prodrug is a compound of the
following formula:
##STR00011##
wherein: X may be OH or
##STR00012##
where R.sub.1 is H or an alkyl, such as C.sub.1-C.sub.4 alkyl; each
of R.sub.2 and R.sub.3 may be independently selected from H,
C.sub.1-4 alkyl,
##STR00013##
wherein Y may be OR.sub.4 or NR.sub.4R.sub.5, each of R.sub.4 and
R.sub.5 is independently selected from H and C.sub.1-4 alkyl, with
a proviso that when X is OH, both of R.sub.2 and R.sub.3 are not H;
or a pharmaceutically acceptable salt thereof.
[0071] Examples of C.sub.1-4 alkyl may include methyl, ethyl,
propyl, isopropyl, n-butyl, sec-butyl, isobutyl or t-butyl.
[0072] Examples of C.sub.1-4 alkyl substituted with a terminal
hydroxyl group may include hydroxymethyl; hydroxyl ethyl;
hydroxypropyl; 4-hydroxybutyl; 2-methyl-3-hydroxy propyl.
[0073] Examples of C.sub.1-4 alkyl substituted with a terminal
carboxy group may include carboxymethyl, carboxyethyl,
carboxypropyl, 4-carboxybutyl, 2-methyl-3-carboxy propyl.
[0074] In some embodiments, X may be OH. In such a case, in certain
embodiments, each of R.sub.2 and R.sub.3 may be each independently
selected from a C.sub.1-4 alkyl. R.sub.2 and R.sub.3 may be the
same or different. In some cases, R.sub.2 and R.sub.3 may be the
same. For example, both of R.sub.2 and R.sub.3 may be ethyl. Yet in
some other cases, R.sub.2 and R.sub.3 may be different. For
example, R.sub.2 may be methyl and R.sub.3 may be ethyl or vice
versa.
[0075] In some embodiments, when X is OH, each of R.sub.2 and
R.sub.3 may be independently selected from H and
##STR00014##
In some cases, one of R.sub.2 and R.sub.3 may be
##STR00015##
while the other is H. Yet in some other cases, both of R.sub.2 and
R.sub.3 may be represented as
##STR00016##
while being the same or different. In some embodiments, Y may be
OR.sub.4. In such a case, R.sub.4 may be H or C.sub.1-4 alkyl, such
as methyl. In some cases, Y may be NR.sub.4R.sub.5. In such a case,
each of R.sub.4 and R.sub.5 may be independently selected from H
and C.sub.1-4 alkyl, such as methyl. In some embodiments, R.sub.4
and R.sub.5 may be the same. For example, in some embodiments, both
of R.sub.4 and R.sub.5 may be H or both of R.sub.4 and R.sub.5 may
be methyl. Yet in some embodiments, R.sub.4 and R.sub.5 may be
different. For example, one of R.sub.4 and R.sub.5 may be H, while
the other may be methyl.
[0076] In some embodiments, when X is OH, at least one R.sub.2 and
R.sub.3 may be phosphate. In certain cases, both of R.sub.2 and
R.sub.3 may be phosphate. In certain other cases, one of R.sub.2
and R.sub.3 may be phosphate and the other may be H.
[0077] In some embodiments, when X is OH, at least one of R.sub.2
or R.sub.3 may be a group, in which OR.sub.2 (or OR.sub.3) forms an
ester of an amino acid. In certain embodiments, one of R.sub.2 or
R.sub.3 may be a group, in which OR.sub.2 (or OR.sub.3) forms an
ester of an amino acid, while the other may be H. For example,
OR.sub.2 may form an ester of an amino acid, while R.sub.3 is H; or
OR.sub.3 may form an ester of an amino acid, while R.sub.2 is H. In
certain embodiments, R.sub.2 and R.sub.3 may be such that OR.sub.2
and OR.sub.3 each form an ester of an amino acid. In certain cases,
OR.sub.2 and OR.sub.3 may form an ester of the same amino acid. Yet
in certain cases, OR.sub.2 may form an ester of a first amino acid,
while OR.sub.3 may form an ester of a second amino acid, which is
different from the first amino acid.
[0078] Amino acid(s) may be a D-isomer amino acid or an L-isomer
amino acid. In certain embodiments, an amino acid may be a
naturally occurring amino acid. Yet, in some embodiments, an amino
acid may be an artificial amino acid. Examples of amino acids
include, but not limited to, carbamic acid, glycine, alanine,
valine, leucine, isoleucine, methionine, proline, phenylalanine,
tryptophan, serine, threonine, asparagine, glutamine, tyrosine,
cysteine, lysine, arginine, histidine, asparatice acid, glutamic
acid. When OR.sub.2 (OR.sub.3) forms an ester of an amino acid,
R.sub.2 (R.sub.3) may have
##STR00017##
(R.sub.4 and R.sub.5 as defined above) or
##STR00018##
where R.sub.10 is selected from the group consisting of amino acid
side chains, R.sub.11 and R.sub.12 may be H. In the embodiments
wherein the amino acid is proline, R.sub.11 together with R.sub.10
forms a pyrrolidine ring structure, while R.sub.12 is H. R.sub.10
may be, for example, one the naturally occurring amino acid side
chains, for example --CH.sub.3 (alanine),
--(CH.sub.2).sub.3HCNH.sub.2NH (arginine), --CH.sub.2CONH.sub.2
(asparagine), --CH.sub.2COOH (aspartic acid), --CH.sub.3SH
(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), --CH.sub.2Ph
(phenylalanine), --CH.sub.2OH (serine), --CHOHCH.sub.3 (threonine),
--CH(CH.sub.3).sub.2 (valine),
##STR00019##
--(CH.sub.2).sub.3NHCONH.sub.2 (citrulline) or
--(CH.sub.2).sub.3NH.sub.2 (ornithine). Ph designates a phenyl
group.
[0079] In some embodiments, each of R.sub.2 and R.sub.3 are H. In
such case, in certain embodiments, X may be NR.sub.1R.sub.6.
R.sub.1 may be H or C.sub.1-C.sub.4 alkyl. R.sub.6 may be
##STR00020##
R.sub.7 may be H or C.sub.1-C.sub.4 alkyl, which may be optionally
substituted with a terminal hydroxy or carboxy group, R.sub.8 may
be H or C.sub.1-C.sub.4 alkyl. In certain embodiments, R.sub.1 and
R.sub.6 are such that NR.sub.1R.sub.6 may form an amide of an amino
acid.
[0080] In certain embodiments, R.sub.1 may be H. In such case, in
some embodiments, R.sub.6 may be
##STR00021##
where R.sub.7 may be H or C.sub.1-C.sub.4 alkyl, which may be
optionally substituted with a terminal hydroxy or carboxy
group.
[0081] In certain embodiments, R.sub.1 may be H and R.sub.6 may
##STR00022##
where R.sub.8 may be H or C.sub.1-C.sub.4 alkyl, such as methyl or
ethyl.
[0082] In certain embodiments, when R.sub.2 and R.sub.3 are each H,
NR.sub.1R.sub.6 may form an amide of an amino acid, which may be an
amino acid discussed above. NR.sub.1R.sub.6 may be, for example,
or
##STR00023##
In certain cases, R.sub.1 may be H and R.sub.10 may be as defined
above. In case of proline being the amino acid, R.sub.1 and
R.sub.10 may form together a pyrrolidine ring structure.
[0083] In certain cases, when R.sub.2 and R.sub.3 are each H, X may
be OR.sub.9, R.sub.9 may be C.sub.1-C.sub.4 alkyl, which may be
optionally substituted with a terminal hydroxyl or carboxy group.
When R.sub.9 is C.sub.1-C.sub.4 alkyl is substituted with a
terminal carboxy group, R.sub.9 may be carboxymethyl, carboxyethyl,
carboxypropyl, 4-carboxybutyl, 2-methyl-3-carboxy propyl.
[0084] In some embodiments, the prodrug may be a compound having
one of the following formulas:
##STR00024## ##STR00025## ##STR00026## ##STR00027##
[0085] These prodrugs may have one or more advantages compared to
treprostinil in addition to or alternative to reduction in site
pain compared to administration of treprostinil or a salt thereof.
For example, some of these prodrugs may have improved stability or
greater tolerance in at least some patient populations.
[0086] At least some of these prodrugs may have half-life in human
plasma of less than 150 minutes or less than 120 minutes or less
than 90 minutes or less than 60 minutes or less than 50 minutes or
less than 45 minutes or less than 40 minutes or less than 30
minutes or less than 20 minutes or less than 15 minutes or less
than 12 minutes or about 10 minutes.
[0087] In certain embodiments, a prodrug of treprostinil may have
equilibrium water solubility of at least 1 mg/mL, or at least 2
mg/mL or at least 3 mg/mL or at least 4 mg/mL or at least 5 mg/mL
or at least 6 mg/mL. In certain embodiments, a prodrug of
treprostinil may have equilibrium water solubility from 3 to 40
mg/mL or from 3 to 35 mg/mL or from 5 to 15 mg/mL or any value or
subrange within these ranges. The solubility of the prodrug may be
greater if pH is increased in a vehicle used in solubility
measurement and/or if one or more salts are removed from the
vehicle.
[0088] Although Remodulin is approved by FDA for subcutaneous
administration, some patients experience site pain as the result of
such administration. Although the present invention is not bound by
any particular theory, this site pain may be the result of the
presence of treprostinil itself as opposed to inactive ingredients,
such as m-cresol, or treprostinil in combination with any inactive
ingredient. FIG. 9 reports withdrawal time at t=0, 15 min, and 90
min due to site pain by tested rats using the rat paw pain model,
in which rats were administered one of the following: a) saline, b)
a placebo formulation that contained citrate buffer, sodium
chloride, and m-cresol but no treprostinil (shown as "Remodulin
Placebo" in FIG. 9); c) a Remodulin formulation with a treprostinil
concentration of 1 .mu.g/mL containing treprostinil, citrate
buffer, sodium chloride and m-cresol (shown as "Treprostinil 1
.mu.g/mL" in FIG. 9); and d) a Remodulin formulation with a
treprostinil concentration of 100 .mu.g/mL containing treprostinil,
citrate buffer, sodium chloride and m-cresol (shown as
"Treprostinil 100 .mu.g/mL" in FIG. 9). The vertical bars in FIG. 9
show how quickly the tested rats withdrew their paws in response to
a heat stimulus following administration of the formulations at
t=0, 15 min and 90 min. The data indicates that tested rats were
more sensitive to the heat stimulus and withdrew their paws more
quickly in the case of the formulations that contained
treprostinil, whereas the Remodulin Placebo (containing the
inactive ingredients of Remodulin but no treprostinil) did not
increase their sensitivity.
[0089] Although the present invention is not limited by its theory
of operation, site pain during subcutaneous administration may be
due to treprostinil binding to one or more of the IP, DP or EP
receptors at the site of the injection. Treprostinil may bind to
these receptors at three functional locations, which correspond to
three hydroxyl groups on the molecule, see e.g., Tsai and Wu,
Eicosanoids, 2(3): 131-43 (1989). Accordingly, prodrugs of
treprostinil with one or more groups attached to treprostinil's
hydroxyl group(s), or other modifications that reduce binding to
these receptors, may have less affinity for the receptors locally
at the site of administration than treprostinil.
[0090] The phrase "prodrug of treprostinil" (also referred to
"treprostinil prodrug" or just "prodrug" depending on context) as
used herein refers to any derivative of treprostinil that converts
in whole or in part to treprostinil in vivo following
administration. The prodrug of treprostinil may have reduced
affinity for one or more of the IP, DP or EP receptors locally at
the site of injection as compared to treprostinil. In some
embodiments, a "prodrug of treprostinil" can be a treprostinil
derivative with one or more hydroxyl groups of the treprostinil
structure modified to have reduced affinity for one or more of the
IP, DP or EP receptors as compared to treprostinil, but which can
be converted in vivo into active treprostinil following
subcutaneous administration and subsequent diffusion into the
blood. In some embodiments, the prodrug of treprostinil is
completely or substantially converted in vivo to treprostinil
outside the subcutaneous space, such as in the bloodstream.
Preferred prodrugs include the compounds of formula I above. Other
preferred prodrugs of treprostinil include amide, carbonate, or
carbamate esters of treprostinil. In some embodiments, the prodrug
of treprostinil has greater than 50%, 75%, 85%, 90%, 95%, or 98%
conversion to treprostinil in vivo following administration. In
some embodiments, this conversion takes place in 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hour, or 3 hours following
administration. Prodrugs of treprostinil include pharmaceutically
acceptable salts of such prodrugs.
[0091] Preferably, the prodrug of treprostinil is stable during
storage, for example, by not hydrolyzing into treprostinil
spontaneously in a solution before administering or during initial
injection and at the site of injection. Preferably, the prodrug
formulations of the present invention are free of treprostinil or
substantially free of treprostinil in free acid form. In some
embodiments, less than 10%, 5%, 2%, 1%, or 0.1% of the prodrug of
treprostinil converts to treprostinil during a defined storage
period. In some embodiments, that defined storage period can be 1,
2, 3, 6, or 12 months.
[0092] The prodrug of treprostinil when administered subcutaneously
is preferably bioequivalent to subcutaneous administration of
Remodulin. In one embodiment, the administered prodrug provides a
plasma concentration of treprostinil that is between 80-125% of the
C.sub.max and AUC for subcutaneous Remodulin. See, e.g.,
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformatio-
n/Guidances/UCM377465.pdf. In another embodiment, the C.sub.max and
AUC values are between 95% and 105% of the subcutaneous Remodulin
C.sub.max and AUC levels. Remodulin is preferably infused
subcutaneously at 1.25 ng/kg/min, but if this initial dose cannot
be tolerated due to side effects, the FDA approved label provides
for reducing the infusion rate to 0.625 ng/kg/min.
[0093] FIG. 8 presents plasma concentration of treprostinil as a
function of time. The lower and upper limits in FIG. 8 correspond
respectively to 75% and 125% of the plasma concentration for
subcutaneously administered Remodulin, which represents one
preferred range of bioequivalent plasma concentrations for
targeting with the prodrugs of the invention. FIG. 8 shows a plot
for one possible subcutaneously administered prodrug formulation of
treprostinil, which fits between the lower and upper limits, and
therefore is bioequivalent to subcutaneously administered Remodulin
in terms of plasma concentrations of treprostinil measured over a
certain time period.
[0094] The disclosed treprostinil prodrugs, such as amide,
carbamate, and carbonate prodrugs, may have one or more advantages
over common ester prodrugs especially for parenteral administering,
including subcutaneous administration. For example, the disclosed
treprostinil prodrugs, such as amide, carbamate and carbonate
prodrugs, may be more stable than common ester prodrugs, which may
have a tendency to hydrolyze, thereby prematurely converting to
treprostinil when it is not desired, e.g., in a solution or at an
injection site.
[0095] A "pharmaceutically acceptable salt" includes a salt with an
inorganic base, organic base, inorganic acid, organic acid, or
basic or acidic amino acid. A salt of an inorganic base may be a
salt of an alkali metal such as sodium or potassium; a salt of an
alkaline earth metal such as calcium and magnesium or aluminum; and
a salt of ammonia. A salt of an organic base may be, for example,
trimethylamine, triethylamine, pyridine, picoline, ethanolamine,
diethanolamine, and triethanolamine. A salt of an inorganic acid
may be, for example, a salt of hydrochloric acid, a salt of
hydroboric acid, a salt of nitric acid, a salt of sulfuric acid, or
a salt of phosphoric acid. A salt of an organic acid may be, for
example, a salt of one of the following acids: 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. A salt of a basic amino acid may be, for example, for
example, a salt of arginine, lysine or ornithine. A salt of an
acidic amino acid may be, for example, a salt of aspartic acid or
glutamic acid.
[0096] "Pulmonary hypertension" refers to all forms of pulmonary
hypertension, WHO Groups 1-5. Pulmonary arterial hypertension, also
referred to as PAH, refers to WHO Group 1 pulmonary hypertension.
PAH includes idiopathic, heritable, drug- or toxin-induced, and
persistent pulmonary hypertension of the newborn (PPHN).
[0097] Treprostinil prodrugs of the invention may be provided in a
form of a pharmaceutical composition, which may also comprise a
pharmaceutically acceptable carrier, excipient, binder, diluent or
the like. Such pharmaceutical composition may be manufactured by
methods known in the art such as granulating, mixing, dissolving,
encapsulating, lyophilizing, emulsifying or levigating processes,
among others. The composition may be in the form of, for example,
granules, powders, tablets, capsules, syrup, suppositories,
injections, emulsions, elixirs, suspensions and solutions. The
composition may be formulated for a number of different
administration routes, such as, for oral administration,
transmucosal administration, rectal administration, transdermal or
subcutaneous administration, as well as intrathecal, intravenous,
intramuscular, intraperitoneal, intranasal, intraocular or
intraventricular injection. The treprostinil prodrug may be
administered by any of the above routes, for example in a local
rather than a systemic administration, including as an injection or
as a sustained release formulation.
[0098] In one embodiment, the pharmaceutical composition can
compromise a prodrug of treprostinil and a carrier, such as sterile
water. In some embodiments, the prodrug of treprostinil is
formulated for subcutaneous administration, and such formulation
may or may not include m-cresol or another preservative.
[0099] For oral, buccal, and sublingual administration, powders,
suspensions, granules, tablets, pills, capsules, gelcaps, and
caplets may be acceptable as solid dosage forms. These can be
prepared, for example, by mixing one or more treprostinil prodrugs,
or pharmaceutically acceptable salts thereof, with at least one
additive or excipient such as a starch or other additive. Suitable
additives or excipients may be 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 may 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.
[0100] 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.
[0101] 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. 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.
[0102] 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. 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.
[0103] A treprostinil prodrug may be formulated in a formulation
suitable for parenteral administration that may comprise sterile
aqueous preparations of a treprostinil prodrug, or a
pharmaceutically acceptable salt thereof, where the preparations
may be isotonic with the blood of the intended recipient. These
preparations may be administered by means of subcutaneous
injection, although administration may also be effected
intravenously or by means of intramuscular or intradermal
injection. Such preparations may conveniently be prepared by
admixing the compound with water or a glycine or citrate buffer and
rendering the resulting solution sterile and isotonic with the
blood. Injectable formulations according to the invention may
contain from 0.1 to 5% w/v based on weight of treprostinil in the
prodrug and may be administered at a rate of 0.1 ml/min/kg.
Alternatively, the invention may administered at a rate of 0.625 to
50 ng/kg/min based on weight of treprostinil in the prodrug.
Alternatively, the invention may be administered at a rate of 10 to
15 ng/kg/min based on weight of treprostinil in the prodrug.
[0104] In some embodiments, a concentration of a treprostinil
prodrug in a formulation for parenteral administration, such as
intravenous infusion or subcutaneous infusion (including continuous
subcutaneous infusion), may be from 0.0005 to 30 mg/mL or from
0.0007 to 50 mg/mL or from 0.001 to 15 mg/mL or any value or
subrange within these ranges. Exemplary concentrations may include
0.1 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL or 10 mg/mL.
[0105] In some embodiments, a formulation of a treprostinil prodrug
for parenteral administration, such as intravenous infusion or
subcutaneous infusion (including continuous subcutaneous infusion),
may be prepared by admixing the prodrug with a vehicle, such as a
buffer. In certain embodiments, the vehicle may be a phosphate
containing vehicle, i.e. at least one phosphate salt, which may be
for example, dibasic phosphate, such as sodium dibasic phosphate or
potassium dibasic phosphate, or tribasic phosphate, such as sodium
tribasic phosphate or potassium phosphate. In certain embodiments,
the vehicle may also contain a halogen salt, such as a chloride
salt, which may be, for example, sodium chloride or potassium
chloride. The halogen salt, such as sodium chloride may be used to
adjust tonicity of the vehicle. In certain embodiments, it may be
preferred that a phosphate and a halogen salt have the same cation.
For example, when a phosphate is sodium phosphate, such as sodium
tribasic phosphate or sodium tribasic phosphate, a halogen salt may
a sodium halogen salt such as sodium chloride.
[0106] Similarly, when a phosphate is potassium phosphate, such as
potassium tribasic phosphate or potassium tribasic phosphate, a
halogen salt may a potassium halogen salt such as potassium
chloride. A solvent in the vehicle may contain water. In certain
embodiments, water may be the only solvent in the vehicle. Yet in
certain embodiments, the vehicle may contain one or more additional
solvent in addition to water. In some embodiments, an additional
solvent may be a preservative, such as m-cresol.
[0107] Preferably, the vehicle is isotonic with blood of a patient,
such as a human being. The term isotonic may mean that the
osmolarity and ion concentrations of the vehicle match those of the
patient, such as human being. Non-limiting example of vehicles
include phosphate-buffered saline, which is a water based salt
solution containing disodium hydrogen phosphate, sodium chloride
and, in some formulations, potassium chloride and potassium
dihydrogen phosphate. Other examples may include a vehicle
containing 20 mM disbasic sodium phosphate with 125 mM sodium
chloride and a vehicle containing 15 mM sodium phosphate tribasic,
125 mM sodium chloride and 0.3% w/w m-cresol.
[0108] In certain embodiments, a treprostinil prodrug may be
administered subcutaneously. In some embodiments, the subcutaneous
administration may be continuous subcutaneous infusion, such as
continuous subcutaneous infusion by an infusion pump, which is
preferably portable or implantable.
[0109] In some embodiments, a treprostinil prodrug may be
administered subcutaneously at a rate (dose) of 0.1 to 100
ng/kg/min or 0.2 to 70 ng/kg/min or 0.3 to 50 ng/kg/min or 0.6 to
10 ng/kg/min based on weight of treprostinil in the prodrug or any
value or subrange within these ranges. In some embodiments, the
infusion may start at an initial rate (dose), which may be later
increased or decreased based on a patient's response to the initial
rate (dose). For example, an initial rate (dose) may be 1.25
ng/kg/min, which may be increased in increments of 1.25 ng/kg/min
per week or 2.5 ng/kg/min per week depending on the patient's
tolerance. If the patient does not tolerate the initial rate (dose)
due to, for example, a side effect, which may be, for example, mild
to moderate hepatic insufficiency and/or headache, the initial rate
(dose) may be reduced down to 0.625 ng/kg/min. After the patient
develops a tolerance to the lower rate (dose), the rate (dose) may
be increased.
[0110] The treprostinil prodrugs may be used for one or more of the
same purposes for which treprostinil is known to be useful. For
example, the treprostinil prodrugs may be used for administering to
a subject, such as a human being, for treating a disease or
disorder, which may be treated with treprostinil, such as pulmonary
hypertension, including pulmonary arterial hypertension and chronic
thromboembolic pulmonary hypertension. For therapeutic purposes,
such as treating pulmonary hypertension, a treprostinil prodrug may
be administered to a subject, such a human being, in a
therapeutically effective amount, which may be an amount of the
treprostinil prodrug, which is sufficient to ameliorate one or more
symptoms of a disease or disorder, which may be treated with
treprostinil, such as pulmonary hypertension.
[0111] The treprostinil prodrugs may be used therapeutically,
including in cytoprotection, reducing cell proliferation, promoting
vasodilation and/or inhibiting platelet aggregation. In some
embodiments, the treprostinil prodrugs may be used in treatment of
a vascular disease, such as pulmonary hypertension, heart failure
(including congestive heart failure), or peripheral vascular
disease. The treprostinil prodrugs may have vasodilating effects so
that they may be used for treating pulmonary hypertension, which
may, for example, result from one or more forms of connective
tissue disease, such as lupus, scleroderma or mixed connective
tissue disease.
[0112] The treprostinil prodrugs may be also used in cancer,
coagulation disorders, and inflammatory diseases. Use of
treprostinil for inhibiting metastasis of cancer cells is disclosed
in US 2003-0108512 and U.S. Pat. No. 6,803,386, which are both
incorporated herein in their entirety.
[0113] Treprostinil prodrugs may be prepared according to methods
illustrated in FIGS. 1-6 and as demonstrated in examples below.
[0114] Scheme 2 illustrates synthesis of acetate amide Prodrug VII.
This synthesis may start with treprostinil reacted with
NH.sub.2CH.sub.2COOBn to form a protected acetato amide compound of
the following formula:
##STR00028##
In some embodiments, an alkyl, such as C.sub.1-C.sub.4 alkyl, e.g.
methyl or ethyl, may be used instead of benzyl. In such case,
NH2CH2COOR.sub.4, where R.sub.4 is an alkyl, may be used for
reacting with treprostinil while forming a protected acetato amide
compound instead of NH2CH2COOBn.
[0115] The protected acetato amide compound may then transferred
into acetato amide Prodrug VII. In case of Bn, such reaction may
involve using Pd/C and H.sub.2. In case of an alkyl, such as methyl
or ethyl, the protected acetato amide compound may be reacted with
a base, such as NaOH or KOH, to be transferred into acetato amide
Prodrug VII.
[0116] FIG. 6 illustrates synthesis of a starting compound of the
following formula:
##STR00029##
where P.sub.1 may be a hydroxyl protecting group, such as
2-tetrahydropyranyl (THP) or a silyl protecting group, such as
tert-Butyldimethylsilyl ether (TBDMS/TBS), Trimethylsilyl (TMS)
Triethylsilyl (TES), tert-Butyldiphenylsilyl (TBDPS),
Triisopropylsilyl (TIPS). This compound may be used as an important
starting compound for synthesizing several treprostinil prodrugs.
The process in FIG. 6 corresponds to the first five reaction of
Scheme 2 of U.S. Pat. No. 8,940,930, which is incorporated herein
in its entirety. Besides the process disclosed in FIG. 6, the
starting compound may also be synthesized for example, using
methods disclosed U.S. Pat. Nos. 6,756,117 and 6,809,223. The
synthesis of the starting compound may start a compound of the
following formula:
##STR00030##
where P.sub.0 is a hydroxyl protecting group, such as benzyl or a
substituted benzyl. A substituted benzyl group may be optionally
substituted at one or more meta, ortho or para positions with one
or more substituents, which may be independently selected from the
group consisting of --NO.sub.2, --CN, halogen (e.g., --F, --Cl,
--Br or --I), (C1-C3)alkyl, halo(C1-C3)alkyl, (C1-C3)alkoxy and
halo(C1-C3)alkoxy. This compound
##STR00031##
may be reacted with
##STR00032##
in the presence of (+)-N-methylephedrine, Zn(OTf.sub.2)/Et.sub.3N
or using
(1S,2S)-3-(tertiary-butyldimethylsilyloxy)-2-N,N-dimethylamino-L-(p-
ara-nitrophenyl)-propane-1-ol to form a compound of the following
formula
##STR00033##
This compound may then transferred into the key starting compound
using the reactions disclosed in U.S. Pat. No. 6,940,930.
[0117] The starting compound of the following formula
##STR00034##
may be used for synthesizing cyclopentyl ring prodrugs of
treprostinil, i.e. compounds with X being OH and R.sub.3 being H,
or side chain prodrugs of treprostinil, i.e. compounds with X being
OH and R.sub.2 being H. Synthesis of cyclopentyl ring prodrugs is
illustrated in FIGS. 1 and 3, while synthesis of side chain
prodrugs is shown in FIGS. 2 and 4.
[0118] For synthesizing the cyclopentyl ring prodrugs, the starting
compound of the following formula
##STR00035##
may be converted into a double-protected compound of the following
formula:
##STR00036##
where P.sub.0 is a hydroxyl protecting group, such as benzyl,
substituted benzyl or al alkyl, such as C.sub.1-C.sub.4 alkyl,
including methyl and ethyl. For example, the intermediate compound
of the following formula
##STR00037##
may be reacted with
##STR00038##
to form a double protected compound
##STR00039##
The double protected compound
##STR00040##
may be converted into a double protected prodrug compound of the
following formula
##STR00041##
where R.sub.2 may be
##STR00042##
wherein Y is OR.sub.4 or NR.sub.4R.sub.5, each of R.sub.4 and
R.sub.5 is independently selected from H and C.sub.1-4 alkyl, or Y
is Cl, Br or OCCl.sub.3. This may be accomplished by reacting the
double protected compound with
##STR00043##
wherein Z is Cl, Br or OCCl.sub.3. For example, when R.sub.2 is
##STR00044##
the double protected compound may be reacted with
##STR00045##
to form a respective double protected prodrug compound. When
R.sub.2 is
##STR00046##
the double protected compound may be reacted
##STR00047##
or a mix of
Cl.sub.3CO--C(.dbd.O)--OCCl.sub.3/HN(CH.sub.3)--CH.sub.3 to form a
respective double protected prodrug compound.
[0119] Each of P.sub.0 and P.sub.1 protecting groups may be then
replaced with H to deprotect the carboxy group's hydroxyl and the
side chain's hydroxyl. In some embodiments, such as the one
illustrated in FIG. 1, deprotection of the carboxy group's hydroxyl
and the side chain's hydroxyl (replacement of each of P.sub.0 and
P.sub.1) may be performed in a single reaction. Yet in some other
embodiments, deprotection of the carboxy group's hydroxyl and the
side chain's hydroxyl in two separate reactions. In some cases, as
illustrated in FIG. 3, the side chain's hydroxyl group may be
deprotected first followed by deprotection of the carboxy group's
hydroxyl. Yet in some other cases, the carboxy group's hydroxyl
group may be deprotected first followed by deprotection of the side
chain's hydroxyl. Deprotection of the side chain's hydroxyl may be
performed in the presence of a lewis acid, such as MgBr.sub.2,
salts of copper, such as copper sulfate, acidic resins, such as
amberlyst, mineral acids, such as HCl and H.sub.2SO.sub.4.
Deprotection of hydroxyl protecting groups is disclosed "Green's
protecting groups in organic synthesis" ISBN 978-0-471-69754-1, 4th
edition, 2007, page 62; John Wiley and Sons). Deprotection of the
carboxy group's hydroxyl may be performed for example, in the
presence of one or more of a palladium carbon catalyst, platinum
oxide and hydrogen gas.
[0120] For synthesizing the side chain prodrugs, the starting
compound
##STR00048##
may be converted into a triple protected triol compound of the
following formula:
##STR00049##
wherein P'.sub.0 and P.sub.2 may the same or different hydroxyl
protecting group, which may be for example, a silyl protecting
group, such as tert-Butyldimethylsilyl ether (TBDMS/TBS),
Trimethylsilyl (TMS) Triethylsilyl (TES), tert-Butyldiphenylsilyl
(TBDPS), Triisopropylsilyl (TIPS). In some embodiments, it may be
preferred to have P'.sub.0 and P.sub.2 to be the same. For example,
in FIGS. 2 and 4, the key intermediate compound is reacted with
TBDMSCl to form a triple protected triol compound with both
P'.sub.0 and P.sub.2 being TBDMS.
[0121] The triple protected triol compound may be then converted
into a double protected triol compound of the following formula
##STR00050##
by deprotecting the conjugated ring's hydroxyl. Such conversion may
be performed in the presence of a Li containing compound, such
LiOAc or LiOH.
[0122] The double protected triol compound may be converted then
into a triple protected carboxy acid compound of the following
formula
##STR00051##
where P.sub.0 is a hydroxyl protecting group, such as benzyl or a
substituted benzyl. The double protected triol compound may be
converted into a triple protected carboxy acid compound by reacting
with
##STR00052##
[0123] The triple protected carboxy acid compound may be then
converted into a double protected carboxy acid compound of the
following formula:
##STR00053##
by deprotecting the side chain's hydroxyl's group. Deprotection of
the side chain's hydroxyl may be performed in the presence of a
lewis acid, such as MgBr.sub.2, salts of copper, such as copper
sulfate, acidic resins, such as amberlyst, mineral acids, such as
HCl and H.sub.2SO.sub.4. Deprotection of hydroxyl protecting groups
is disclosed "Green's protecting groups in organic synthesis" ISBN
978-0-471-69754-1, 4th edition, 2007, page 62; John Wiley and
Sons).
[0124] The double protected carboxy acid compound may be then
converted into a double protected prodrug compound of the following
formula:
##STR00054##
where R.sub.3 may be
##STR00055##
wherein Y is OR.sub.4 or NR.sub.4R.sub.5, each of R.sub.4 and
R.sub.5 is independently selected from H and C.sub.1-4 alkyl, or Y
is Cl, Br or OCCl.sub.3. This may be accomplished by reacting the
double protected carboxy acid compound with
##STR00056##
where Z is Cl, Br or OCCl.sub.3. For example, when R.sub.3 is
##STR00057##
the double protected carboxy acid compound may be reacted with
##STR00058##
to form a respective double protected prodrug compound. When
R.sub.3 is
##STR00059##
the double protected compound may be reacted
##STR00060##
or a mix of
Cl.sub.3CO--C(.dbd.O)--OCCl.sub.3/HN(CH.sub.3)--CH.sub.3 to form a
respective double protected prodrug compound.
[0125] The double protected prodrug compound may converted into a
side chain prodrug
##STR00061##
by deprotecting the cyclopentyl ring's hydroxyl and the carboxy
group's hydroxyl. Deprotections of the cyclopentyl ring's hydroxyl
and the carboxy group's hydroxyl may be performed in a single
reaction or two separate reactions. In the latter case,
deprotection of the cyclopentyl ring's hydroxyl may follow or
precede deprotection of the carboxy group's hydroxyl. In FIGS. 2
and 4, deprotection of the cyclopentyl ring's hydroxyl and
deprotection of the carboxy group's hydroxyl are performed as two
separate reactions with the latter following the former.
Deprotection of the carboxy group's hydroxyl may be performed in
the presence of one or more of palladium, carbo, platinum oxide and
hydrogen cgas. Deprotection of the cyclopentyl ring's hydroxyl may
be performed in the presence of tetra-n-butylammonium fluoride
(TBAF and n-Bu.sub.4NF) or a mineral acid, such as HCl or
H.sub.2SO.sub.4.
[0126] Treprostinil Amino Acid amide prodrugs, such as prodrugs J,
K, L or M, may be prepared by reacting treprostinil with a
protected amino acid, which is an amino acid in which hydrogen in
its carboxy group is replaced with a hydroxyl protecting group,
such as benzyl. As the result of such reaction a protected amino
acid amide prodrug may be formed. The hydroxyl protecting group may
be then removed from the protected amino acid amide prodrug to form
a treprostinil amino acid amide prodrug, such as prodrugs J, K, L
or M.
[0127] Embodiments described herein are further illustrated by,
though in no way limited to, the following working examples.
WORKING EXAMPLES
Example 1
Synthesis of Treprostinil Carbamate Pro-Drugs
##STR00062##
[0128] Synthesis of Treprostinil Mono-TES Benzyl Esters (2a and
2b)
##STR00063##
[0130] To a solution of treprostinil benzyl ester (1) (100 g, 20.80
mmol) in dichloromethane (DCM) (200 mL) was added imidazole (1.41
g, 20.80 mmol) and 4-dimethylaminopyridine (0.25 g, 2.08 mmol). To
this mixture, while stirring, chlorotriethylsilane (3.5 mL, 20.80
mmol) was added using a syringe under argon atmosphere. After 1 h
the reaction was found to be complete based on TLC (Note 1). The
reaction was quenched with water (150 mL) and the organic layer was
separated, washed with brine (100 mL), dried over sodium sulfate
and evaporated in vacuo to obtain crude product. The crude material
was purified by column chromatography using ethyl acetate:hexanes
(0-11%) as mobile phase to obtain both mono-protected compound 2a
(RD-UT-1160-185-I, 6.68 g) in 54.04% yield and 2b
(RD-UT-1160-185-III, 0.48 g) in 3.88% yield. The pure products were
characterized by .sup.1H NMR.
[0131] Note 1: A silica gel TLC was used to monitor the progress of
the reaction using 20% EtOAc: Hexanes as mobile phase.
Experimental for Synthesis of Side Chain Carbamate Treprostinil
Pro-Drug (5a)
Synthesis of TES Side Chain Carbamate Benzyl Ester (3a)
##STR00064##
[0133] To a solution of treprostinil mono-TES benzyl ester (2a)
(0.5 g, 0.841 mmol) in 15 mL of toluene was added pyridine (0.14
mL, 1.682 mmol) and stirred under argon. To this an ice-cold
solution of triphosgene (0.37 g, 1.261 mmol) in toluene (12 mL) was
added drop-wise over a period of 0.5 h. After stirring for
additional 0.5 h, reaction was found to be complete based on TLC
(Note 1). The dropping funnel was charged with dimethylamine
solution (2.0 M in THF) (6.0 mL) and added to the reaction mixture
over a period of 0.5 h. After stirring for additional 1 h the
reaction was found to be complete based on TLC (Note 1). The
reaction was quenched with water (20 mL). The organic layer was
separated and aqueous layer was extracted with MTBE (2.times.20
mL). The combined organic layers were washed with brine (20 mL),
dried over sodium sulfate and evaporated in vacuo to obtain crude
product. This was purified by column chromatography using ethyl
acetate:hexanes (0 to 12%) as mobile phase to obtain pure TES side
chain carbamate benzyl ester (3a) (RD-UT-1160-188, 0.32 g) and
impure product (RD-UT-1160-188-Fr-22-23, 0.20 g) with a total yield
of 93.4%. The pure product was characterized by .sup.1H NMR.
[0134] Note 1: A silica gel TLC was used to monitor the progress of
the reaction using 30% EtOAc:Hexanes as mobile phase.
Synthesis of Side Chain Carbamate Benzyl Ester (4a)
##STR00065##
[0136] To a solution of TES side chain carbamate benzyl ester (3a)
(0.295 g, 0.443 mmol) in THF (20 mL) and water (4 mL) was added a
2N HCl aqueous solution (0.22 mL, 0.443 mmol). This was stirred at
ambient temperature for 1 h upon which TLC showed completion of the
reaction (Note 1). This reaction mixture was extracted with ethyl
acetate (2.times.40 mL) and the combined organic layers were washed
with water (20 mL), brine (20 mL), dried over sodium sulfate and
evaporated in vacuo to obtain crude material. This was purified by
column chromatography using ethyl acetate:hexanes (0 to 40%) as
mobile phase to obtain pure side chain carbamate benzyl ester (4a)
(RD-UT-1160-194, 0.26 g) a yield of 106.5% (with residual
solvents). The pure product was characterized by .sup.1H NMR and
.sup.13C NMR.
[0137] Note 1: A silica gel TLC was used to monitor the progress of
the reaction using 60% EtOAc:Hexanes as mobile phase.
Synthesis of Side Chain Carbamate Treprostinil Pro-Drug (5a)
##STR00066##
[0139] To a solution of side chain carbamate benzyl ester (4a)
(0.25 g, 0.443 mmol) in ethyl acetate (15 mL) was added palladium
on carbon (25 mg) and the reaction system was evacuated using
vacuum and replaced with hydrogen gas under balloon pressure. This
was stirred for 6 h at room temperature and the reaction was found
to be complete based on TLC (Note 1). The reaction mixture was
filtered through Celite and the filterate was evaporated in vacuo
to obtain side chain carbamate treprostinil pro-drug (5a) (0.18 g)
(RD-UT-1160-198) with 86.1% yield and 98.62% chemical purity
(HPLC). The product was characterized by .sup.1H NMR, .sup.13C NMR,
IR and LC-MS.
[0140] Note 1: A silica gel TLC was used to monitor the progress of
the reaction using 60% EtOAc:Hexanes as mobile phase.
Experimental for Synthesis of Cyclopentyl Carbamate Treprostinil
Pro-Drug (5b)
Synthesis of TES Cyclopentyl Carbamate Benzyl Ester (3b)
##STR00067##
[0142] To a solution of treprostinil mono-TES benzyl ester (2b)
(0.45 g, 0.757 mmol) in toluene (15 mL) was added pyridine (0.12
mL, 1.513 mmol) and stirred under argon. To this an ice cold
solution of triphosgene (0.33 g, 1.135 mmol) in toluene (15 mL) was
added drop-wise over a period of 1 h. After stirring for additional
1 h, reaction was found to be complete based on TLC (Note 1). The
dropping funnel was charged with dimethylamine solution (2.0 M in
THF) (6.0 mL) and added to the reaction mixture over a period of
0.5 h. After stirring for additional 1 h the reaction was found to
be complete based on TLC (Note 1). The reaction was quenched with
water (20 mL). The organic layer was separated and aqueous layer
was extracted with MTBE (2.times.20 mL). The combined organic
layers were washed with brine (20 mL), dried over sodium sulfate
and evaporated in vacuo to obtain crude product. This was purified
by column chromatography using ethyl acetate:hexanes (0 to 14%) as
mobile phase to obtain pure TES cyclopentyl carbamate benzyl ester
(3b) (RD-UT-1160-195, 0.44 g) with a yield of 87.5%. The pure
product was characterized by .sup.1H NMR.
[0143] Note 1: A silica gel TLC was used to monitor the progress of
the reaction using 30% EtOAc:Hexanes as mobile phase.
Synthesis of Cyclopentyl Carbamate Benzyl Ester (4b)
##STR00068##
[0145] To a solution of TES cylcopentyl carbamate benzyl ester (3b)
(0.42 g, 0.631 mmol) in THF (12 mL) and water (3 mL) was added a 2N
HCl aqueous solution (0.31 mL, 0.631 mmol). This was stirred at
ambient temperature for 1 h upon which TLC showed completion of the
reaction (Note 1). This reaction mixture was extracted with ethyl
acetate (2.times.30 mL) and the combined organic layers were washed
with brine (20 mL), dried over sodium sulfate and evaporated in
vacuo to obtain crude material. This was purified twice by column
chromatography using ethyl acetate:hexanes (0 to 40%) as mobile
phase to obtain pure cyclopentyl carbamate benzyl ester (4b)
(RD-UT-1160-205, 0.32 g) a yield of 93.4%. The pure product was
characterized by .sup.1H NMR, .sup.13C NMR.
[0146] Note 1: A silica gel TLC was used to monitor the progress of
the reaction using 60% EtOAc:Hexanes as mobile phase.
Synthesis of Cyclopentyl Carbamate Treprostinil Pro-Drug (5b)
##STR00069##
[0148] To a solution of cyclopentyl carbamate benzyl ester (4b)
(0.31 g, 0.562 mmol) in 15 mL of ethyl acetate was added palladium
on carbon (30 mg) and the reaction system was evacuated using
vacuum and replaced with hydrogen gas under balloon pressure. This
was stirred for 6 h at room temperature and the reaction was found
to be complete based on TLC (Note 1). The reaction mixture was
filtered through Celite and evaporated in vacuo to obtain
cyclopentyl carbamate treprostinil pro-drug (5b) (0.24 g)
(RD-UT-1160-198) in 92.6% yield and 99.39% chemical purity (HPLC).
The product was characterized by .sup.1H NMR, .sup.13C NMR, IR and
LC-MS.
[0149] Note 1: A silica gel TLC was used to monitor the progress of
the reaction using 60% EtOAc:Hexanes as mobile phase.
Example 2
Synthesis of Treprostinil Glycolamide Prodrug (Prodrug VII)
##STR00070##
[0150] Discussion
[0151] Two methods for the synthesis of glycolamide prodrug (Scheme
2) were explored: first, via the reaction of treprostinil (UT-15)
and glycine methyl ester to obtain amide intermediate Amide I
followed by NaOH hydrolysis; second, via the reaction of UT-15 with
glycine benzyl ester p-toluenesulfonate to form the amide
intermediate Amide II followed by hydrogenation. The first route
involved strong basic conditions for the hydrolysis step and caused
the hydrolysis of both ester bond and amide bond and lead to the
formation of UT-15. The second route involved non-basic
hydrogenolysis for the de-benzylation of Amide II and provided
clean desired product without any amide bond cleavage and did not
lead to the formation of UT-15. Finally, the second route was used
to make glycolamide prodrug of treprostinil (Prodrug VII).
##STR00071##
Step 1: Synthesis of Amide II
##STR00072##
[0153] A 50 ml round bottom flask equipped with magnetic stir bar
was charged with a solution of UT-15 (0.5 g, 1.28 mmol) in
anhydrous DCM (20 ml) under argon. To this solution was added
Bop-Cl (0.49 g, 1.92 mmol) followed by glycine benzyl ester
p-toluenesulfonate (0.43 g, 1.28 mmol) at room temperature under
argon. The reaction mixture was stirred for 20 minutes, then the
triethylamine (0.39 g, 3.84 mmol) was added. The reaction mixture
was stirred overnight until the reaction was complete. The progress
of reaction was checked by tlc. The reaction was quenched with 0.1N
HCl (10 ml), the DCM layer was separated and washed with 10%
NaHCO.sub.3 (10 ml), water (10 ml) and brine (10 ml), dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo to
obtain crude product (0.9 g, RD-UT-1161-101). The crude product was
purified on silica gel using a gradient solvent of 30-60% EtOAc in
hexanes to obtain pure product (Amide II) (0.396 g,
RD-UT-1161-101B). The compound was characterized by .sup.1H
NMR.
Step 2: Synthesis of Glycolamide Prodrug (Prodrug VII)
##STR00073##
[0155] A 50 ml round bottom flask equipped with magnetic stir bar
was charged with a solution of amide II (250 mg, 0.465 mmol) in
ethanol (30.0 ml). The reaction mixture was evacuated air with
argon for three times, then 5% Pd--C (75 mg) was added and replaced
argon with H.sub.2 for three times. The reaction mixture was
pressured with H.sub.2 using balloon and left at room temperature.
The reaction mixture was stirred for 1 hr and the progress of
reaction was checked by tlc (EtOAc). The reaction mixture was
passed through a Celite pad, and the Celite pad was washed with
ethanol (50 ml). The combined ethanol solution was concentrated
under vacuo to obtain product Glycolamide (191 mg, RD-UT-1161-121)
as white foam. The compound was characterized by .sup.1H NMR,
.sup.13C NMR, IR and MS. The HPLC purity was 98.77% and no free
UT-15 was observed.
Example 3
Syntheses of Prodrugs of Treprostinil: Side Chain and Cyclopentyl
Methyl Carbonate of Treprostinil
##STR00074##
[0157] Side chain methyl carbonate prodrug of treprostinil (left)
and cyclopentyl methyl carbonate of treprostinil (right) are
presented above.
##STR00075## ##STR00076##
##STR00077##
Experimental
Syntheses of Mono-TES Protected Treprostinil Benzyl Ester (2 and
3)
##STR00078##
[0159] To a solution of treprostinil benzyl ester (1) (5.06 g,
10.53 mmol) in anhydrous dichloromethane (100 mL) was added
imidazole (0.72 g, 10.57 mmol) and 4-(dimethylamino)pyridine (DMAP)
(0.13 g, 1.06 mmol) at room temperature. To this clear solution was
added dropwise a solution of chlorotriethylsilane (1.59 g, 1.77 mL,
10.55 mmol) in anhydrous dichloromethane (30 mL) over period of 1 h
at room temperature under argon. After complete addition, the
reaction mixture was stirred for 4.5 h and checked tlc
(EtOAc/Hexane, 1:4). The reaction mixture was quenched with water
(50 mL) and separated the dichloromethane layer. The
dichloromethane layer was washed with water (1.times.50 mL), brine
(1.times.20 mL), dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo to give a light yellow viscous liquid (6.95
g) (Lot# D-1166-160). The crude product was chromatographed on
silica gel (265 g) column using ethyl acetate in hexane (2-20%) to
give di-TES protected treprostinil benzyl ester (1.59 g, Lot#
D-1166-160-A), cyclopentyl-TES protected treprostinil benzyl ester
(2) (2.95 g, Lot# D-1166-160-B) and side chain-TES protected
treprostinil benzyl ester (3) (0.55 g, Lot# D-1166-160-D). Both
mono-TES protected compounds (2 and 3) were characterized by
spectral data (IR, .sup.1H NMR and MS).
Synthesis of Cyclopentyl-TES Side Chain Methyl Carbonate
Treprostinil Benzyl Ester (4)
##STR00079##
[0161] To a solution of cyclopentyl-TES protected treprostinil
benzyl ester (2) (0.84 g, 1.41 mmol) in anhydrous pyridine (4.0 mL)
was added dropwise a solution of methyl chloroformate (1.33 g, 1.09
mL, 14.1 mmol) in anhydrous dichloromethane (4.0 mL) at 0.degree.
C. to 5.degree. C. over a period of 5 min under argon. After
complete addition, the reaction mixture was stirred at 0.degree. C.
to room temperature overnight. After 20 h, the reaction mixture was
checked by tlc (EtOAc/Hexane, 1:4) and the reaction was complete.
The mixture was treated with water (20 mL) and then extracted with
dichloromethane (3.times.25 mL). The combined dichloromethane
extracts were washed with water (1.times.20 mL), brine (1.times.10
mL), dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo
to give crude product as a light pink viscous liquid (0.88 g) (Lot#
D-1166-189). The crude product was chromatographed on silica gel
(35 g) column using ethyl acetate in hexane (2-10%) to give
cyclopentyl-TES side chain methyl carbonate treprostinil benzyl
ester (4) as a pale yellow viscous liquid (0.69 g) (Lot#
D-1166-189-B). The pure compound was characterized by spectral data
(IR, .sup.1H NMR, .sup.13C NMR and MS) and purity (94.58%, AUC) by
HPLC.
Synthesis of Side Chain Methyl Carbonate Treprostinil Benzyl Ester
(5)
##STR00080##
[0163] To a solution of cyclopentyl-TES side chain methyl carbonate
treprostinil benzyl ester (4) (0.307 g, 0.470 mmol) in a mixture of
tetrahydrofuran (10 mL) and water (2 mL) (ratio of
THF:H.sub.2O=5:1) was added 1N hydrochloric acid (0.71 mL, 0.71
mmol) at room temperature. The reaction mixture was stirred at room
temperature for 30 min and checked tlc (EtOAc/Hexane, 3:7). The
reaction was complete and mixture was neutralized with saturated
sodium bicarbonate (1 mL) to pH 7-8 and then diluted with water (10
mL). The mixture was extracted with MTBE (3.times.15 mL). The
combined MTBE extracts were washed with water (2.times.10 mL),
brine (1.times.10 mL), dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo to give a clear viscous liquid (0.37 g) (Lot#
D-1166-203). The crude product (0.031 g) from other reaction (Lot#
D-1166-201) was combined with this lot for purification. The
combined crude product was chromatographed on silica gel (16 g)
column using ethyl acetate in hexane (5-30%) to give side chain
methyl carbonate treprostinil benzyl ester (5) as a clear viscous
liquid (0.252 g) (Lot# D-1166-203-B). The pure compound was
characterized by spectral data (IR, .sup.1H NMR, .sup.13C NMR and
MS) and purity (99.83%, AUC) by HPLC.
Synthesis of Side Chain Methyl Carbonate of Treprostinil (6)
##STR00081##
[0165] To a solution of side chain methyl carbonate treprostinil
benzyl ester (5) (0.23 g, 0.427 mmol) in ethyl acetate (10 mL) was
added palladium on carbon (5 wt %, 50% wet) (0.12 g). The mixture
was stirred and evacuated under house vacuum and replaced by
hydrogen (filled in balloon). The process was repeated three times.
The mixture was stirred at room temperature under the atmosphere of
hydrogen for 2 h and checked tlc (EtOAc/Hexane, 3:7 and EtOAc,
100%). The reaction was complete. The reaction mixture was treated
with Celite (1.0 g) and the filtered through a pad of Celite (2.0
g) in a disposable polyethylene frit with Whatman filter No. 50,
and the solid was washed with ethyl acetate (3.times.10 mL). The
combined ethyl acetate filtrate was evaporated in vacuo to give
side chain methyl carbonate of treprostinil (6) as a gray-white
foamy solid (0.188 g) (Lot# D-1166-206). The compound was fully
characterized by spectral data (IR, .sup.1H NMR, .sup.13C NMR and
MS) and purity (99.64%, AUC) by HPLC.
Synthesis of Side Chain-TES Cyclopentyl Methyl Carbonate
Treprostinil Benzyl Ester (7)
##STR00082##
[0167] To a solution of side chain-TES protected treprostinil
benzyl ester (3) (0.28 g, 0.47 mmol) in anhydrous pyridine (2.0 mL)
was added dropwise a solution of methyl chloroformate (0.44 g, 0.36
mL, 4.66 mmol) in anhydrous dichloromethane (2.0 mL) at 0.degree.
C. to 5.degree. C. over a period of 5 min under argon. After
complete addition, the reaction mixture was stirred at 0.degree. C.
to room temperature overnight. After 17 h, the reaction mixture was
checked by tlc (EtOAc/Hexane, 1:4) and the reaction was complete.
The mixture was treated with water (10 mL) and MTBE (15 mL). The
organic layer was separated and washed with water (2.times.15 mL),
5% citric acid (2.times.10 mL), water (1.times.10 mL), brine
(1.times.5 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated
in vacuo to give side chain-TES cyclopentyl methyl carbonate
treprostinil benzyl ester (7) as a pale yellow viscous liquid
(0.285 g) (Lot# D-1166-187). The compound was characterized by
spectral data (IR, .sup.1H NMR, .sup.13C NMR and MS) and purity
(74.27%, AUC) by HPLC.
Synthesis of Cyclopentyl Methyl Carbonate Treprostinil Benzyl Ester
(8)
##STR00083##
[0169] To a solution of side chain-TES cyclopentyl methyl carbonate
treprostinil benzyl ester (7) (0.27 g, 0.413 mmol) in a mixture of
tetrahydrofuran (10 mL) and water (2 mL) (ratio of
THF:H.sub.2O=5:1) was added 1N hydrochloric acid (0.62 mL, 0.62
mmol) at room temperature. The reaction mixture was stirred at room
temperature for 20 min and checked tlc (EtOAc/Hexane, 1:4 and 3:7).
The reaction was complete and mixture was neutralized with
saturated sodium bicarbonate (1 mL) to pH 7-8 and then diluted with
water (10 mL). The mixture was extracted with MTBE (3.times.15 mL).
The combined MTBE extracts were washed with water (2.times.10 mL),
brine (1.times.10 mL), dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo to give a clear viscous liquid (0.24 g) (Lot#
D-1174-001). The crude product was chromatographed on silica gel
(18 g) column using ethyl acetate in hexane (5-30%) to give
cyclopentyl methyl carbonate treprostinil benzyl ester (8) as a
clear viscous liquid/white semi-solid (0.19 g) (Lot# D-1174-001-B).
The pure compound was characterized by spectral data (IR, .sup.1H
NMR, .sup.13C NMR and MS) and purity (98.45%, AUC) by HPLC.
Synthesis of Cyclopentyl Methyl Carbonate of Treprostinil (9)
##STR00084##
[0171] To a solution of cyclopentyl methyl carbonate treprostinil
benzyl ester (8) (0.16 g, 0.297 mmol) in ethyl acetate (10 mL) was
added palladium on carbon (5 wt %, 50% wet) (0.08 g). The mixture
was stirred and evacuated under house vacuum and replaced by
hydrogen (filled in balloon). The process was repeated three times.
The mixture was stirred at room temperature under the atmosphere of
hydrogen for 2 h and checked tlc (EtOAc/Hexane, 3:7 and EtOAc,
100%). The reaction was complete. The reaction mixture was treated
with Celite (1.0 g) and the filtered through a pad of Celite (2.0
g) in a disposable polyethylene frit with Whatman filter No. 50,
and the solid was washed with ethyl acetate (3.times.10 mL). The
combined ethyl acetate filtrate was evaporated in vacuo to give
cyclopentyl methyl carbonate of treprostinil (9) as a white viscous
liquid/semi-solid (0.138 g) (Lot# D-1174-004). The compound was
fully characterized by spectral data (IR, .sup.1H NMR, .sup.13C NMR
and MS) and purity (98.97%, AUC) by HPLC.
Example 4
In Vitro Receptor Activity of Treprostinil and its Prodrugs
[0172] Prodrugs I, II, III, IV, VII, and IX (for structures see
FIGS. 7 and 39) as well as treprostinil were tested for 3
G-protein-coupled receptors (GPCRs), namely DP1, EP2, and IP, using
cyclin adenosine monophosphate (cAMP) assay.
[0173] Materials. Cells and control agonists: Cells and control
agonists used in the study are summarized in Table 1.
TABLE-US-00001 TABLE 1 Cell Lines and Control Agonists Used in the
Study Species Target Parental Catalog # Assays Control agonist
Human DP1 HEK293T C1200 cAMP PGD2 Human EP2 HEK293T C1202 cAMP
Iloprost Human IP1 CHO-K1 C1206-1 cAMP Iloprost
[0174] Compounds were provided in powder form. The compounds were
reconstituted in DMSO at a concentration of 10 mM.
[0175] Cyclic AMP assay kit: HTRF cAMP HiRange Kit (CisBio,
Cat#62AM6PEC).
[0176] Instruments: FlexStation III (Molecular Devices).
Methods
[0177] Cyclic AMP (cAMP) Assay: cAMP assays were performed
according to the manufacturer's protocol using CisBio's HTRF cAMP
HiRange Kit. For Gs pathway assay in agonist mode, cells were
incubated with compounds in 384 well plates for 20 minutes at
37.degree. C. The reaction was terminated by sequentially adding
D2-labeled cAMP and cryptate-labeled anti-cAMP antibody in lysis
buffer. The plate was then incubated at room temperature for 60
minutes before reading fluorescent emissions at 620 nm and 668 nm
with excitation at 314 nm on FlexStation III (Molecular
Devices).
Data Analysis
[0178] Cyclic AMP (cAMP) assays: Cyclic AMP assay results are shown
as "Ratio 668/620.times.10,000" (ratio of fluorescence at 668 nm
and 620 nm.times.10,000). Data in graphs are represented in
Mean.+-.SD. Dose-dependent responses were fitted with sigmoidal
dose-response curves allowing variable slopes using GraphPad Prism
versions 4, 5 or 6 (Graphpad Prism).
Results
[0179] All compounds and control agonists displayed dose response
activity in DP1, EP2, and IP1 receptor expressing cells. EC50 (a
drug concentration that gives half-maximal response) values
determined from the dose-dependent responses is presented in Table
2.
TABLE-US-00002 TABLE 2 Receptor activity (EC50, M) for treprostinil
and its prodrugs. Compound IP receptor EP receptor DP receptor
Treprostinil 2.28 .times. 10.sup.-9 6.75 .times. 10.sup.-9 .sup.
7.07 .times. 10.sup.-10 Prodrug I 8.07 .times. 10.sup.-6 1.80
.times. 10.sup.-6 4.85 .times. 10.sup.-7 Prodrug II 3.05 .times.
10.sup.-7 1.64 .times. 10.sup.-6 1.18 .times. 10.sup.-7 Prodrug III
4.79 .times. 10.sup.-7 4.35 .times. 10.sup.-7 7.34 .times.
10.sup.-8 Prodrug IV 4.0 .times. 10.sup.-7 4.55 .times. 10.sup.-6
1.43 .times. 10.sup.-7 Prodrug VII 2.0 .times. 10.sup.-6 7.4
.times. 10.sup.-4 4.22 .times. 10.sup.-7 Prodrug IX 1.58 .times.
10.sup.-6 1.35 .times. 10.sup.-6 1.62 .times. 10.sup.-7
[0180] The data in Table 2 demonstrates that each of the studied
prodrugs was significantly less potent than treprostinil against
DP1, EP2 and IP1. Although the present invention is not limited by
a theory of its operation, the site pain observed during
subcutaneous administering of treprostinil may be due to
treprostinil affecting one or more of the IP receptor, the DP
receptor and the EP receptor in the subcutaneous tissue. Because
the studied prodrugs are less potent against each of the IP
receptor, the DP receptor and the EP receptor than treprostinil,
these prodrugs may cause less site pain when administered
subcutaneously.
Example 5
Evaluation of Prodrug XV Against Human G Protein Coupled
Receptors
[0181] Prodrug XV (for structure see FIG. 10) as well as
treprostinil were tested for 3 G-protein-coupled receptors (GPCRs),
namely DP1, EP2, and IP, using cyclic adenosine monophosphate
(cAMP) assay.
[0182] Materials. Cells and control agonists: Cells and control
agonists used in the study are summarized in Table 1.
[0183] Compounds were provided 2 compounds in powder form. The
compounds were reconstituted in DMSO at a concentration of 10
mM.
[0184] Cyclic AMP assay kits: Multiscreen.TM. TR-FRET cAMP 1.0 No
Wash Assay Kit (Multispan, Inc., Cat# MSCM01-25) and HTRF cAMP
HiRange Kit (CisBio, Cat#62AM6PEC). Instruments: FlexStation III
(Molecular Devices).
Methods
[0185] Cyclic AMP (cAMP) Assay: cAMP assays were performed
according to the manufacturer's protocol using Multiscreen.TM.
TR-FRET cAMP 1.0 No Wash Assay Kit or HTRF cAMP HiRange Kit. For
agonist mode testing, cells were preincubated with customer
compounds for 5 minutes at room temperature prior to the addition
of forskolin, and the plate was then incubated at 37.degree. C. for
20 minutes. The reaction was terminated by sequentially adding
sequentially adding trFluor.TM. Eu-labeled cAMP and trFluor.TM.
650-labeled anti-cAMP antibody or D2-labeled cAMP and
cryptate-labeled anti-cAMP antibody in lysis buffer. The plate was
then incubated at room temperature for 60 minutes before reading
fluorescent emissions at 620 nm and 665 or 668 nm with excitation
at 314 nm on FlexStation III (Molecular Devices).
Data Analysis
[0186] Cyclic AMP (cAMP) assays: Cyclic AMP assay results are shown
as "Ratio 665/620.times.10,000" (ratio of fluorescence at 665 nm
and 620 nm.times.10,000) or "Ratio 668/620.times.10,000" (ratio of
fluorescence at 668 nm and 620 nm.times.10,000). Data in graphs
were represented in Mean.+-.SD. Dose-dependent responses were
fitted with sigmoidal dose-response curves allowing variable slopes
using GraphPad Prism versions 4, 5 or 6 (Graphpad Prism).
Results and Discussion
[0187] Control agonists for all 3 GPCRs showed dose-dependent
stimulation in the receptor expressing cells with expected EC50b (a
drug concentration that gives half-maximal response) values. All
compounds and control agonists displayed dose response activity in
DP1, EP2, and IP1 receptor expressing cells. EC50 values determined
from the dose-dependent responses is presented in Table 3.
TABLE-US-00003 TABLE 3 Compound IP receptor EP receptor DP receptor
Treprostinil 7.79 .times. 10.sup.-11 5.37 .times. 10.sup.-10 7.80
.times. 10.sup.-11 Prodrug XV 4.28 .times. 10.sup.-9 4.15 .times.
10.sup.-8 6.99 .times. 10.sup.-9
Conclusions
[0188] The data in Table 3 demonstrate that Prodrug XV was
significantly less potent than treprostinil against DP1, EP2 and
IP1. Although the present invention is not limited by a theory of
its operation, the site pain observed during subcutaneous
administering of treprostinil may be due to treprostinil affecting
one or more of the IP receptor, the DP receptor and the EP receptor
in the subcutaneous tissue. Because Prodrug XV is less potent
against each of the IP receptor, the DP receptor and the EP
receptor than treprostinil, it may cause less site pain when
administered subcutaneously.
Example 6
Evaluation of Prodrug XIV Against Human G Protein Coupled
Receptors
[0189] Treprostinil Prodrug XIV (for structure see FIG. 10) as well
as treprostinil were tested for 3 G-protein-coupled receptors
(GPCRs), namely DP1, EP2, and IP, using cyclin adenosine
monophosphate (cAMP) assay.
[0190] Materials. Cells and control agonists: Cells and control
agonists used in the study are summarized in Table 1.
[0191] Compounds were provided 2 compounds in powder form. The
compounds were reconstituted in DMSO at a concentration of 10
mM.
[0192] Cyclic AMP assay kits: Multiscreen.TM. TR-FRET cAMP 1.0 No
Wash Assay Kit (Multispan, Inc., Cat# MSCM01-25) and HTRF cAMP
HiRange Kit (CisBio, Cat#62AM6PEC).
[0193] Instruments: FlexStation III (Molecular Devices).
Methods
[0194] Cyclic AMP (cAMP) Assay: cAMP assays were performed
according to the manufacturer's protocol using Multiscreen.TM.
TR-FRET cAMP 1.0 No Wash Assay Kit or HTRF cAMP HiRange Kit. For
agonist mode testing, cells were preincubated with customer
compounds for 5 minutes at room temperature prior to the addition
of forskolin, and the plate was then incubated at 37.degree. C. for
20 minutes. The reaction was terminated by sequentially adding
sequentially adding trFluor.TM. Eu-labeled cAMP and trFluor.TM.
650-labeled anti-cAMP antibody or D2-labeled cAMP and
cryptate-labeled anti-cAMP antibody in lysis buffer. The plate was
then incubated at room temperature for 60 minutes before reading
fluorescent emissions at 620 nm and 665 or 668 nm with excitation
at 314 nm on FlexStation III (Molecular Devices).
Data Analysis
[0195] Cyclic AMP (cAMP) assays: Cyclic AMP assay results are shown
as "Ratio 665/620.times.10,000" (ratio of fluorescence at 665 nm
and 620 nm.times.10,000) or "Ratio 668/620.times.10,000" (ratio of
fluorescence at 668 nm and 620 nm.times.10,000). Data in graphs
were represented in Mean.+-.SD. Dose-dependent responses were
fitted with sigmoidal dose-response curves allowing variable slopes
using GraphPad Prism versions 4, 5 or 6 (Graphpad Prism).
Results and Discussion
[0196] Control agonists for all 3 GPCRs showed dose-dependent
stimulation in the receptor expressing cells with expected EC50 (a
drug concentration that gives half-maximal response) values. All
compounds and control agonists displayed dose response activity in
DP1, EP2, and IP1 receptor expressing cells. EC50 values determined
from the dose-dependent responses is presented in Table 4.
TABLE-US-00004 TABLE 4 Compound IP receptor EP receptor DP receptor
Treprostinil 1.69 .times. 10.sup.-11 2.35 .times. 10.sup.-10 2.77
.times. 10.sup.-10 Prodrug XIV 3.33 .times. 10.sup.-9 6.70 .times.
10.sup.-8 3.88 .times. 10.sup.-8
Conclusions
[0197] The data in Table 4 demonstrate that Prodrug XIV was
significantly less potent than treprostinil against DP1, EP2 and
IP1. Although the present invention is not limited by a theory of
its operation, the site pain observed during subcutaneous
administering of treprostinil may be due to treprostinil affecting
one or more of the IP receptor, the DP receptor and the EP receptor
in the subcutaneous tissue. Because Prodrug XIV is less potent
against each of the IP receptor, the DP receptor and the EP
receptor than treprostinil, it may cause less site pain when
administered subcutaneously.
Example 7
Development of HPLC Analytical Methods and Determination of
Equilibrium Solubility and Solution Stability for Eight Prodrugs of
Treprostinil in a Chosen Vehicle
1. Objective and Summary
[0198] The objective of this study was to develop analytical method
suitable for the analysis multiple prodrugs of treprostinil and to
determine the equilibrium solubility and solution stability of
eight prodrugs in a chosen vehicle (20 mM dibasic sodium phosphate
with 125 mM sodium chloride).
[0199] Eight prodrugs of treprostinil including Prodrug III, IV,
VIII, X, XI, XII, XIII, and XIV were used for this study. The
analytical method previously developed for prodrug VII was utilized
for other prodrugs after minor modification of the method
parameters to improve specificity. Once adequate specificity was
achieved, an equilibrium solubility study was conducted for each
prodrug. The solubility study was evaluated across multiple time
points to assess solution stability of the prodrugs in the selected
vehicle.
2. Experimental
2.1 Equipment
[0200] All studies were conducted on Waters UPLC H-Class system
equipped with a photodiode array detector (PDA). All columns
evaluated were 2.1.times.100 mm, 1.7 .mu.m.
2.2 Development of Method Conditions
2.2.1 Evaluation of Prodrug VII Method Conditions
[0201] A previously developed analytical method for treprostinil
Prodrug VII was the starting place for developing conditions for
chromatographic specificity of Prodrugs III, IV, VIII, X, XI, XII,
XIII, and XIV. The Prodrug VII method conditions are provided in
Table 5.
TABLE-US-00005 TABLE 5 Nominal Starting conditions for method
optimization Parameter Final Method Condition Detection UV @ 217 nm
(4.8 nm resolution) Sampling Rate: 5 pts/s Flow Rate 0.4 mL/min
Column ACE Excel2 C18, 2.1 .times. 100 mm 1.7 .mu.m Column
Temperature 40.degree. C. Sample Concentration 1 mg/mL Diluent
50:50 Acetonitrile:20 mM Sodium Phosphate Buffer pH 6.2 Injection
Volume 1.0 .mu.L Column Temp: 40.degree. C. Run Time 20 min MPA %
MPB % Time, min (0.1% phosphoric acid in H.sub.2O) (Acetonitrile)
0.0 70 30 0.50 70 30 14.0 25 75 16.0 5 95 17.0 5 95 17.1 70 30 20.0
70 30
[0202] The goal of the specificity study was to achieve a single
chromatographic condition that resolves each prodrug from
treprostinil. Each prodrug was individually prepared and then
separately spiked with 10% nominal treprostinil to evaluate
specificity. A chromatographic overlay the prodrugs and
treprostinil analyzed by the Prodrug VII analytical method is
provided in FIG. 11.
[0203] The results of the specificity study using the Prodrug VII
method indicate that treprostinil is well separated from all
prodrugs except Prodrug XIV which co-elutes.
[0204] Three additional columns were screened to evaluate
specificity of prodrugs using the Prodrug VII method conditions. A
chromatographic overlay of treprostinil and the eight prodrugs on a
Waters BEH C18 column is provided in FIG. 12. A chromatographic
overlay of treprostinil and the eight prodrugs on a ACE C18-AR
column is provided in FIG. 13. A chromatographic overlay of
treprostinil and the eight prodrugs on a Waters CSH Phenyl Hexyl
column is provided in FIG. 14.
[0205] The results of the column screening indicated that all
prodrugs including Prodrug XIV can be adequately resolved from
treprostinil on either the ACE Excel 2 C18-AR column (Rs=2.8,
Result ID 2949) or the Waters CSH Phenyl Hexyl column (Rs=2.9,
Result ID 3028). While the resolution was essentially equivalent
between columns the CSH Phenyl Hexyl column was selected for
equilibrium solubility and solution stability studies.
3. Equilibrium Solubility and Solution Stability
3.1. Study Design
[0206] Each prodrug was dissolved in a vehicle containing 20 mM
sodium phosphate dibasic with 125 mM sodium chloride. Prodrugs were
prepared at saturation concentrations by weighing 15-30 mg of
prodrug in to a 4 mL clear glass vial and dissolving in an
appropriate volume of vehicle (0.5-1.0 mL) to achieve a nominal
saturation concentration of 30 mg/mL. The solutions were mixed for
23 hours on a rotating mixer. All solutions except Prodrug XIV
exhibited solids after mixing. The resultant supernatant solutions
were isolated from undissolved prodrug by centrifugation at 15000
RPM for 15 min. Supernatants were transferred to clear glass vials
and stored at ambient temperature. To evaluate solubility and
solution stability, supernatants were diluted 21.7-fold by
transferring 15 .mu.L supernatant to a micro vial and adding 210 uL
of diluent (25:75 Acetonitrile: 20 mM sodium phosphate pH 6.2) and
100 .mu.L acetonitrile. The resultant samples were assayed against
treprostinil to determine prodrug concentration. At each testing
interval (0, 24, 72 hours) the supernatant was assayed for prodrug
concentration and purity by area %.
3.2. Study Results
[0207] The results of the equilibrium solubility study across the
three stability testing intervals is summarized in Table 6. The
results of the solution stability for prodrugs is summarized in
Table 7 and Table 8.
TABLE-US-00006 TABLE 6 Equilibrium Solubility of Treprostinil
Prodrugs evaluated over 72 hours in vehicle containing 20 mM sodium
phosphate dibasic with 125 mM sodium chloride T0 hours T24 hours
T72 hours Concentration Concentration Concentration Compound
(mg/mL) (mg/mL) (mg/mL) Prodrug 6.1 6.0 6.2 III Prodrug 12.5 12.2
12.9 IV Prodrug 11.0 10.5 10.9 VIII Prodrug X 10.0 10.6 11.3
Prodrug 11.5 11.3 11.5 XI Prodrug 7.4 7.5 7.4 XII Prodrug 3.2 3.7
3.8 XIII Prodrug 29.5 27.5 26.6 XIV
TABLE-US-00007 TABLE 7 Solution stability of Prodrug (area %
purity) in vehicle containing 20 mM sodium phosphate dibasic with
125 mM sodium chloride T0 T24 hours T72 hours Compound Purity (%
Area) Purity (% Area) Purity (% Area) Prodrug 94.48 94.31 94.61 III
Prodrug 99.19 99.02 99.15 IV Prodrug 96.41 96.40 96.34 VIII Prodrug
X 96.59 96.32 96.54 Prodrug 99.49 99.39 98.92 XI Prodrug 98.70
98.68 98.58 XII Prodrug 97.66 97.81 97.58 XIII Prodrug 94.20 94.25
94.56 XIV
TABLE-US-00008 TABLE 7 Solution stability of Prodrug (area %
residual treprostinil) in vehicle containing 20 mM sodium phosphate
dibasic with 125 mM sodium chloride T0 Residual T24 Residual T72
Residual Treprostinil Treprostinil Treprostinil Compound (% Area)
(% Area) (% Area) Prodrug 0.15 0.14 0.15 III Prodrug ND ND ND IV
Prodrug 0.11 0.11 0.15 VIII Prodrug X 0.08 0.08 0.08 Prodrug ND ND
ND XI Prodrug ND ND ND XII Prodrug ND ND ND XIII Prodrug 0.08 0.13
0.23 XIV
3.3. Discussion
[0208] The equilibrium solubility study for eight prodrugs of
treprostinil indicates a wide range of solubility across the
compounds in the selected vehicle. Prodrug XIII was the least
soluble (approx. 3.5 mg/mL) and prodrug-N was the most soluble
(approx. 30 mg/mL). The prodrugs were demonstrated to be stable in
the vehicle up to 72 hours with little to no change in both prodrug
purity and residual treprostinil. Prodrug XIV showed the largest
treprostinil formation in the vehicle across all prodrugs evaluated
however only 0.15% was formed for this prodrug over 72 hours.
4. Conclusion
[0209] An analytical method was developed for evaluation of eight
prodrugs of treprostinil by UPLC-UV. The PRODRUG VII method
conditions were used as a starting place for method optimization
minor modification to change column chemistry (Waters CSH Phenyl
Hexyl instead of ACE Excel 2 C18). The change was required to
resolve each prodrug adequately from treprostinil. Equilibrium
solubility studies indicated a wide range of solubility for
prodrugs in the selected vehicle across the eight compounds. Six of
the eight prodrugs had solubility in vehicle between 6-13 mg/mL
while one low solubility prodrug was observed (Prodrug-M, approx.
3.5 mg/mL) and one high solubility prodrug was observed (Prodrug-N,
approx. 30 mg/mL). All prodrugs showed were stable up to 72 hours
in the vehicle based on minimal formation of treprostinil and minor
changes in area % purity.
Example 8
Objective and Summary
[0210] The objective of this study was to determine the in vitro
metabolic stability of nine prodrugs (III, IV, VII, VIII, X, XI,
XII, XIII, and XIV) in human, Beagle dog and Sprague Dawley rat
liver microsomes, as well as four prodrugs (III, IV, VII and XIV)
in Cynomolgus monkey liver microsomes. Another goal was to study
the release of parent compound (treprostinil) over the time
course.
[0211] Test articles were incubated with liver microsomes in the
presence and absence of NADPH. At selected time points, aliquots of
the incubation reaction were removed, quenched, and analyzed using
liquid chromatography tandem mass spectrometry (LC-MS/MS). Both the
prodrug and treprostinil concentrations were determined, and
half-lives of prodrugs were calculated.
[0212] The half-lives of prodrugs are tabulated in FIG. 15.
Half-lives longer than three times testing duration (120 min) are
reported as ">360".
Materials
[0213] SigmaFAST.TM. protease inhibitor cocktail tablets
(Sigma-Aldrich P/N: S8830); HPLC water (Fisher); Acetonitrile, HPLC
grade, (Fisher); Formic Acid, Optima LCMS grade, (Fisher P/N:
A117); Dimethyl Sulfoxide (Fisher P/N D159-4); Human Liver
Microsomes, mixed gender, pool of 50 (XenoTech P/N: H0610); Dog
Liver Microsomes, Beagle, male, pool of 8 (XenoTech P/N: D1000);
Rat Liver Microsomes, Sprague Dawley, male, pool of 500 (XenoTech
P/N: R1000); Monkey Liver Microsomes, Cynomolgus, male, pool of 12
(XenoTech P/N: P2000; Lot#1110090); Matrix tubes, 1.4 ml (Fisher
P/N 50823825); Matrix tube racks (Fisher P/N 50823921); Sepraseal
Caps for matrix tubes (Fisher P/N NC9995413)
Equipment
[0214] AB SCIEX API 4000.TM. LC-MS/MS system; Agilent 1100 Binary
HPLC Pump, Model G1312A; Leap HTS PAL Autosampler Equipped with a
Cold Stack; Ascentis Express.RTM. Phenyl Hexyl 2.7 .mu.m Column,
100 mm.times.3 mm (Sigma-Aldrich P/N: 53345-U); Ascentis
Express.RTM. Phenyl-Hexyl 2.7 .mu.m Guard Cartridge, 5 mm.times.3
mm (Sigma-Aldrich P/N: 53526-U); Ascentis Express.RTM. Guard
Cartridge Holder, (Sigma-Aldrich P/N: 53500-U); Aquasil C18 Dash
HTS column, 5 .mu.m, 20.times.2.1 mm (Thermo P/N: 77505-022150);
Beckman Allegra 25R Centrifuge (P/N 36934); Rainin Pipettes: 0.2-2
.mu.L, 2-20 .mu.L, 10-100 .mu.L, 20-200 .mu.L, and 100-1000 .mu.L;
Repeater (Eppendorf); Rainin Multi-channel Pipettes: 1-20 .mu.L,
20-200 .mu.L, and 100-1000 .mu.L
Incubation
[0215] Dosing solutions of Prodrugs III, IV, VII, VIII, X, XI, XII,
XIII, and XIV were made in 20 mM Dibasic Potassium Phosphate.
Concentration of prodrugs were 5 mM.
[0216] Liver microsomes (Xenotech) were diluted to a final protein
(enzyme) concentration of 0.5 mg/mL in a buffer with the following
constituents: 100 mM potassium phosphate (pH 7.4), 5 mM Magnesium
Chloride (MgCl.sub.2), and 1 mM .beta.-Nicotinamide adenine
dinucleotide 2'-phosphate (NADPH). The microsomal solution was
aliquoted into glass tubes and incubated at 37.degree. C. for about
three minutes. An aliquot of each compound was diluted 50-fold into
the pre-warmed microsome solutions and mixed to initiate the
reaction. Final concentration of prodrugs in incubation solution
was 100 .mu.M.
[0217] In the first assay, all nine prodrugs (III, IV, VII, IX, X,
XI, XII, XIII and XIV) were tested in human, Beagle dog and Sprague
Dawley rat liver microsomes. In the second assay, four prodrugs
(III, IV, VII and XIV) were tested in Cyno monkey liver
microsomes.
[0218] In addition to the test compounds, three quality control
compounds (7-ethoxycoumarin, propranolol, and verapamil) were
included to ensure the microsomes were active. Quality control
compound stock solution were made at 25 .mu.M in 25% methanol,
while the concentration in final incubation solution was 500
nM.
[0219] Negative controls were also included; these reactions
contained all of the components listed above except NADPH.
[0220] All tests were performed in duplicate. All replicates were
tested in separate reaction vials.
[0221] Time points of 0, 15, 30, 60 and 120 minutes were evaluated
with the +NADPH conditions described above, while time points of 0
and 120 min were evaluated with the -NADPH conditions. At specified
time points, a 100 .mu.L aliquot of each reaction was removed from
the reaction and added to 200 .mu.L of ice cold acetonitrile in a
deep 96-well plate. This step both quenched the reaction and
precipitated proteins in preparation for LC/MS/MS analysis. When
the time course was complete, the plates were sealed, mixed, and
centrifuged at 4500 g and 4.degree. C. for 15 minutes. 200 .mu.l of
the resulting supernatant was frozen in matrix tubes at -80.degree.
C. until analysis.
Bioanalytical Preparation Procedure
Test Articles
[0222] Solution preparation. Primary stock solutions of the
Prodrugs III, IV, VII, IX, X, XI, XII, XIII and XIV, and
treprostinil were made in 90% DMSO. The 9 prodrug stock solutions
were combined and serially diluted to 9-in-1 standard spike-in
solutions and 9-in-1 QC spike-in solutions. On the other hand,
treprostinil stock solution was serially diluted to treprostinil
standard spike-in solutions and QC spike-in solutions. After
preparation, all solutions were stored at 4.degree. C. Dilution QCs
(QC-dilu) were tested for individual compounds to ensure the
compounds do not crosstalk. Individual primary stock solutions of
prodrugs and treprostinil were used as spike-in solution for
QC-dilu.
[0223] Blank matrix. Blank matrix was prepared by preparing liver
microsomal solution (100 mM potassium phosphate buffer pH7.4, 5 mM
MgCl.sub.2, 0.5 mg/ml enzyme, 1 mM NADPH), followed by
heat-inactivation in boiling water bath for 5 min. Unknown samples
were analyzed separately for different species. The species of
liver microsomes used in preparation of blank matrix was the same
as the unknown samples, e.g., human liver microsomes were used to
prepare the blank matrix for analysis of human samples only.
[0224] Blank extract. Blank extract was prepared by combining two
volumes of acetonitrile and one volume of blank matrix. The mixture
was then centrifuged at 4000 g and the supernatant was taken.
[0225] Standard, QC and undiluted unknown samples. Samples were
extracted using a protein precipitation procedure. 5 .mu.l of
standard or QC (including QC-dilu) spike-in solution were spiked
into 95 .mu.l of blank matrix in a deep-well plate, followed by
addition of 200 .mu.l acetonitrile. Undiluted unknown samples
(thawed at room temperature and mixed well) were added to a
deep-well plate, followed by addition of 200 .mu.l acetonitrile.
The standard/QC and undiluted sample plates were then sealed, mixed
and centrifuged at 5500 g and 4.degree. C. for 15 min. 100 .mu.l of
supernatant (excluding QC-dilu) was combined with 100 .mu.l of
internal standard working solution (ISWS, 20 ng/ml
d.sub.4-treprostinil in water) on final microtiter plates.
[0226] Dilution QC additional steps. After protein precipitation
and centrifugation, 10 .mu.l supernatant was added to an
intermediate row loaded with 90 .mu.l blank extract (10.times.
dilution), mixed with pipette, and then 10 .mu.l of diluted samples
were added to a row on the final microtiter plate loaded with 90
.mu.l blank extract (another 10.times. dilution). 100 .mu.l ISWS
was added.
[0227] Diluted unknown samples. 10 .mu.l unknown sample was added
to an intermediate plate loaded with 90 .mu.l blank extract
(10.times. dilution), mixed with pipette, and then 10 .mu.l of
diluted samples were added to the final microtiter plates loaded
with 90 .mu.l blank extract (another 10.times. dilution). 100 .mu.l
ISWS was added.
[0228] Double blanks. 100 .mu.l blank extract was combined with 100
.mu.l water.
[0229] All plates were sealed, mixed, centrifuged at 5500 g and
4.degree. C. for 5 min, and ready for LC-MS/MS.
Quality Control Compounds
[0230] Unknown samples. All quality control compound samples were
thawed at room temperature and mixed well. 60 .mu.l of sample was
added to microtiter plates loaded with 120 .mu.l water and 40 .mu.l
ISWS (50 ng/ml labetalol in methanol).
[0231] Double blanks. 60 .mu.l blank extract (blank extracts from
the test article runs were used) was combine with 120 .mu.l water
and 40 .mu.l methanol. Separate double blanks were made for each
species.
[0232] The plates are sealed, mixed, centrifuged at 5000 g and
10.degree. C. for 10 min, and ready for LC-MS/MS.
LC-MS/MS
[0233] The LC-MS/MS system consisted of a Leap HTS PAL autosampler,
an Agilent 1100 series liquid chromatography pump, and a Sciex
API4000 mass spectrometer operated in triple quadrupole mode. An
Ascentis Express.RTM. Phenyl Hexyl column (2.7 .mu.m, 100
mm.times.3 mm) or an Aquasil C18 Dash HTS column (5 .mu.m,
20.times.2.1 mm) was used at 40.degree. C. with 0.1% formic acid as
mobile phase A and neat acetonitrile as mobile phase B.
[0234] The mass spectrometer (MS) was operated in negative or
positive Turbo IonSpray.TM. mode with Multiple Reaction Monitoring
(MRM). The MS parameters are also shown in Appendix 2.
Quantitation
Test Articles
[0235] To quantify the prodrugs and treprostinil in the unknown
samples, all the unknown samples ran with calibration curves of the
9-in-1 prodrugs and treprostinil in separate batches.
[0236] Automatic integration algorithm was used to integrate the
chromatographic peaks. Integrations were adjusted only as needed to
ensure integrations are consistent for all standards, quality
controls, and samples within a run.
[0237] Peak area ratios were calculated (analyte peak area divided
by internal standard peak area). Standard curves were created by
generating least squares fitting plots of peak area ratio versus
nominal concentration. Sample concentrations were calculated from
the results of the least squares fits. When calculated sample
concentration is lower than the LLOQ (Lower Limit of Quantitation),
a "BQL" (Below Quantitation Limit) is reported.
[0238] Acceptance criteria: The back-calculated accuracy should be
within .+-.20% of the nominal concentration for at least 75% of all
standards. The accuracy of at least two-thirds of all the quality
control samples should be within .+-.20% of the nominal
concentration, and at least 50% at each level must meet the above
criteria. Correlation coefficient of final curve must be
>0.99.
[0239] A 1/(x.sup.2) weighted, quadratic or linear regression was
used. Calibration standards that do not meet acceptance criteria
for back-calculated accuracy were removed.
[0240] Test injections were made to estimate the concentrations of
representative samples, and this was used to decide whether diluted
or undiluted samples would be included in the formal batches. The
calculated concentration of either the undiluted sample or diluted
sample must be within the test range; otherwise, samples with lower
concentration than LLOQ (Lower Limit of Quantitation) were reported
as BQL (Below Quantitation Limit), while samples with higher
concentration than ULOQ (Upper Limit of Quantitation) were
re-tested with a higher dilution factor.
Quality Control Compounds
[0241] The peak area ratio at time zero was set to 100% and percent
remaining at the remainder of time points was calculated. Plots of
percent remaining versus time were ultimately used to calculate
t.sub.1/2 values for each compound.
Kinetic Analysis
[0242] Rate constant (k): k=-.beta.
[0243] Where .beta. is the slope obtained from fitting semi-log
plots of concentration versus time. For quality control compounds,
when concentration is not available, % remaining is used
instead.
Half - life ( t 1 / 2 ) : t 1 / 2 = - 0.692 .beta. ##EQU00001##
Results
Test Articles
[0244] Half-lives of the prodrugs are tabulated in Table 9.
Quality Control Compounds
[0245] Half-lives of quality control compounds are tabulated in
Table 9.
TABLE-US-00009 TABLE 9 Half-lives of quality control compounds.
Half-life (min) 7-EC Propranolol Verapamil +NADPH -NADPH +NADPH
-NADPH +NADPH -NADPH Human 5.40 >360 86 >360 7.46 >360 Dog
2.05 >360 12.7 >360 10.1 >360 Rat 5.89 >360 2.16
>360 6.09 >360 Monkey 2.00 >360 10.9 >360 1.26
>360
[0246] Quality control compounds had t1/2 values that were
comparable to historical data, indicating that the microsomes used
in these tests were active.
Discussions
[0247] According to the earlier aqueous solution stability study,
all prodrugs are stable in 20 mM Sodium Phosphate dibasic with 125
mM NaCl at saturation concentrations (3-30 mg/ml). Also, in the
method development work, it was also observed that the most
unstable prodrugs III, IV and XIV were stable in 200 mM potassium
phosphate buffer (pH 7.4) at a low concentration of 1 .mu.M. These
suggest that the metabolism of prodrugs in this study are not due
to chemical or aqueous solution instability.
[0248] It was observed that in some cases the half-life was similar
with or without NADPH. This indicates that some NADPH-independent
enzymes, such as esterase and amidase, may be mainly responsible
for metabolism of the prodrugs. In other cases, when the half-life
with NADPH was much shorter than the half-life without NADPH, the
reaction was likely mediated by (or partly mediated by) Cytochrome
P450 enzymes.
Conclusions
[0249] The stability of the prodrugs are species-dependent, with
IV, III, and XIV being the least stable across all species.
Example 9
Evaluation of Treprostinil Prodrugs in the Rat Intraplantar
Injection Model
1. Summary
[0250] Treprostinil, a synthetic prostacyclin analog, is the active
pharmaceutical ingredient in Remodulin. Subcutaneous administration
of treprostinil is associated with pain at the site of injection,
and the objective of this study was to evaluate alternative
prodrugs of treprostinil to assess a pain response in the rat paw
pain model.
[0251] Male Sprague Dawley rats (n=112) were allocated into 14
groups of 8/group. The study was run in 2 cycles, on consecutive
days, 7 groups per cycle. Each cycle was composed of a Saline
group, PBS group, which served as the control, and also
treprostinil at a dose of 100 .mu.g/mL or 1 .mu.g/mL. In addition,
the test items treprostinil ring carbamate (Prodrug I),
treprostinil side-chain carbamate (Prodrug II), treprostinil amide
(Prodrug VII) and treprostinil methyl ether (Prodrug VIII) all
formulated in PBS, were tested at 2 doses (100 .mu.g/mL or 1
.mu.g/mL, one dose per cycle).
[0252] Animals were administered 0.1 mL of test material by
subcutaneous injection into the paw pad (Intraplantar injection) at
time zero. Animals were subsequently evaluated for their response
to mechanical (von Frey filaments) and thermal stimuli, 15 and 90
minutes post-injection. The von Frey test was conducted prior to
the thermal test, which followed immediately within minutes for
each animal. In addition, clinical observation scoring of the
animal's reaction to injection was conducted. FIG. 16 schematically
illustrates the study design.
1.1 Measurement of Mechanical Pain Sensitivity Using the Von Frey
Test (FIGS. 17 and 18)
[0253] Mechanical pain sensitivity was tested using the von Frey
test, which measures the withdrawal force threshold of the animals.
The lower the force applied represents a greater sensitivity to the
stimulus. The von Frey test was performed 15 and 90 minutes
post-Test Item injection.
[0254] Animals treated with treprostinil at a dose of 1 .mu.g/mL or
100 .mu.g/mL had a reduced withdrawal force threshold (higher
sensitivity) at 15 and 90 minutes post-injection. This increased
sensitivity was statistically significantly greater than the
PBS-treated group (p<0.05).
[0255] Animals treated with treprostinil side-chain carbamate
(Prodrug II) or treprostinil methyl ether (Prodrug VIII), at both
doses, showed no statistically significant difference when compared
to the PBS-treated group at both time points post-injection.
[0256] Animals treated with treprostinil ring carbamate (Prodrug I)
at a dose of 1 .mu.g/mL showed no statistically significant
difference when compared to the PBS-treated group at both time
points post-injection. Animals treated with 100 .mu.g/mL
treprostinil ring carbamate (Prodrug I) showed no statistically
significant difference when compared to the PBS-treated group at 15
minutes post-injection; however, a statistically significant
increased sensitivity was observed when compared to the PBS-treated
group at 90 minutes post-injection.
[0257] Animals treated with treprostinil amide (Prodrug VII) at a
dose of 1 .mu.g/mL showed no statistically significant difference
when compared to the PBS group at both time points post-injection.
Animals treated with treprostinil amide (Prodrug VII) at a dose of
100 .mu.g/mL had a statistically significant increased sensitivity
when compared to the PBS treated group at both time points
post-injection.
1.2 Measurement of Thermal Pain Sensitivity (FIGS. 19 and 20)
[0258] The sensitivity of animals to a thermal pain stimulus was
assessed immediately following the von Frey test. The time until
withdrawal of the right-injected leg from a heat source was
measured, and the lower (faster) the time of response represents a
greater sensitivity to the stimulus. The test was performed 15 and
90 minutes post-Test Item injection.
[0259] Animals treated with treprostinil at a dose of 1 .mu.g/mL or
100 .mu.g/mL had a statistically significantly faster paw
withdrawal time (increased sensitivity) when compared to the PBS
treated group at 15 minutes post-administration (p<0.05).
[0260] Animals treated with all prodrugs at both doses showed no
statistically significant difference in response to the thermal
stimuli when compared to the PBS group. However, animals treated
with the prodrug treprostinil amide (Prodrug VII) at a dose of 100
.mu.g/mL or with the prodrug treprostinil methyl ether (Prodrug
VIII) at both doses showed a trend toward reduction in the time of
response, compared to the PBS group at both time points, although
with no statistical significance.
1.3 Clinical Observation Score (FIGS. 21 and 22)
[0261] A Clinical observation score was assigned by assessing for
redness, swelling and paw placement.
[0262] Treatment with treprostinil at a dose of 1 .mu.g/mL or 100
.mu.g/mL resulted in a statistically significant increase in the
clinical score at 15 and 90 minutes post-injection when compared to
the PBS treated group (p<0.05).
[0263] Treatment with all prodrugs at a dose of 1 .mu.g/mL showed
no clinical score, similar to the PBS group, at both time points.
Animals treated with the prodrugs treprostinil side-chain carbamate
(Prodrug II) or treprostinil methyl ether (Prodrug VIII), at a dose
of 100 .mu.g/mL were not statistically significantly different from
the PBS group at both time-points. Animals treated with the prodrug
treprostinil ring carbamate (Prodrug I) at a dose of 100 .mu.g/mL
showed a statistically significant increase in the clinical score
when compared to PBS group at 90 minutes post-injection. Animals
treated with the prodrug treprostinil amide (Prodrug VII) at a dose
of 100 .mu.g/mL showed a statistically significant increase in the
clinical score when compared to the PBS group at both time points
post-injection.
2. Conclusions
[0264] In view of the findings obtained under the conditions of
this study, and confined to the in-life data, administration of the
alternative prodrugs of treprostinil showed a reduced pain response
when compared with similar doses of treprostinil, although some
differences were noted between the individual prodrugs and the
different tests.
[0265] For example, treprostinil side-chain carbamate (Prodrug II)
or treprostinil methyl ether (Prodrug VIII), at both doses and at
both time points, were generally associated with a statistically
significant reduced sensitivity of the animals to mechanical
stimulation and reduced clinical score when compared to a similar
doses of treprostinil.
[0266] Whereas, the prodrugs treprostinil ring carbamate (Prodrug
I) and treprostinil amide (Prodrug VII) were generally associated
with a reduced sensitivity of the animals to mechanical stimulation
and reduced clinical score at both time points post-injection for
only the 1 .mu.g/mL dose when compared to a similar dose of
treprostinil.
[0267] In addition, all prodrugs showed a reduced sensitivity to a
thermal stimulation at both doses and at both time points
post-injection when compared to similar doses of treprostinil.
Example 10
Evaluation of Treprostinil Prodrugs in the Rat Intraplantar
Injection Model
1. Summary
[0268] Treprostinil, a synthetic prostacyclin analog, is the active
pharmaceutical ingredient in Remodulin. Subcutaneous administration
of treprostinil is associated with pain at the site of injection,
and the objective of this study was to evaluate alternative
prodrugs of treprostinil to assess a pain response in the rat paw
pain model.
[0269] Male Sprague Dawley rats (n=56) were allocated into 7 groups
of 8 animals per group. Animals were treated with treprostinil at a
dose of 100 .mu.g/mL or 1 .mu.g/mL, or with the test items Prodrug
VII and Prodrug XV, at both doses. Each group was compared to the
phosphate buffer (50-mM phosphate buffer with 50-mM sodium chloride
at pH=7.4) treated group, which served as the control (Group
1).
[0270] Animals were administered 0.1 mL of test material by
subcutaneous injection into the paw pad (Intraplantar injection) at
time zero. Then, the animals were subsequently evaluated for their
response to mechanical (von Frey filaments) and thermal stimuli, 15
and 90 minutes post-injection. The von Frey test was conducted
prior to the thermal test, which followed immediately within
minutes for each animal. In addition, clinical observation scoring
of the animal's reaction to injection was conducted. The study
design is schematically illustrated in FIG. 23
1.1 Measurement of Mechanical Pain Sensitivity Using the Von Frey
Test (FIG. 24)
[0271] Mechanical pain sensitivity was tested using the von Frey
test, which measures the withdrawal force threshold of the animals.
The lower the force applied represents a greater sensitivity to the
stimulus. The von Frey test was performed 15 and 90 minutes
post-test item injection.
[0272] Animals treated with treprostinil at a dose of 1 .mu.g/mL or
100 .mu.g/mL had a reduced withdrawal force threshold (higher
sensitivity) at 15 and 90 minutes post-injection. This increased
sensitivity was statistically significantly greater than the
phosphate buffer group (p<0.001 and p<0.0001,
respectively).
[0273] Animals treated with Prodrug VII or Prodrug XV at a dose of
1 .mu.g/mL or 100 .mu.g/mL also showed a reduced withdrawal force
threshold (higher sensitivity) at 15 and 90 minutes post-injection.
This increased sensitivity was statistically significantly greater
than the phosphate buffer group (p<0.001 or p<0.0001). No
difference in the withdrawal force threshold was found in the test
items treated groups when compared to the treprostinil treated
groups.
1.2 Measurement of Thermal Pain Sensitivity (FIG. 25)
[0274] The sensitivity of animals to a thermal pain stimulus was
assessed immediately following the von Frey test. The time until
withdrawal of the right-injected leg from a heat source was
measured, and the lower (faster) the time of response represents a
greater sensitivity to the stimulus. The test was performed 15 and
90 minutes post-test item injection.
[0275] Animals treated with treprostinil at a dose of 1 .mu.g/mL or
100 .mu.g/mL had a statistically significantly faster paw
withdrawal time (increased sensitivity) when compared to the
phosphate buffer treated group, 15 minutes post-administration
(p<0.0001). 90 minutes post-test item injection, only animals
treated with the higher dose of treprostinil (100 .mu.g/mL; group
2) showed a statistically significantly increased sensitivity to
the heat stimulation, compared to the vehicle (phosphate buffer)
group; p<0.05.
[0276] Animals treated with Prodrug VII at a dose of 100 .mu.g/mL
or Prodrug XV at a both doses (1 .mu.g/mL or 100 .mu.g/mL) had a
statistically significantly faster paw withdrawal time (increased
sensitivity) when compared to the phosphate buffer treated group,
15 minutes post-administration (p<0.01 or p<0.001 or
p<0.0001).
[0277] Interestingly, animals treated with Prodrug VII at a dose of
1 .mu.g/mL (Group 5) had similar response time to the baseline, 15
minutes post-administration.
1.3 Clinical Observation Score (FIG. 26)
[0278] A clinical observation score was assigned by assessing for
redness, swelling and paw placement.
[0279] All treated animals, except animals in group 5 that were
treated with Prodrug VII at a dose of 1 .mu.g/mL, showed
statistically significant increase in the clinical score, 15
minutes post-injection when compared to the phosphate buffer
treated group (p<0.0001). At 90 minutes time point, only animals
treated with treprostinil at both doses, and animals treated with
both test items at the higher dose (100 .mu.g/mL) showed the same
effect (p<0.0001).
2. Conclusions
[0280] In view of the findings obtained under the conditions of
this study, and confined to the in-life data, administration of the
alternative prodrugs of treprostinil did not show a significant
effect in reducing the pain response when compared with similar
doses of treprostinil. However, it is worth mentioning that
treatment with the test item Prodrug VII, at a dose of 1 .mu.g/mL,
resulted in reduced sensitivity to thermal stimulation and lower
values of clinical score, at both time-points post
administration.
Example 11
Evaluation of Treprostinil Prodrugs in the Rat Intraplantar
Injection Model
[0281] The study design is schematically illustrated in FIG. 16.
FIGS. 27 and 28 present results for Von Frey Response test of
Cycles 1 and 2 respectively. FIGS. 29 and 30 present results for
Thermal Response test of Cycles 1 and 2 respectively. FIGS. 31 and
32 present mean clinical score for Cycles 1 and 2 respectively.
Example 12
Prodrug VII and Treprostinil: Cardiovascular Assessment Following
Subcutaneous Injection to Sprague Dawley Rats
1. Objective
[0282] The objective of this study was to assess the potential
acute effects of Prodrug VII or treprostinil (a prostacyclin
analog) on heart rate, blood pressure (systolic, diastolic, and
mean), and body temperature following subcutaneous injection in
conscious Crl:CD(SD) rats instrumented with a radiotelemetry
transmitter.
2. Methodology
[0283] Treprostinil in the vehicle (phosphate buffered saline [PBS]
1.times.) or Prodrug VII in the vehicle (20 mM phosphate buffer
with 125 mM sodium chloride) was administered as a single dose via
subcutaneous injection to 9 groups (Groups 1 through 9) of 4 male
Sprague Dawley rats/group according to a dose escalation design
(up/down procedure). The study design is shown in Table 10.
TABLE-US-00010 TABLE 10 Dose Dose Dose Number Group Level
Concentration Volume of Number Test Article (mg/kg).sup.a (mg/mL)
(mL/kg).sup.a Males 1 Treprostinil 0.000125 0.000125 1 4.sup.b 2
Treprostinil 0.00125 0.00125 1 4.sup.b 3 Treprostinil 0.030 0.030 1
4.sup.b 4 Treprostinil 0.3 0.3 1 .sup. 4.sup.b,d 5 Prodrug VII 1
0.1 10 4.sup.c 6 Prodrug VII 10 1 10 4.sup.c 7 Prodrug VII 30 3 10
4.sup.c 8 Prodrug VII 3 0.3 10 4.sup.c 9 Treprostinil 0.1 0.1 1
.sup. 4.sup.b,e .sup.a= Dose calculated from body weight. .sup.b=
The same 4 animals received each treatment with approximately 3
days between doses. .sup.c= The same 4 animals received each
treatment with approximately 3 days between doses. .sup.d= Due to
the probe failure of Male No. 1172 during the Group 4 dosing
session, an additional animal from the stock colony was selected
and evaluated at this dose level. .sup.e= The 3 animals
successfully evaluated in Group 4 and an additional animal from the
stock colony were evaluated at this dose level. Heart rate,
arterial blood pressure (systolic, diastolic, and mean arterial
pressure), pulse pressure, and body temperature were collected
continuously for at least 2 hours prior to administration of
treprostinil or Prodrug VII, and continuously for at least 24 hours
postdosing. Clinical observations were performed at approximately 6
hours prior to dosing, at the completion of dosing and at
approximately 4 hours postdosing.
3. Results
3.1. Clinical Observations
[0284] Following administration of 0.1 and 0.3 mg/kg treprostinil,
clinical observations of flushed extremities were noted in some
animals at approximately 4 hours postdosing. Clinical observations
of flushed body and/or extremities, piloerection, hypoactivity,
reddened forelimb(s) and/or hindlimb(s), wet yellow material on the
urogenital area and ventral trunk, and dried red material around
nose were noted in some animals approximately 4 hours following
administration of 3, 10, or 30 mg/kg Prodrug VII.
3.2. Hemodynamic Data
3.2.1. Heart Rate (FIG. 33)
[0285] Higher heart rates were observed following administration of
treprostinil and Prodrug VII.
[0286] While the magnitude of change was generally similar for
treprostinil groups (.gtoreq.0.030 mg/kg), the duration of change
increased with increasing dose. Heart rate changes were considered
resolved by approximately 5 hours postdosing.
[0287] Similar magnitude of higher heart rate was observed
following administration of 1 and 3 mg/kg; and 10 and 30 mg/kg
Prodrug VII, respectively, with slightly higher heart rates noted
at 10 and 30 mg/kg. Changes in heart rate persisted longer in
comparison to treprostinil groups, with recovery (compared to
predose baseline) for all groups observed at approximately 19 to 20
hours postdosing.
[0288] Increased heart rate following administration of
treprostinil or Prodrug VII was considered to be a compensatory
increase in response to reduced systemic blood pressure.
3.2.2. Systolic Blood Pressure (FIG. 34)
[0289] No meaningful changes in systolic blood pressure were
observed following administration of <0.030 mg/kg treprostinil,
although marginally lower systolic blood pressure was observed
following administration of 0.03 mg/kg treprostinil. Significantly
lower systolic blood pressure was observed as early as the 10
minute time point following administration of 0.1 and 0.3 mg/kg
treprostinil, with the nadir at 30 and 20 minutes, and persisted
through 90 and 120 minutes postdosing, respectively.
[0290] No meaningful change in systolic blood pressure was observed
following administration of 0.1 mg/kg Prodrug VII. Marginally lower
systolic blood pressure was observed following administration of 3
mg/kg Prodrug VII, with the nadir at 50 minutes. Significantly
lower systolic blood pressure was noted following administration of
10 and 30 mg/kg as early as the 20 minute time point, with the
nadir at 40 minutes and hypotension persisting through
approximately 4 and 6 hours, respectively.
3.2.3. Diastolic Blood Pressure (FIG. 35)
[0291] Changes in diastolic blood pressure largely mirrored the
observed changes in systolic blood pressure.
[0292] No meaningful changes in diastolic blood pressure were
observed following administration of .ltoreq.0.030 mg/kg
treprostinil, although marginally lower diastolic blood pressure
was observed following administration of 0.03 mg/kg treprostinil.
Significantly lower diastolic blood pressure was observed as early
as the 10 minute time point following administration of 0.1 and 0.3
mg/kg treprostinil, with the nadir at 30 and 20 minutes, and
persisted through 90 and 180 minutes postdosing, respectively.
[0293] No meaningful change in diastolic blood pressure was
observed following administration of 0.1 mg/kg Prodrug VII.
Marginally lower diastolic blood pressure was observed following
administration of 3 mg/kg Prodrug VII, with the nadir at 50 minutes
and persisted through 80 minutes. Significantly lower diastolic
blood pressure was noted as early as the 20 minute time point
following administration of 10 and 30 mg/kg, with the nadir at 40
minutes and hypotension persisting through approximately 4 and 7 to
8 hours, respectively.
3.2.4. Mean Arterial Pressure (FIG. 36)
[0294] Observed trends in mean arterial pressure mirrored magnitude
and duration of observed changes in systolic and diastolic blood
pressure. FIG. 36 illustrates the approximately 100-fold decrease
in potency for Prodrug VII and the 10-20 minute delay in time to
initial and maximal vasodilator effect, which may suggest that
Prodrug VII conversion to treprostinil governs its vasodilatory
properties. In addition, the sustained vasopressor response of
Prodrug VII over approximately 2-6 hours (compared to 1-2 hours for
treprostinil) suggests maintained conversion to pharmacodynamic
concentrations of treprostinil.
[0295] No meaningful changes in mean blood pressure were observed
following administration of <0.030 mg/kg treprostinil, although
marginally lower mean blood pressure was observed following
administration of 0.03 mg/kg treprostinil, with the nadir at 30
minutes. Significantly lower mean blood pressure was observed as
early as the 10 minute time point following administration of 0.1
and 0.3 mg/kg treprostinil, with the nadir at 30 and 20 minutes,
and persisted through 90 and 180 minutes postdosing,
respectively.
[0296] No meaningful change in mean blood pressure was observed
following administration of 0.1 mg/kg Prodrug VII. Marginally lower
mean blood pressure was observed following administration of 3
mg/kg Prodrug VII, with the nadir at 50 minutes. Significantly
lower mean blood pressure was noted following administration of 10
and 30 mg/kg, with the nadir at 40 minutes and hypotension
persisting through approximately 4 and 7 to 8 hours,
respectively.
3.2.5. Pulse Pressure (FIG. 37)
[0297] Changes in pulse pressure were variable and lacked
consistent direction and magnitude of response, and further lacked
dose response relationship.
[0298] Marginally lower pulse pressure was observed from 20 to 40
minutes postdosing following administration of 0.3 mg/kg
treprostinil. There were no other consistent trends observed
following administration of treprostinil at any other dose levels
investigated.
[0299] Marginally higher pulse pressure was noted following
administration of 10 mg/kg Prodrug VII. No other consistent trends
were observed following any other doses of Prodrug VII.
[0300] The pulse pressure is a function of the systolic and
diastolic blood pressure. As the magnitude and direction of change
for systolic and diastolic were similar, the overall net change
(difference between systolic and diastolic) was largely
unaltered.
3.2.6. Body Temperature (FIG. 38)
[0301] No significant changes in body temperature were observed
following administration of 0.000125 mg/kg or 0.00125 mg/kg
treprostinil. Marginally lower body temperature was observed
following 0.1 mg/kg treprostinil. This change persisted through
approximately 3 hours postdosing. Significantly lowered body
temperature was observed following administration of 0.1 and 0.3
mg/kg treprostinil. These changes were considered resolved by
approximately 3 and 4 hours postdosing, respectively.
[0302] No significant changes in body temperature were observed
following administration of 1 mg/kg Prodrug VII. Significantly
lower body temperature was observed following administration of 3,
10, and 30 mg/kg. Hypothermic response persisted through 4 hours
and 11 to 12 hours following administration of 3 and 10 mg/kg,
respectively. Body temperature did not recover within 24 hours
following administration of 30 mg/kg Prodrug VII.
[0303] Changes in body temperature were secondary to changes in
blood pressure. Body temperature decreases were directly related to
vasodilation.
4. Conclusions
[0304] Administration of treprostinil resulted in higher heart rate
(all doses), and significantly lower systolic, diastolic, mean
arterial blood pressure (.gtoreq.0.1 mg/kg), and body temperature
(.gtoreq.0.030 mg/kg). Administration of Prodrug VII resulted in
higher heart rate (all doses), and significantly lower systolic,
diastolic, mean arterial), and body temperature (>3 mg/kg).
Example 13
Synthesis of Dimethyl Ether of Treprostinil (Prodrug IX)
Discussion
[0305] The synthesis of dimethyl ether of treprostinil (1) was
achieved by O-methylation using NaH and methyl iodide in THF at
room temperature. This method involved short reaction time and
simple work-up as compared to other reaction conditions studied to
obtain the Prodrug IX.
##STR00085##
Experimental Procedure
[0306] A 50-ml round bottom flask was charged with sodium hydride
(0.61 g, 15.36 mmol, 60% in mineral oil) and this was washed with
hexane (2.times.20 ml) to remove the mineral oil. To this solid
NaH, anhydrous THF (10 ml) was added and stirred at ambient
temperature under argon. To this suspension, treprostinil (1) (0.5
g, 1.26 mmol) in THF (5.0 ml) was added dropwise, followed by
methyl iodide (3.0 ml). The reaction mixture was stirred for 5 h
and the progress of the reaction was monitored by TLC
(DCM/methanol, 9:1). The reaction was quenched with aq. saturated
NH.sub.4Cl solution (1.0 ml), diluted with water (10.0 ml). The pH
was adjusted to 1-2 with 2N HCl. The organic layer was separated
and aqueous layer was extracted with EtOAc (3.times.20 ml). The
extracts were combined and dried over Na.sub.2SO.sub.4. The solvent
was removed in vacuo to obtain crude product. The crude product was
purified by silica gel chromatography using a gradient solvent
(0-10% methanol in DCM) to give product dimethyl ether of
treprostinil (Prodrug IX) (230 mg).
Example 14
Synthesis of Treprostinil Monomethyl Carbamate (Prodrug VIII)
[0307] The treprostinil monomethyl carbamate (Prodrug VIII) (5) was
synthesized from mono-TES treprostinil benzyl ester (1). The
mono-TES treprostinil benzyl ester (1) was treated with
p-nitrophenyl chloroformate to generate the carbonate of
p-nitrophenyl (2). The carbonate (2), without isolation, was
treated with methylamine in tetrahydrofuran to give TES
treprostinil benzyl monomethyl carbamate (3) in good yield. The
desilylation of compound (3) with hydrochloric acid in aqueous
tetrahydrofuran afforded treprostinil benzyl ester monomethyl
carbamate (4). The debenzylation of pure carbamate (4) with
palladium on carbon under the atmosphere of hydrogen gave
treprostinil monomethyl carbamate (Prodrug VIII) (5).
Synthesis of Treprostinil Monomethyl Carbamate (Prodrug VIII)
(5)
##STR00086##
[0308] Experimental
Synthesis of TES-Treprostinil Benzyl Ester Monomethyl Carbamate
(3)
##STR00087##
[0310] To a solution of mono-TES-treprostinil benzyl ester (1)
(1.11 g, 1.87 mmol) in anhydrous tetrahydrofuran (12 mL) was added
pyridine (0.44 g, 0.45 mL, 5.56 mmol) at room temperature under
argon. The clear solution was cooled to 0.degree. C. (ice/water
bath) and then added dropwise a solution of 4-nitrophenyl
chloroformate (0.56 g, 2.78 mmol) in anhydrous tetrahydrofuran (4
mL) over a period of 5 min keeping the temperature below 5.degree.
C. under argon. After complete addition, the reaction mixture
(white turbid) was stirred at 0.degree. C. to room temperature for
2 h. After 2 h, the reaction mixture was checked by tlc
(EtOAc/Hexane, 1:4) and the reaction was complete. The reaction
mixture was cooled to 0.degree. C. and then added a solution of
methylamine in tetrahydrofuran (2.0 M) (3.8 mL, 7.60 mmol) over a
period of 3 min. The reaction mixture was stirred at 0.degree. C.
for 1 h and checked tlc (EtOAc/Hexane, 1:4). The reaction was
complete. The mixture was filtered and the yellow solid was washed
with MTBE (2.times.20 mL). The filtrate was concentrated in vacuo
to give light yellow viscous liquid (1.70 g). The chromatography of
the crude product on silica gel (31 g) column using 5-15%
EtOAc/Hexane afforded pure TES-treprostinil benzyl ester monomethyl
carbamate (3) (1.17 g)
Synthesis of Treprostinil Benzyl Ester Monomethyl Carbamate (4)
##STR00088##
[0312] To a solution of TES-treprostinil benzyl ester monomethyl
carbamate (3) (1.10 g, 1.69 mmol) in a mixture of tetrahydrofuran
(20 mL) and water (4 mL) was added hydrochloric acid solution (2 N)
(0.85 mL, 1.70 mmol) at room temperature under argon. The reaction
mixture was stirred at room temperature for 2 h and checked tlc
(EtOAc/Hexane, 1:1). The reaction was complete. The reaction
mixture was neutralized with triethylamine (0.25 mL) and then
evaporated off all organic volatiles and the residue was dissolved
in EtOAc (25 mL) and washed with water (2.times.20 mL), brine
(1.times.10 mL), dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo to give clear viscous liquid (1.07 g). The
crude product was chromatographed on silica gel (30 g) column using
5-70% EtOAc/Hexane) to give pure treprostinil benzyl ester
monomethyl carbamate (4) as a colorless viscous liquid (0.90
g).
Synthesis of Treprostinil Monomethyl Carbamate (5)
##STR00089##
[0314] To a solution of treprostinil benzyl ester monomethyl
carbamate (4) (0.84 g, 1.56 mmol) in ethyl acetate (13 mL) was
added palladium on carbon (5 wt %, 50% water) (0.15 g). The mixture
was stirred and evacuated under house vacuum and replaced by
hydrogen (filled in a balloon). The process was repeated three
times. The mixture was stirred at room temperature under the
atmosphere of hydrogen for 16 h and checked tlc (EtOAc/Hexane,
1:1). The reaction was complete. The reaction mixture was filtered
through a pad of Celite (1.0 g) in a disposable polyethylene filter
funnel, and the solid was washed with ethyl acetate (3.times.10
mL). The filtrate was concentrated in vacuo at 30.degree. C. (water
bath temperature) to give treprostinil monomethyl carbamate
(Prodrug VIII) (5) as an off-white foamy solid (0.71 g)
Example 15
Synthesis of Treprostinil Amino Acid Amide Prodrugs
Discussion
[0315] Treprostinil was subjected to amidation with various amino
acids using coupling agents to form the treprostinil amides as
prodrugs, as shown in schemes below.
Synthesis of Treprostinil Alanine Amide (Prodrug X)
##STR00090##
[0316] Step 1
[0317] To a suspension of treprostinil (1) (1.0 g, 2.561 mmol) and
L-alanine benzyl ester p-toluenesulfonate salt (0.9 g, 2.561 mmol)
in dichloromethane (30 mL) was added triethylamine (0.89 mL, 6.401
mmol). To this mixture
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
(0.59 g, 3.073 mmol) and 1-hydroxybenzotriazole hydrate (0.42 g,
3.073 mmol) were added. The reaction mixture was stirred at ambient
temperature under argon for 2.5 h. Based on TLC (eluent: ethyl
acetate) the reaction was found to be complete. The reaction
mixture was quenched with water (30 mL) and the organic layer was
separated, dried over sodium sulfate and evaporated in vacuo to
obtain crude product. The crude product was purified using silica
gel column chromatography using 0-70% ethyl acetate in hexane to
obtain pure treprostinil alanine amide benzyl ester (2) (1.34 g,
97.8% yield).
Step 2
[0318] To a solution of treprostinil alanine amide benzyl ester (2)
(1.3 g) in ethyl acetate was added a 5% palladium on carbon (50%
w/w water) (130 mg). This was evacuated three times using vacuum,
replaced with hydrogen gas and stirred under hydrogen atmosphere
for 1.5 h. Based on TLC (eluent: ethyl acetate) the reaction was
found to be complete. The reaction mixture was filtered through
Celite to remove palladium on carbon. The filtrate was evaporated
in vacuo to obtain treprostinil alanine amide prodrug (Prodrug X)
(1.03 g, 91.7% yield).
Synthesis of Treprostinil Valine Amide (Prodrug XI)
##STR00091##
[0319] Step 1
[0320] To a suspension of treprostinil (1) (1.0 g, 2.561 mmol) and
L-valine benzyl ester p-toluenesulfonate salt (0.97 g, 2.561 mmol)
in dichloromethane (30 mL) was added triethylamine (0.89 mL, 6.401
mmol). To this mixture
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
(0.59 g, 3.073 mmol) and 1-hydroxybenzotriazole hydrate (0.42 g,
3.073 mmol) were added. The reaction mixture was stirred at ambient
temperature under argon for 2 h. Based on TLC (eluent: ethyl
acetate) the reaction was found to be complete. The reaction
mixture was quenched with water (30 mL) and stirred for 15 min. The
organic layer was separated, dried over sodium sulfate and
evaporated in vacuo to obtain crude product. The crude product was
purified using silica gel column chromatography using 0-50% ethyl
acetate in hexane to obtain pure treprostinil valine amide benzyl
ester (2) (1.3 g, 90.3% yield).
Step 2
[0321] To a solution of treprostinil valine amide benzyl ester (2)
(1.3 g) in ethyl acetate (15 mL) was added a 5% palladium on carbon
(50% w/w water) (130 mg). This was evacuated three times using
vacuum, replaced with hydrogen gas and stirred under hydrogen
atmosphere for 2 h. Based on TLC (eluent: ethyl acetate) the
reaction was found to be complete. The reaction mixture was
filtered through Celite to remove palladium on carbon. The filtrate
was evaporated in vacuo to obtain treprostinil valine amide prodrug
(Prodrug XI) (1.1 g, 97.1% yield with residual solvent).
Synthesis of Treprostinil Aspartic Acid Amide (Prodrug XII)
##STR00092##
[0322] Step 1
[0323] To a suspension of treprostinil (1) (1.0 g, 2.561 mmol) and
L-aspartic acid dibenzyl ester p-toluenesulfonate salt (1.24 g,
2.561 mmol) in dichloromethane (30 mL) was added triethylamine
(0.89 mL, 6.401 mmol). To this mixture
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
(0.59 g, 3.073 mmol) and 1-hydroxybenzotriazole hydrate (0.42 g,
3.073 mmol) were added. The reaction mixture was stirred at ambient
temperature under argon for 2 h. Based on TLC (eluent: ethyl
acetate) the reaction was found to be complete. The reaction
mixture was quenched with water (30 mL) and stirred for 15 min. The
organic layer was separated, dried over sodium sulfate and
evaporated in vacuo to obtain crude product. The crude product was
purified using silica gel column chromatography using 0-50% ethyl
acetate and hexane as a mobile to obtain pure treprostinil aspartic
acid amide benzyl ester (2) (1.63 g, 97.6% yield).
Step 2
[0324] To a solution of treprostinil aspartic acid amide benzyl
ester (2) (0.57 g) in ethyl acetate (20 mL) was added a 5%
palladium on carbon (50% w/w water) (57 mg). This was evacuated
three times using vacuum, replaced with hydrogen gas and stirred
under hydrogen atmosphere for 5 h. Based on TLC (eluent: ethyl
acetate) the reaction was found to be complete. The reaction
mixture was filtered through Celite to remove palladium on carbon.
The filtrate was evaporated in vacuo to obtain treprostinil
aspartic acid amide prodrug (Prodrug XII) (0.4 g, 90.9% yield).
Synthesis of Treprostinil Serine Amide (Prodrug XIII)
##STR00093##
[0325] Step 1
[0326] To a suspension of treprostinil (1) (1.0 g, 2.561 mmol) and
L-serine benzyl ester benzenesulfonate salt (0.9 g, 2.561 mmol) in
dichloromethane (30 mL) was added triethylamine (0.89 mL, 6.401
mmol). To this mixture
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
(0.59 g, 3.073 mmol) and 1-hydroxybenzotriazole hydrate (0.42 g,
3.073 mmol) were added. The reaction mixture was stirred at ambient
temperature under argon for 2 h. Based on TLC (eluent: ethyl
acetate) the reaction was found to be complete. The reaction
mixture was quenched with water (30 mL) and stirred for 15 min. The
organic layer was separated, dried over sodium sulfate and
evaporated in vacuo to obtain crude product. This was purified
using silica gel column chromatography using 0-100% ethyl acetate
and hexane as a mobile to obtain pure treprostinil serine amide
benzyl ester (2) (0.62 g, 49.3% yield).
Step 2
[0327] To a solution of treprostinil serine amide benzyl ester (2)
(0.57 g) in ethyl acetate (120 mL) was added a 5% palladium on
carbon (50% w/w water) (57 mg). This was evacuated three times
using vacuum, replaced with hydrogen gas and stirred under hydrogen
atmosphere for 2 h. Based on TLC (eluent: ethyl acetate) the
reaction was found to be complete. The compound crashed out of the
solution after the reaction. To solubilize and isolate the product
from palladium on carbon, isopropyl alcohol (30 mL) was added. The
reaction mixture was then filtered through Celite to remove
palladium on carbon. The filtrate was evaporated in vacuo to obtain
treprostinil serine amide prodrug (Prodrug XIII) (0.54 g, 100%
yield).
Example 16
Synthesis of Treprostinil Methanesulfonamide (Prodrug XIV)
[0328] The treprostinil methanesulfonamide (Prodrug XIV) (7) was
synthesized from benzindene triol (1). The treprostinil benzyl
ester (2) was prepared from triol (1). The ester (2) was silylated
with tert-butyldimethyl trifluoromethanesulfonamide (TBDMSOTf) to
give di-TBDMS treprostinil benzyl ester (3). The debenzylation of
compound (3) in ethyl acetate with 5% palladium on carbon in the
atmosphere of hydrogen provided di-TBDMS treprostinil (4). The
activation of acid (4) with CDI followed by reaction with
methanesulfonamide in the presence of DBU gave di-TBDMS
treprostinil methanesulfonamide (6) and purified by silica gel
column. The deprotection of TBDMS from sulfonamide (6) using
hydrogen chloride in methanol afforded the desired treprostinil
methanesulfonamide (Prodrug XIV) (7).
Synthesis of Treprostinil Methanesulfonamide (Prodrug XIV)
##STR00094## ##STR00095##
[0329] Experimental
Synthesis of Treprostinil Benzyl Ester (2)
##STR00096##
[0331] To a solution of benzindene triol (1) (240.0 g, 0.72 mol) in
acetone (3.0 L) was added powdered potassium carbonate (199.5 g,
1.44 mol) and bromo benzylacetate (190.2 g, 0.83 mol) at room
temperature under argon. The reaction mixture was stirred at room
temperature and the progress of the reaction was monitored by tlc.
After 72 h, the reaction was complete. The reaction mixture was
filtered and the filtrate was evaporated in vacuo to give the
treprostinil benzyl ester (2) (346.0 g, 99%) as an off white
solid.
Synthesis of Di-TBDMS Treprostinil Benzyl Ester (3)
##STR00097##
[0333] To a solution of treprostinil benzyl ester (2) (15.26 g,
31.75 mmol) in anhydrous dichloromethane (150 mL) was added
2,6-lutidine (13.61 g, 14.75 mL, 127.01 mmol) at room temperature.
The clear solution was cooled to 0.degree. C. (ice/water bath) and
then added dropwise a solution of tert-butyldimethyl
trifluoromethanesulfonate (TBDMSOTf) (20.98 g, 18.23 mL, 79.37
mmol) in anhydrous dichloromethane (30 mL) over a period of 20 min
keeping the temperature below 5.degree. C. under argon. After
complete addition, the reaction mixture was stirred at 0-5.degree.
C. for 2 h. After 2 h, the reaction mixture was checked by tlc
(EtOAc/Hexane, 1:4) and the reaction was complete. The mixture was
treated with hexane (360 mL, twice the volume of dichloromethane
used) and stirred for 10 min at room temperature. The mixture was
passed through silica gel (230-400 mesh) (293 g) column and the
compound was eluted with ethyl acetate in hexane (2-6%) to give
pure di-TBDMS treprostinil benzyl ester (3) (21.7 g, 96.4%).
Synthesis of Di-TBDMS Treprostinil (4)
##STR00098##
[0335] To a solution of di-TBDMS treprostinil benzyl ester (3)
(21.6 g, 30.46 mmol) in ethyl acetate (320 mL) was added palladium
on carbon (5 wt %, 50% water) (2.16 g). The mixture was stirred and
evacuated under house vacuum and replaced by hydrogen (filled in a
balloon). The process was repeated three times. The mixture was
stirred at room temperature under the atmosphere of hydrogen for 2
h and checked tlc (EtOAc/Hexane, 1:4 and EtOAc, 100%). The reaction
was complete. The reaction mixture was treated with Celite (7.0 g)
and the filtered through a pad of silica gel (22 g) in a disposable
polyethylene filter funnel, and the solid was washed with ethyl
acetate (3.times.50 mL). The filtrate contained some carbon
particles and therefore the filtrate was filtered again through a
pad of Celite (10.0 g) to get clear filtrate. The clear filtrate
was passed through silica gel (30 g) column and washed the silica
gel with ethyl acetate (2.times.70 mL). The filtrate was clear and
the filtrate was concentrated in vacuo at 30.degree. C. (water bath
temperature) to give di-TBDMS treprostinil (4) as a colorless
viscous liquid (18.6 g, 98.7%).
Synthesis of Di-TBDMS Treprostinil Methanesulfonamide (6)
##STR00099##
[0337] To a solution of di-TBDMS treprostinil (4) (18.5 g, 29.88
mmol) in anhydrous tetrahydrofuran (190 mL) was added
1,1'-carbonyldiimidazole (CDI) (7.27 g, 44.83 mmol) in one portion
at room temperature under argon. The clear reaction mixture was
stirred at room temperature for 30 min and then at 75.degree. C.
(oil bath temperature) for 30 min. The reaction mixture was cooled
to room temperature. To this in situ generated CDI intermediate of
di-TBDMS treprostinil (5) was added methansulfonamide (8.53 g,
89.68 mmol) in one portion and stirred at room temperature for 10
min until clear solution was obtained. To this clear solution was
added a solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (22.74
g, 149.37 mmol) in anhydrous tetrahydrofuran (40 mL) under argon.
After complete addition, the reaction mixture was stirred at room
temperature and monitored by tlc (EtOAc, 100% and
MeOH/CH.sub.2Cl.sub.2, 1:9). After 2 h, the reaction was complete.
The mixture was quenched with water (200 mL) and then extracted
with EtOAc (1.times.200 mL), (2.times.100 mL). The combined EtOAc
extracts were washed with water (3.times.100 mL), brine (1.times.30
mL), dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo
to give off-white foamy solid (21.44 g). The crude product was
chromatographed on silica gel (230-400 mesh) (296 g) using
CH.sub.2Cl.sub.2 and 1-30% MeOH/CH.sub.2Cl.sub.2 to give di-TBDMS
treprostinil methanesulfonamide (6) as a white foamy solid (15.4 g,
74.0%).
Synthesis of Treprostinil Methanesulfonamide (7)
##STR00100##
[0339] A solution of di-TBDMS treprostinil methanesulfonamide (6)
(13.4 g, 19.25 mmol) in anhydrous methanol (135 mL) was cooled to 0
to 5.degree. C. (ice/water bath). To this cold solution was added a
solution of hydrogen chloride in methanol (1.25 M) (38.5 mL, 48.13
mmol) in anhydrous methanol (135 mL) over a period of 3 min under
argon. The reaction mixture was stirred at 0 to 5.degree. C.
(ice/water bath) for 30 min and checked tlc (MeOH/CH.sub.2Cl.sub.2,
1:9). The argon was bubbled slowly through the reaction mixture for
5 min at 0 to 5.degree. C. to remove excess hydrogen chloride.
Then, the reaction mixture was evaporated in vacuo at 25.degree. C.
(water bath temperature) to remove the organic volatiles to give
crude sulfonamide product (7) as a pale yellow foamy solid (11.03
g). This compound was combined with other crude product (0.80 g) to
give a total weight of 11.83 g. The combined crude product was
chromatographed on silica gel (175 g) column using 25-100%
EtOAc/Hexane and 1-20% MeOH/EtOAc to give pure treprostinil
methanesulfonamide (7) as an off-white foamy solid (6.28 g).
Example 17
Synthesis of Starting Material: Treprostinil Mono-TES Benzyl Ester
(2a) Required for Various Prodrugs
##STR00101##
[0341] To a solution of treprostinil benzyl ester (1) (100 g, 20.80
mmol) in acetone (200 mL) was added imidazole (1.41 g, 20.80 mmol)
and 4-dimethylaminopyridine (0.25 g, 2.08 mmol). To this mixture,
while stirring, chlorotriethylsilane (3.5 mL, 20.80 mmol) was added
using a syringe under argon atmosphere. After 1 h the reaction was
found to be complete based on TLC (eluent: 20% ethyl
acetate/hexane). The reaction was quenched with water (150 mL) and
the organic layer was separated, washed with brine (100 mL), dried
over sodium sulfate and evaporated in vacuo to obtain crude
product. The crude material was purified by column chromatography
using ethyl acetate: hexanes (0-11%) as mobile phase to obtain both
mono-protected compound 2a (6.68 g) in 54.04% yield and 2b (0.48 g)
in 3.88% yield.
Example 18
[0342] Mean Metabolite-to-Parent Ratios in Male Sprague Dawley Rats
Following Single Administration of Prodrugs I, II, III and XV are
presented in Table 11.
TABLE-US-00011 TABLE 11 SC Injection 6-Hour SC Infusion IV
100.sup.a/200.sup.b 100.sup.a/200.sup.b Injection Prodrug 1 mg/kg
50 mg/kg mg/kg 1 mg/kg 50 mg/kg mg/kg 1 mg/kg I 0.0000826 0.00035
0.000546.sup.b NC 0.000277 0.000493.sup.b NC II 0.00058 0.000711
0.000805.sup.b 0.00039 0.000866 0.000651.sup.b 0.000363 III 0.0371
0.2134 NC.sup.a 0.0342 0.124 NC.sup.b 0.158 XV 0.329 0.251
0.352.sup.a 0.262 0.39 0.308.sup.a 0.211 NC = Not calculated.
.sup.aAnimals were administered 100 mg/kg. .sup.bAnimals were
administered 200 mg/kg.
Additional Embodiments
[0343] 1. A compound having the following formula:
##STR00102##
[0343] wherein:
[0344] X is OH or
##STR00103##
where R.sub.1 is H or C.sub.1-C.sub.4 alkyl; and
[0345] each of R.sub.2 and R.sub.3 is independently selected from
H, C.sub.1-4 alkyl, or
##STR00104##
wherein Y is OR.sub.4 or NR.sub.4R.sub.5, wherein each of R.sub.4
and R.sub.5 is independently selected from H and C.sub.1-4 alkyl;
with a proviso that when X is OH, both of R.sub.2 and R.sub.3 are
not H; or
[0346] a pharmaceutically acceptable salt of the compound. [0347]
2. The compound of embodiment 1, wherein X is OH. [0348] 3. The
embodiment of claim 2, wherein each of R.sub.2 and R.sub.3 is
independently selected from C.sub.1-4 alkyl. [0349] 4. The compound
of embodiment 2, wherein each of R.sub.2 and R.sub.3 is methyl.
[0350] 5. The compound of embodiment 2, wherein each of R.sub.2 and
R.sub.3 is independently selected from H, and
[0350] ##STR00105## [0351] 6. The compound of embodiment 5, wherein
one of R.sub.2 and R.sub.3 is
##STR00106##
[0351] and the other of R.sub.2 and R.sub.3 is H. [0352] 7. The
compound of embodiment 6, wherein Y is OR.sub.4. [0353] 8. The
compound of embodiment 7, wherein R.sub.4 is methyl or H. [0354] 9.
The compound of embodiment 6, wherein Y is NR.sub.4R.sub.5. [0355]
10. The compound of embodiment 9, wherein each of R.sub.4 and
R.sub.5 is independently selected from H or methyl. [0356] 11. The
compound of embodiment 9, wherein both of R.sub.4 and R.sub.5 are H
or methyl. [0357] 12. A pharmaceutical composition, comprising (A)
the compound of any one of embodiments 1-11 and (B) a
pharmaceutically acceptable carrier. [0358] 13. The pharmaceutical
composition of embodiment 12, which is an oral pharmaceutical
composition. [0359] 14. The pharmaceutical composition of
embodiment 12, which is a subcutaneous pharmaceutical composition.
[0360] 15. A method of treating pulmonary hypertension comprising
administering to a subject in need thereof an effective amount of
the compound of any one of embodiments 1-11. [0361] 16. The method
of embodiment 15, wherein the administering is performed orally.
[0362] 17. The method of embodiment 15, wherein the subject is a
human being. [0363] 18. The method of embodiment 15, wherein the
administering is performed by an injection. [0364] 19. The method
of embodiment 18, wherein the administering is performed
subcutaneously. [0365] 20. The method of embodiment 19, wherein
said administering is continuous subcutaneous administering. [0366]
21. The method of embodiment 18, wherein said administering results
in no or less pain at a site of the injection compared to
administering treprostinil. [0367] 22. A method of treating
pulmonary hypertension comprising administering subcutaneously to a
patient suffering from pulmonary hypertension an effective amount
of a prodrug of treprostinil. [0368] 23. A method of treating
pulmonary hypertension comprising selecting a patient who has
experienced site pain upon subcutaneous administration of
treprostinil or a pharmaceutically salt thereof and administering
subcutaneously to a patient suffering from pulmonary hypertension
an effective amount of a prodrug of treprostinil.
[0369] Although the foregoing refers to particular preferred
embodiments, it will be understood that the present invention is
not so limited. It will occur to those of ordinary skill in the art
that various modifications may be made to the disclosed embodiments
and that such modifications are intended to be within the scope of
the present invention.
[0370] All of the publications, patent applications and patents
cited in this specification are incorporated herein by reference in
their entirety.
* * * * *
References