U.S. patent application number 16/576720 was filed with the patent office on 2020-04-30 for treating sickle cell disease with a pyruvate kinase r activating compound.
The applicant listed for this patent is FORMA Therapeutics, Inc.. Invention is credited to Anna Ericsson, Neal Green, Gary Gustafson, David R. Lancia, JR., Gary Marshall, Lorna Mitchell, Madhu Mondal, Schroeder Patricia, Kelly J. Patrick, Maria Ribadeneira, David Richard, Forsyth Sanjeev, Zhongguo Wang.
Application Number | 20200129485 16/576720 |
Document ID | / |
Family ID | 69774634 |
Filed Date | 2020-04-30 |
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United States Patent
Application |
20200129485 |
Kind Code |
A1 |
Ericsson; Anna ; et
al. |
April 30, 2020 |
TREATING SICKLE CELL DISEASE WITH A PYRUVATE KINASE R ACTIVATING
COMPOUND
Abstract
Compounds that activate pyruvate kinase R can be used for the
treatment of sickle cell disease (SCD). Methods and compositions
for the treatment of SCD are provided herein, including a
therapeutic compound designated as Compound 1.
Inventors: |
Ericsson; Anna; (Shrewsbury,
MA) ; Green; Neal; (Newton, MA) ; Gustafson;
Gary; (Ridgefield, CT) ; Lancia, JR.; David R.;
(Boston, MA) ; Marshall; Gary; (Watertown, MA)
; Mitchell; Lorna; (West Beach, AU) ; Richard;
David; (Littleton, MA) ; Wang; Zhongguo;
(Lexington, MA) ; Sanjeev; Forsyth; (Milton,
MA) ; Patrick; Kelly J.; (Concord, MA) ;
Mondal; Madhu; (Winchester, MA) ; Ribadeneira;
Maria; (Cambridge, MA) ; Patricia; Schroeder;
(Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FORMA Therapeutics, Inc. |
Watertown |
MA |
US |
|
|
Family ID: |
69774634 |
Appl. No.: |
16/576720 |
Filed: |
September 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62733558 |
Sep 19, 2018 |
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|
62733562 |
Sep 19, 2018 |
|
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|
62782933 |
Dec 20, 2018 |
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62789641 |
Jan 8, 2019 |
|
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|
62811904 |
Feb 28, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/436 20130101;
A61P 7/06 20180101; A61K 9/0053 20130101 |
International
Class: |
A61K 31/436 20060101
A61K031/436; A61P 7/06 20060101 A61P007/06; A61K 9/00 20060101
A61K009/00 |
Claims
1. A method of reducing levels of 2,3-diphosphoglycerate (2,3-DPG)
in a patient's red blood cells, the method comprising orally
administering to the patient in need thereof a therapeutically
effective amount of the compound
(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-
-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-
-1 in a pharmaceutical composition each day.
2. The method of claim 1, comprising administering a total of 25
mg-1,500 mg of the compound
(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6--
tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1
to the patient per day.
3. The method of claim 1, wherein the compound is administered in a
single dose each day.
4. The method of claim 1, wherein the compound is administered in a
divided dose each day.
5. The method of claim 2, wherein the compound is administered in a
single dose each day.
6. The method of claim 2, wherein the compound is administered in a
divided dose each day.
7. A method of reducing levels of 2,3-diphosphoglycerate (2,3-DPG)
in a patient's red blood cells, the method comprising administering
to the patient in need thereof a therapeutically effective amount
of Compound 1: ##STR00026## in a pharmaceutical composition
comprising Compound 1 and a pharmaceutically acceptable
carrier.
8. The method of claim 7, comprising administering a total of 25
mg-1,500 mg of the Compound 1 to the patient per day.
9. The method of claim 7, wherein the Compound 1 is administered in
a single dose each day.
10. The method of claim 7, wherein the Compound 1 is administered
in a divided dose each day.
11. The method of claim 8, wherein the Compound 1 is administered
in a single dose each day.
12. The method of claim 8, wherein the Compound 1 is administered
in a divided dose each day.
13. A method of reducing levels of 2,3-diphosphoglycerate (2,3-DPG)
in a patient's red blood cells, the method comprising orally
administering to the patient in need thereof a total of 25 mg-1,500
mg of the Compound 1 to the patient per day in a single or divided
dose: ##STR00027## in a pharmaceutical composition comprising
Compound 1 and a pharmaceutically acceptable carrier.
14. The method of claim 13, wherein the Compound 1 is administered
in a single dose each day.
15. The method of claim 13, wherein the Compound 1 is administered
in a divided dose each day.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/733,558, filed on Sep. 19, 2018, U.S.
Provisional Application No. 62/733,562, filed on Sep. 19, 2018,
U.S. Provisional Application No. 62/782,933, filed on Dec. 20,
2018, U.S. Provisional Application No. 62/789,641, filed on Jan. 8,
2019, and U.S. Provisional Application No. 62/811,904, filed on
Feb. 28, 2019, each of which is incorporated by reference in its
entirety.
TECHNICAL FIELD
[0002] This disclosure relates to the treatment of sickle cell
disease (SCD), including the treatment of patients diagnosed with
SCD by the administration of a compound that activates pyruvate
kinase R (PKR).
BACKGROUND
[0003] Sickle cell disease (SCD) is a chronic hemolytic anemia
caused by inheritance of a mutated form of hemoglobin (Hgb), sickle
Hgb (HgbS). It is the most common inherited hemolytic anemia,
affecting 70,000 to 80,000 patients in the United States (US). SCD
is characterized by polymerization of HgbS in red blood cells
(RBCs) when HgbS is in the deoxygenated state (deoxy-HgbS),
resulting in a sickle-shaped deformation. Sickled cells aggregate
in capillaries precipitating vaso-occlusive events that generally
present as acute and painful crises resulting in tissue ischemia,
infarction, and long-term tissue damage. RBCs in patients with SCD
tend to be fragile due to sickling and other factors, and the
mechanical trauma of circulation causes hemolysis and chronic
anemia. Finally, damaged RBCs have abnormal surfaces that adhere to
and damage vascular endothelium, provoking a
proliferative/inflammatory response that underlies large-vessel
stroke and potentially pulmonary-artery hypertension. Collectively,
these contribute to the significant morbidity and increased
mortality associated with this disease.
[0004] Currently, therapeutic treatment of SCD is inadequate. The
only known cure for SCD is hematopoietic stem cell transplantation
which has serious risks, is typically recommended for only the most
serious cases, and is largely offered only to children with
sibling-matched donors. Gene therapy is also under investigation
with promising preliminary results; however, there are market
access hurdles, mainly high cost and treatment complexities, that
are likely to limit its broad use in the near term. There have been
few advances in therapies for SCD over the past two decades.
Hydroxyurea (HU) induces HgbF which interrupts the polymerization
of HgbS, and thereby has activity in decreasing the onset of
vaso-occlusive crises and pathological sequelae of SCD. While HU is
in wide use as a backbone therapy for SCD, it remains only
partially effective, and is associated with toxicity, such as
myelosuppression and teratogenicity. Patients receiving HU still
experience hemolysis, anemia, and vaso-occlusive crises, suggesting
a need for more effective therapies, either as a replacement or in
combination with HU. Beyond HU, therapeutic intervention is largely
supportive care, aimed at managing the symptoms of SCD. For
instance, blood transfusions help with the anemia and other SCD
complications by increasing the number of normal RBCs. However,
repeated transfusions lead to iron overload and the need for
chelation therapies to avoid consequent tissue damage. In addition
to these approaches, analgesic medications are used to manage
pain.
[0005] Given the current standard of care for SCD, there is a clear
medical need for a noninvasive, disease-modifying therapy with
appropriate safety and efficacy profiles.
SUMMARY
[0006] One aspect of the disclosure relates to methods of treating
SCD comprising the administration of a therapeutically effective
amount of a pyruvate kinase R (PKR) activator to a patient in need
thereof diagnosed with SCD. Pyruvate kinase R (PKR) is the isoform
of pyruvate kinase expressed in RBCs, and is a key enzyme in
glycolysis. The invention is based in part on the discovery that
the activation of PKR can target both sickling, by reducing
deoxy-HgbS, and hemolysis. Targeting hemolysis may be achieved by
improving RBC membrane integrity. One aspect of the disclosure is
the recognition that activation of PKR can reduce
2,3-diphosphoglycerate (2,3-DPG), which leads to decreased
deoxy-HgbS (and, therefore, sickling), as well as can increase ATP,
which promotes membrane health and reduces hemolysis. Another
aspect of the disclosure is the recognition that activation of PKR
can reduce 2,3-diphosphoglycerate (2,3-DPG), which inhibits Hgb
deoxygenation/increases oxygen affinity of HgbS and leads to
decreased deoxy-HgbS (and, therefore, sickling), as well as can
increase ATP, which promotes membrane health and reduces hemolysis.
Accordingly, in one embodiment, PKR activation (e.g., by
administration of a therapeutically effective amount of a PKR
Activating Compound to a patient diagnosed with SCD) reduces RBC
sickling via a reduction in levels of 2,3-diphosphoglycerate
(2,3-DPG), which in turn reduces the polymerization of sickle Hgb
(HgbS) into rigid aggregates that deform the cell. Furthermore, in
some embodiments, PKR activation may contribute to overall RBC
membrane integrity via increasing levels of adenosine triphosphate
(ATP), which is predicted to reduce vaso-occlusive and hemolytic
events which cause acute pain crises and anemia in SCD
patients.
[0007] Preferably, a patient diagnosed with SCD is treated with a
compound that is a PKR Activating Compound. The PKR activator can
be a compound identified as a PKR Activating Compound or a
composition identified as a PKR Activating Composition having an
AC.sub.50 value of less than 1 M using the Luminescence Assay
described in Example 2, or a pharmaceutically acceptable salt
and/or other solid form thereof. For example, the PKR Activating
Compound can be the compound
(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6--
tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one
(Compound 1):
##STR00001##
or a pharmaceutically acceptable salt thereof. Compound 1 is a
selective, orally bioavailable PKR Activating Compound that
decreases 2,3-DPG, increases ATP, and has anti-sickling effects in
disease models with a wide therapeutic margin relative to
preclinical toxicity.
[0008] PKR Activating Compounds can be readily identified as
compounds of Formula I:
##STR00002##
or a pharmaceutically acceptable salt thereof, (e.g., Compound 1
and mixtures of Compound 1 with its stereoisomer) having an
AC.sub.50 value of less than 1 M using the Luminescence Assay
described in Example 2.
[0009] In other embodiments, the PKR Activating Compound can be any
of the compounds listed in FIG. 1, or a pharmaceutically acceptable
salt thereof.
[0010] PKR Activating Compounds, such as
1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetr-
ahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one,
or a pharmaceutically acceptable salt thereof, are useful in
pharmaceutical compositions for the treatment of patients diagnosed
with SCD. PKR Activating Compounds, such as any of the compounds
listed in FIG. 1, or a pharmaceutically acceptable salt thereof,
are useful in pharmaceutical compositions for the treatment of
patients diagnosed with SCD. The compositions comprising a compound
of Formula I (e.g., Compound 1), or a pharmaceutically acceptable
salt thereof, can be obtained by certain processes also provided
herein. The compositions comprising any of the compounds listed in
FIG. 1, or a pharmaceutically acceptable salt thereof, can be
obtained by certain processes also provided herein.
[0011] The methods of treating SCD provided herein can offer
greater protection against vaso-occlusive crises and hemolytic
anemia, as compared to existing and emerging therapies. Therefore,
use of a PKR Activating Compound, such as Compound 1, provides a
novel and improved therapeutic approach either alone or in
combination with drugs that act through alternative mechanisms,
such as hydroxyurea (HU). In addition, use of a PKR Activating
Compound, such as any of the compounds listed in FIG. 1, provides a
novel and improved therapeutic approach either alone or in
combination with drugs that act through alternative mechanisms,
such as hydroxyurea (HU).
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a table of PKR Activating Compounds.
[0013] FIG. 2 is a schematic showing the relationship of PKR
activation to the reduction of the clinical consequences of sickle
cell disease (SCD).
[0014] FIG. 3 is a graph showing the oxyhemoglobin dissociation
curve and modulating factors by plotting the relationship between
hemoglobin saturation (percent) vs. partial pressure of oxygen
(mmHg).
[0015] FIG. 4A is a chemical synthesis scheme for compounds of
Formula I, including a synthesis of Compound 1 (separately provided
in FIG. 4B).
[0016] FIG. 4B is a chemical synthesis scheme for Compound 1.
[0017] FIG. 4C is a general chemical synthesis of the compounds
listed in FIG. 1.
[0018] FIG. 5 is a graph showing activation of recombinant
PKR-R510Q with Compound 1, plotting the normalized rate vs.
concentration of phosphoenolpyruvate (PEP) (Example 3).
[0019] FIG. 6 is a graph of data showing activation of recombinant
PKR-R510Q by Compound 1 in the enzyme assay of Example 3.
[0020] FIG. 7 is a graph of data showing PKR activation in human
red blood cells treated with Compound 1 (Example 4).
[0021] FIG. 8A (Study 1) and FIG. 8B (Study 2) are each graphs
showing the observed changes in 2,3-DPG levels in blood from mice
following 7 days of once daily (QD) oral treatment with Compound 1
(Example 5).
[0022] FIG. 9 is a graph showing observed changes in 2,3-DPG levels
in blood from mice following 7 days of once daily (QD) oral
treatment with Compound 1 (Example 5, Study 2).
[0023] FIG. 10A (Study 1) and FIG. 10B (Study 2) are graphs of data
measuring ATP concentrations in red blood cells of mice following 7
days of once daily (QD) oral treatment with Compound 1 (Example
5).
[0024] FIG. 11 is a graph of the blood 2,3-DPG levels measured over
time in healthy volunteers who received a single dose of Compound 1
or placebo.
[0025] FIG. 12 is a graph of the blood 2,3-DPG levels measured 24
hours post-dose in healthy volunteers who received a single dose of
Compound 1 or placebo.
[0026] FIG. 13 is a graph of the blood 2,3-DPG levels measured over
time in healthy volunteers who received daily doses of Compound 1
or placebo for 14 days.
[0027] FIG. 14 is a graph of the blood 2,3-DPG levels measured on
day 14 in healthy volunteers who received daily doses of Compound 1
or placebo for 14 days.
[0028] FIG. 15 is a graph of the blood ATP levels measured on day
14 in healthy volunteers who received daily doses of Compound 1 or
placebo for 14 days.
[0029] FIG. 16 is a graph plotting the blood concentration of
Compound 1 (ng/mL) measured in healthy volunteer (HV) patients on a
first (left) axis and the concentration of 2,3-DPG (micrograms/mL)
measured in these HV patients on a second (right) axis after
administration of a single dose of Compound 1 (400 mg).
DETAILED DESCRIPTION
[0030] Methods of treating SCD preferably include administration of
a therapeutically effective amount of a compound (e.g., Compound 1)
that reduces HgbS polymerization, for example by increasing HgbS
affinity for oxygen. Methods of treating SCD also preferably
include administration of a therapeutically effective amount of a
compound (e.g., any of the compounds listed in FIG. 1) that reduces
HgbS polymerization, for example by increasing HgbS affinity for
oxygen. Methods of lowering 2,3-DPG and/or increasing ATP levels in
human RBCs comprise administering a PKR Activating Compound, such
as Compound 1. Methods of lowering 2,3-DPG and/or increasing ATP
levels in human RBCs also comprise administering a PKR Activating
Compound, such as any of the compounds listed in FIG. 1. Together
these effects are consistent with providing therapies to reduce
HgbS sickling and to improve RBC membrane health, presenting a
unique disease-modifying mechanism for treating SCD.
[0031] A PKR Activating Compound, such as Compound 1, is useful to
promote activity in the glycolytic pathway. A PKR Activating
Compound, such as any of the compounds listed in FIG. 1, also is
useful to promote activity in the glycolytic pathway. As the enzyme
that catalyzes the last step of glycolysis, PKR directly impacts
the metabolic health and primary functions of RBCs. PKR Activating
Compounds (e.g., Compound 1), are useful to decrease 2,3-DPG and
increase ATP. PKR Activating Compounds (e.g., any of the compounds
listed in FIG. 1), are useful to decrease 2,3-DPG and increase ATP.
PKR Activating Compounds (e.g., Compound 1 or any of the compounds
listed in FIG. 1, preferably Compound 1) are also useful to
increase Hgb oxygen affinity in RBC. The disclosure is based in
part on the discovery that PKR activation is a therapeutic modality
for SCD, whereby HgbS polymerization and RBC sickling are reduced
via decreased 2,3-DPG and increased ATP levels.
[0032] SCD is the most common inherited blood disorder and
clinically manifests with potentially severe pathological
conditions associated with substantial physical, emotional, and
economic burden. For instance, acute vaso-occlusive pain crises can
be debilitating and necessitate rapid medical response. Chronic
hemolytic anemia causes fatigue and often necessitates blood
transfusions and supportive care. Over time, impaired oxygen
transport through microvasculature precipitates organ and tissue
damage. While there are a number of options available for treating
symptoms, overall disease management would benefit from therapies
that target upstream processes to prevent vaso-occlusion and
hemolysis.
[0033] The described clinical symptoms are largely due to
perturbations in RBC membrane shape and function resulting from
aggregation of HgbS molecules. Unlike normal Hgb, HgbS polymerizes
when it is in the deoxygenated state and ultimately causes a
deformed, rigid membrane that is unable to pass through small blood
vessels, thereby blocking normal blood flow through
microvasculature. The loss of membrane elasticity also increases
hemolysis, reducing RBC longevity. Furthermore, decreased cellular
ATP and oxidative damage contribute to a sickle RBC membrane that
is stiffer and weaker than that of normal RBCs. The damaged
membrane has a greater propensity for adhering to vasculature,
leading to hemolysis, increased aggregation of sickled RBCs, and
increased coagulation and inflammation associated with
vaso-occlusive crises.
[0034] The underlying cause of sickling is the formation of rigid
deoxy-HgbS aggregates that alter the cell shape and consequently
impact cellular physiology and membrane elasticity. These
aggregates are highly structured polymers of deoxygenated HgbS; the
oxygenated form does not polymerize. Polymerization is promoted by
a subtle shift in conformation from the oxygen-bound relaxed
(R)-state to the unbound tense (T)-state. In the latter, certain
residues within the 0-chain of HgbS are able to interact in a
specific and repetitive manner, facilitating polymerization.
[0035] The concentration of deoxy-HgbS depends on several factors,
but the predominant factor is the partial pressure of oxygen
(PO.sub.2). Oxygen reversibly binds to the heme portions of the Hgb
molecule. As oxygenated blood flows via capillaries to peripheral
tissues and organs that are actively consuming oxygen, PO.sub.2
drops and Hgb releases oxygen. The binding of oxygen to Hgb is
cooperative and the relationship to PO.sub.2 levels fits a
sigmoidal curve (FIG. 3). This relationship can be impacted by
temperature, pH, carbon dioxide, and the glycolytic intermediate
2,3-DPG. 2,3-DPG binds within the central cavity of the Hgb
tetramer, causes allosteric changes, and reduces Hgb's affinity for
oxygen. Therefore, therapeutic approaches that increase oxygen
affinity (i.e., reduce deoxygenation) of HgbS would presumably
decrease polymer formation, changes to the cell membrane, and
clinical consequences associated with SCD.
[0036] One aspect of this disclosure is targeting PKR activation to
reduce 2,3-DPG levels, based on PKR's role in controlling the rate
of glycolysis in RBCs. A decrease in 2,3-DPG with PKR activation
has been demonstrated in preclinical studies and in healthy
volunteers and patients with pyruvate kinase deficiency.
Additionally, PKR activation would be expected to increase ATP, and
has been observed to do so in these same studies. Given the role of
ATP in the maintenance of a healthy RBC membrane and protection
from oxidative stress, elevating its levels is likely to have broad
beneficial effects. Therefore, activation of PKR offers the
potential for a 2,3-DPG effect (i.e., reduced cell membrane damage
from HgbS polymerization) that is augmented by ATP support for
membrane integrity. It is via these changes that a PKR activator is
could positively impact physiological changes that lead to the
clinical pathologies of SCD (FIG. 2). In another aspect, the
disclosure relates to a method of improving the anemia and the
complications associated with anemia in SCD patients (e.g.,
.gtoreq.12 years of age) with Hgb SS or Hgb
SB.sup.0-thalassemia.
[0037] Compound 1 is a selective, orally bioavailable PKR activator
that has been shown to decrease 2,3-DPG, increase ATP, and have
anti-sickling effects in disease models with a wide therapeutic
margin relative to preclinical toxicity.
[0038] Methods of treatment can comprise administering to a subject
in need thereof a therapeutically effective amount of (i) a PKR
Activating Compound (e.g., a compound disclosed herein), or a
pharmaceutically acceptable salt thereof; or (ii) a PKR Activating
Composition (e.g., a pharmaceutical composition comprising a
compound disclosed herein, or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier). The
pharmaceutical composition may be orally administered in any orally
acceptable dosage form. In some embodiments, to increase the
lifetime of red blood cells, a compound, composition, or
pharmaceutical composition described herein is added directly to
whole blood or packed cells extracorporeally or provided to the
subject (e.g., the patient) directly. A patient and/or subject can
be selected for treatment using a compound described herein by
first evaluating the patient and/or subject to determine whether
the subject is in need of activation of PKR, and if the subject is
determined to be in need of activation of PKR, then administering
to the subject a PKR Activating Compound in a pharmaceutically
acceptable composition. A patient and/or subject can be selected
for treatment using a compound described herein by first evaluating
the patient and/or subject to determine whether the subject is
diagnosed with SCD, and if the subject is diagnosed with SCD, then
administering to the subject a PKR Activating Compound in a
pharmaceutically acceptable composition. For example,
administration of a therapeutically effective amount of a PKR
Activating Compound can include administration of a total of about
25 mg-1,500 mg of Compound 1 each day, in single or divided doses.
In some embodiments, Compound 1 is administered to patients
diagnosed with SCD in total once daily (QD) doses of 25 mg, 50 mg,
75 mg, 100 mg, 125 mg, 150 mg, and/or higher if tolerated (e.g.,
250 mg, 300 mg, 500 mg, 600 mg, 1000 mg, and/or 1500 mg). In some
embodiments, a human dose of 80 to 130 mg of Compound 1 is
administered once daily (QD) to a patient in need thereof (e.g., a
patient diagnosed with SCD). In some embodiments, a PKR Activating
Compound is administered in an amount of 400 mg per day (e.g., 400
mg QD or 200 mg BID). In some embodiments, Compound 1 or a
pharmaceutically acceptable salt thereof is administered in an
amount of 400 mg per day (e.g., 400 mg QD or 200 mg BID). In some
embodiments, any of the compounds listed in FIG. 1 or a
pharmaceutically acceptable salt thereof is administered in an
amount of 400 mg per day (e.g., 400 mg QD or 200 mg BID). In some
embodiments, a PKR Activating Compound is administered in an amount
of 700 mg per day (e.g., 700 mg QD or 350 mg BID). In some
embodiments, Compound 1 or a pharmaceutically acceptable salt
thereof is administered in an amount of 700 mg per day (e.g., 700
mg QD or 350 mg BID). In some embodiments, any of the compounds
listed in FIG. 1 or a pharmaceutically acceptable salt thereof is
administered in an amount of 700 mg per day (e.g., 700 mg QD or 350
mg BID). In some embodiments, a PKR Activating Compound is
administered in an amount of 100 mg, 200 mg, 400 mg, 600 mg, 700
mg, 1100 mg, or 1500 mg per day, in single or divided doses. In
some embodiments, Compound 1 or a pharmaceutically acceptable salt
thereof is administered in an amount of 100 mg, 200 mg, 400 mg, 600
mg, 700 mg, 1100 mg, or 1500 mg per day, in single or divided
doses. In some embodiments, any of the compounds listed in FIG. 1
or a pharmaceutically acceptable salt thereof is administered in an
amount of 100 mg, 200 mg, 400 mg, 600 mg, 700 mg, 1100 mg, or 1500
mg per day, in single or divided doses.
[0039] Methods of treating a patient diagnosed with SCD can include
administering to the patient in need thereof a therapeutic compound
targeting reduction of deoxy-HgbS, which may or may not directly
improve RBC membrane integrity. Compound 1 has been shown to
decrease 2,3-DPG and increase ATP, and reduced cell sickling has
been demonstrated in disease models. Accordingly, in some
embodiments, the methods of treatment can address not only
sickling, but also hemolysis and anemia.
[0040] Methods of treating a patient diagnosed with sickle cell
disease, and PKR Activating Compounds for use in such methods, can
include administering to the patient the PKR Activating Compound
(e.g., a composition comprising one or more compounds of Formula I,
such as Compound 1 or a mixture of Compound 1 and Compound 2) in an
amount sufficient to reduce 2,3-DPG levels in the patient's red
blood cells. In some embodiments, the amount is sufficient to
reduce 2,3-DPG levels by at least 30% after 24 hours, or greater
(e.g., reducing 2,3-DPG levels in the patient's red blood cells by
at least 40% after 24 hours). In some embodiments, the amount is
sufficient to reduce 2,3-DPG levels by 30-50% after 24 hours. In
some embodiments, the amount is sufficient to reduce 2,3-DPG levels
by 40-50% after 24 hours. In some embodiments, the amount is
sufficient to reduce 2,3-DPG levels by at least 25% after 12 hours.
In some embodiments, the amount is sufficient to reduce 2,3-DPG
levels by 25-45% after 12 hours. In some embodiments, the amount is
sufficient to reduce 2,3-DPG levels by at least 15% after 6 hours.
In some embodiments, the amount is sufficient to reduce 2,3-DPG
levels by 15-30% after 6 hours. In some embodiments, the amount is
sufficient to reduce 2,3-DPG levels by at least 40% on day 14 of
treatment. In some embodiments, the amount is sufficient to reduce
2,3-DPG levels by 40-60% on day 14 of treatment. In some
embodiments, the amount is sufficient to reduce 2,3-DPG levels by
at least 50% on day 14 of treatment. In some embodiments, the
amount is sufficient to reduce 2,3-DPG levels by 50-60% on day 14
of treatment.
[0041] Methods of treating a patient diagnosed with sickle cell
disease, and PKR Activating Compounds for use in such methods, can
also include administering to the patient the PKR Activating
Compound (e.g., a composition comprising one or more compounds of
Formula I, such as Compound 1 or a mixture of Compound 1 and
Compound 2) in a daily amount sufficient to increase the patient's
ATP blood levels. In some embodiments, the amount is sufficient to
increase ATP blood levels by at least 40% on day 14 of treatment,
or greater (e.g., t least 50% on day 14 of treatment). In some
embodiments, the amount is sufficient to increase ATP blood levels
by 40-65% on day 14 of treatment. In some embodiments, the amount
is sufficient to increase ATP blood levels by at least 50% on day
14 of treatment, or greater (e.g., at least 50% on day 14 of
treatment). In some embodiments, the amount is sufficient to
increase ATP blood levels by 50-65% on day 14 of treatment.
[0042] In some examples, a pharmaceutical composition comprising
Compound 1 can be used in a method of treating a patient diagnosed
with sickle cell disease, the method comprising administering to
the patient 400 mg of Compound 1 once per day (QD)
##STR00003##
In some examples, a pharmaceutical composition comprising Compound
1 can be used in a method of treating a patient diagnosed with
sickle cell disease, the method comprising administering to the
patient 200 mg of Compound 1 twice per day (BID)
##STR00004##
[0043] In some embodiments, the present disclosure provides PKR
Activating Compounds of Formula I:
##STR00005##
or a pharmaceutically acceptable salt thereof. In some embodiments,
a PKR Activating Compound is
1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetr-
ahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.
[0044] The compound of Formula I is preferably Compound 1:
##STR00006##
or a pharmaceutically acceptable salt thereof. In some embodiments,
a compound of Formula I is
(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6--
tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.
[0045] In some embodiments, the present disclosure provides a PKR
Activating Compound that is any of the compounds listed in FIG. 1,
or a pharmaceutically acceptable salt thereof.
[0046] The present disclosure also provides compositions (e.g.
pharmaceutical compositions) comprising a compound of Formula I. In
some embodiments, a provided composition containing a compound of
Formula I comprises a mixture of Compound 1 and Compound 2:
##STR00007##
or a pharmaceutically acceptable salt thereof. The present
disclosure also provides compositions (e.g. pharmaceutical
compositions) comprising any of the compounds listed in FIG. 1, or
a pharmaceutically acceptable salt thereof.
[0047] Pharmaceutical compositions comprising a PKR Activating
Composition containing a compound of Formula (I) can be formulated
for oral administration (e.g., as a capsule or tablet). For
example, Compound 1 can be combined with suitable compendial
excipients to form an oral unit dosage form, such as a capsule or
tablet, containing a target dose of Compound 1. The drug product
can be prepared by first manufacturing Compound 1 as an active
pharmaceutical ingredient (API), followed by roller
compaction/milling with intragranular excipients and blending with
extra granular excipients. A Drug Product can contain the Compound
1 API and excipient components in Table 1 in a tablet in a desired
dosage strength of Compound 1 (e.g., a 25 mg or 100 mg tablet
formed from a Pharmaceutical Composition in Table 1). The blended
material can be compressed to form tablets and then film
coated.
[0048] The pharmaceutical composition preferably comprises about
30-70% by weight of
(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl-
)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropa-
n-1-one, and a pharmaceutically acceptable excipient in an oral
dosage form.
TABLE-US-00001 TABLE 1 Exemplary Pharmaceutical Compositions of
Compound 1 for Oral Administration % Formulation Function (weight)
Examplary Component API 30-70% Compound 1 Filler 15-40%
Microcrystalline Cellulose Dry binder 2-10% Crospovidone Kollidon
CL Glidant 0.25-1.25% Colloidal Silicon Dioxide Lubricant
0.25-1.00% Magnesium Stearate, Hyqual
[0049] In some embodiments, a provided composition containing a
compound of Formula I comprises a mixture of
(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6--
tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one
and
(R)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,-
5,6-tetrahydropyrro[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.
In some embodiments, a provided composition containing a compound
of Formula I is a mixture of Compound 1 and Compound 2 as part of a
PKR Activating Composition. In some embodiments, a compound of
Formula I is racemic. In some embodiments, a compound of Formula I
consists of about 50% of Compound 1 and about 50% of Compound 2. In
some embodiments, a compound of Formula I is not racemic. In some
embodiments, a compound of Formula I does not consist of about 50%
of Compound 1 and about 50% of Compound 2. In some embodiments, a
compound of Formula I comprises about 99-95%, about 95-90%, about
90-80%, about 80-70%, or about 70-60% of Compound 1. In some
embodiments, a compound of Formula I comprises about 99%, 98%, 95%,
90%, 80%, 70%, or 60% of Compound 1.
[0050] In some embodiments, a PKR Activating Composition comprises
a mixture of Compound 1 and Compound 2. In some embodiments, a PKR
Activating Composition comprises a mixture of Compound 1 and
Compound 2, wherein the PKR Activating Composition comprises a
therapeutically effective amount of Compound 1.
[0051] Compositions comprising a compound of Formula I can be
prepared as shown in FIG. 4A and FIG. 4B. Compounds of Formula I
can be obtained by the general chemical synthesis scheme of FIG.
4A. Compound 1 can be obtained by the chemical synthesis route of
FIG. 4A or FIG. 4B. In brief, compounds of Formula I (FIG. 4A)
and/or Compound 1 (FIG. 4B) can be obtained from a series of four
reaction steps from commercially available starting materials.
Commercially available 7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine
was treated with a mixture of n-butyl lithium and dibutylmagnesium
followed by sulfuryl chloride to give sulfonyl chloride 3.
Treatment of 3 with tert-butyl
1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate in the
presence of triethylamine (TEA) afforded Boc-protected
monosulfonamide 4. Compound 4 was then de-protected in the presence
of trifluoroacetic acid (TFA) to give 5, the free base of the
monosulfonamide. The last step to generate Compound 1 (FIG. 4B) or
Compound 1 and Compound 2 (FIG. 4A) was an amide coupling of 5 and
tropic acid in the presence of
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide hexafluoro-phosphate (HATU).
[0052] The compounds listed in FIG. 1 can be prepared as shown in
FIG. 4C and as described in International Publication No. WO
2018/175474, published Sep. 27, 2018. Generally, the compounds
listed in FIG. 1 can be prepared by acylation and sulfonylation of
the secondary amine groups of hexahydropyrrolopyrrole 6. For
example, sulfonylation of 6 with a suitable sulfonyl chloride 7
affords sulfonyl hexahydropyrrolopyrrole 8, which is then treated
with a suitable carboxylic acid 9 in the presence of an amide
coupling reagent (e.g., HATU) to afford compound 10 (Path 1).
Alternatively, acylation of 6 with a suitable carboxylic acid 9 in
the presence of an amide coupling reagent affords acyl
hexahydropyrrolopyrrole 11, which is then treated with a suitable
sulfonyl chloride 7 to afford compound 10 (Path 2). As a person of
ordinary skill would understand, well-known protecting groups,
functionalization reactions, and separation techniques can be used
in conjunction with Paths 1 and 2 to obtain the specific compounds
listed in FIG. 1.
[0053] Methods of treating SCD also include administration of a
therapeutically effective amount of a bioactive compound (e.g., a
small molecule, nucleic acid, or antibody or other therapy) that
reduces HgbS polymerization, for example by increasing HgbS
affinity for oxygen.
[0054] In other embodiments, the disclosure relates to each of the
following numbered embodiments:
1. A composition comprising a PKR Activating Compound of Formula I,
or a pharmaceutically acceptable salt thereof:
##STR00008##
2. The composition of embodiment 1, wherein the compound of Formula
I is Compound 1, or a pharmaceutically acceptable salt thereof:
##STR00009##
3. The composition of embodiment 2, wherein the composition
comprises a mixture of Compound 1 and Compound 2, or a
pharmaceutically acceptable salt thereof:
##STR00010##
4. The composition of embodiment 1, comprising the compound:
1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfony)-3,4,5,6-tetra-
hydrpyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.
5. The composition of any one of embodiments 1-4, formulated as an
oral unit dosage form. 6. A method of treating a patient diagnosed
with a sickle cell disease (SCD), the method comprising
administering to the patient in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising
(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6--
tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one,
or a pharmaceutically acceptable salt thereof. 7. The method of
embodiment 6, wherein the method comprises oral administration of
the pharmaceutical composition comprising
(S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6--
tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one,
as the only PKR Activating Compound in the pharmaceutical
composition. 8. A method of treating a patient diagnosed with a
sickle cell disease (SCD), the method comprising administering to
the patient in need thereof a therapeutically effective amount of a
pharmaceutical composition comprising Compound 1:
##STR00011##
or a pharmaceutically acceptable salt thereof. 9. A composition
comprising a compound of Formula I obtainable by a process
comprising the step of converting compound 5 into a compound of
Formula I in a reaction described as Step 4:
##STR00012##
10. The composition of embodiment 9, wherein the process further
comprises first obtaining the compound 5 from a compound 4 by a
process comprising Step 3:
##STR00013##
11. The composition of embodiment 10, wherein the process further
comprises first obtaining the compound 4 from a compound 3 by a
process comprising Step 2:
##STR00014##
12. The composition of embodiment 11, wherein the process further
comprises first obtaining the compound 3 from a process comprising
Step 1:
##STR00015##
13. A method of treating a patient diagnosed with sickle cell
disease (SCD), the method comprising administering to the patient
in need thereof a therapeutically effective amount of a PKR
Activating Compound having an AC.sub.50 value of less than 1 M
using the Luminescence Assay described in Example 2. 14. The method
of embodiment 13, wherein the PKR Activating Compound is Compound
1. 15. The method of any one of embodiments 13-14, wherein the PKR
Activating Compound is orally administered to the patient in need
thereof.
16. The use of Compound 1:
##STR00016##
[0055] or a pharmaceutically acceptable salt thereof, for the
treatment of patients diagnosed with sickle cell disease (SCD). 17.
The use of a PKR Activating Compound having an AC.sub.50 value of
less than 1 M using the Luminescence Assay described in Example 2,
in the treatment of patients diagnosed with sickle cell disease.
18. The method of any one of embodiments 6-8 or 13-15, comprising
the administration of Compound 1 once per day. 19. The method of
any one of embodiments 6-8 or 13-15, comprising the administration
of a total of 25 mg-1,500 mg of Compound 1 each day. 20. The method
of any one of embodiments 18-19, comprising the administration of a
total of 25 mg-130 mg of Compound 1 each day.
[0056] In other embodiments, the disclosure relates to each of the
following numbered embodiments:
1. A method for reducing 2,3-diphosphoglycerate (2,3-DPG) levels in
a patient's red blood cells, comprising administering to the
patient a PKR Activating Compound in a therapeutically effective
amount, wherein the PKR Activating Compound is a compound of
Formula I:
##STR00017##
or a pharmaceutically acceptable salt thereof, having an AC.sub.50
value of less than 1 M using the Luminescence Assay described in
Example 2. 2. The method of embodiment 1, wherein the PKR
Activating Compound is Compound 1:
##STR00018##
or a pharmaceutically acceptable salt thereof. 3. The method of
embodiment 1, wherein the PKR Activating Compound is Compound
1:
##STR00019##
4. The method of embodiment 3, wherein the PKR Activating Compound
is administered in an amount of 25-1500 mg per day. 5. The method
of embodiment 3, wherein the PKR Activating Compound is
administered once daily in an amount of 250 mg, 300 mg, 500 mg, 600
mg, 1000 mg, or 1500 mg per day. 6. The method of embodiment 3,
wherein the PKR Activating Compound is administered once daily in
an amount of 100 mg per day. 7. The method of embodiment 3, wherein
the PKR Activating Compound is administered once daily in an amount
of 600 mg per day. 8. The method of embodiment 3, wherein the PKR
Activating Compound is administered once per day. 9. The method of
embodiment 3, wherein the PKR Activating Compound is orally
administered to the patient. 10. The method of embodiment 3,
wherein Compound 1 is the only PKR Activating Compound administered
to the patient.
[0057] In other embodiments, the disclosure relates to each of the
following numbered embodiments:
1. A method for reducing 2,3-diphosphoglycerate (2,3-DPG) levels in
a patient's red blood cells, comprising administering to the
patient the PKR Activating Compound in an amount sufficient to
reduce 2,3-DPG levels in the patient's red blood cells by at least
30% after 24 hours, wherein the PKR Activating Compound is a
compound of Formula I:
##STR00020##
or a pharmaceutically acceptable salt thereof, having an AC.sub.50
value of less than 1 M using the Luminescence Assay described in
Example 2. 2. The method of embodiment 1, wherein the PKR
Activating Compound is Compound 1:
##STR00021##
or a pharmaceutically acceptable salt thereof. 3. The method of
embodiment 1, wherein the PKR Activating Compound is Compound
1:
##STR00022##
4. The method of embodiment 1, wherein Compound 1 is the only PKR
Activating Compound administered to the patient. 5. The method of
any one of embodiments 1-4, wherein the PKR Activating Compound is
orally administered to the patient. 6. The method of any one of
embodiments 1-5, wherein the PKR Activating Compound is
administered once per day. 7. The method of any one of embodiments
1-6, wherein the PKR Activating Compound is administered in an
amount sufficient to reduce 2,3-DPG levels in the patient's red
blood cells by at least 40% after 24 hours. 8. The method of any
one of embodiments 1-7, wherein the PKR Activating Compound is
administered in a daily amount sufficient to increase the patient's
ATP blood levels by at least 40% on day 14 of treatment. 9. The
method of any one of embodiments 1-5, wherein the PKR Activating
Compound is administered in an amount of 100 mg, 200 mg, 400 mg,
600 mg, 700 mg, 1100 mg, or 1500 mg per day. 10. The method of any
one of embodiments 1-5, wherein the PKR Activating Compound is
administered in an amount of 200 mg per day. 11. The method of
embodiment 10, wherein the PKR Activating Compound is administered
in an amount of 200 mg per day once per day (QD). 12. The method of
embodiment 10, wherein the PKR Activating Compound is administered
in an amount of 100 mg per day twice per day (BID). 13. The method
of any one of embodiments 1-5, wherein the PKR Activating Compound
is administered in an amount of 400 mg per day. 14. The method of
embodiment 13, wherein the PKR Activating Compound is administered
in an amount of 400 mg once per day (QD). 15. The method of
embodiment 13, wherein the PKR Activating Compound is administered
in an amount of 200 mg twice per day (BID). 16. The method of any
one of embodiments 1-5, wherein the PKR Activating Compound is
administered in an amount of 600 mg per day. 17. The method of
embodiment 16, wherein the PKR Activating Compound is administered
in an amount of 300 mg twice per day (BID). 18. The method of any
one of embodiments 1-5, wherein the PKR Activating Compound is
administered in an amount of 700 mg per day. 19. The method of
embodiment 18, wherein the PKR Activating Compound is administered
in an amount of 700 mg once per day (QD). 30. The method of
embodiment 18, wherein the PKR Activating Compound is administered
in an amount of 350 mg twice per day (BID).
[0058] The present disclosure enables one of skill in the relevant
art to make and use the inventions provided herein in accordance
with multiple and varied embodiments. Various alterations,
modifications, and improvements of the present disclosure that
readily occur to those skilled in the art, including certain
alterations, modifications, substitutions, and improvements are
also part of this disclosure. Accordingly, the foregoing
description and drawings are by way of example to illustrate the
discoveries provided herein.
EXAMPLES
[0059] As the enzyme that catalyzes the last step of glycolysis,
PKR underlies reactions that directly impact the metabolic health
and primary functions of RBCs. The following Examples demonstrate
how PKR activation by Compound 1 impacts RBCs. The primary effect
of Compound 1 on RBCs is a decrease in 2,3-DPG that is proposed to
reduce Hgb sickling and its consequences on RBCs and oxygen
delivery to tissues. Compound 1 also increases ATP, which may
provide metabolic resources to support cell membrane integrity and
protect against loss of deformability and increased levels of
hemolysis in SCD. With the combination of effects Compound 1 has on
RBCs, it is likely to reduce the clinical sequelae of sickle Hgb
and provide therapeutic benefits for patients with SCD.
[0060] The PKR Activating Compound designated Compound 1 was
prepared as described in Example 1, and tested for PKR activating
activity in the biochemical assay of Example 2.
[0061] The biological enzymatic activity of PKR (i.e., formation of
ATP and/or pyruvate) was evaluated in enzyme and cell assays with
Compound 1, as described in Example 3 and Example 4, respectively.
Results from enzyme assays show that Compound 1 is an activator of
recombinant wt-PKR and mutant PKR, (e.g., R510Q), which is one of
the most prevalent PKR mutations in North America. PKR exists in
both a dimeric and tetrameric state, but functions most efficiently
as a tetramer. Compound 1 is an allosteric activator of PKR and is
shown to stabilize the tetrameric form of PKR, thereby lowering the
K.sub.m (the Michaelis-Menten constant) for PEP.
[0062] In vivo testing in mice (Examples 5) demonstrated PKR
activation in wt mice, and provided an evaluation of effects on
RBCs and Hgb in a murine model of SCD. Compound 1 was well
tolerated up to the highest dose tested, and exposures increased in
a dose-proportional manner. Levels of 2,3-DPG were reduced by
>30% for doses 2120 mg/kg Compound 1 (AUC from 0 to 24 hours
(AUC.sub.0-24>5200 hrng/mL) and levels of ATP were increased by
>40% for 260 mg/kg Compound 1 (AUC.sub.0-24>4000
hrng/mL).
[0063] In some embodiments, a daily dose of between 100 mg to 1500
mg of a PKR Activating Compound is administered to humans. In some
embodiments, a daily dose of between 100 mg to 1500 mg of Compound
1 is administered to humans. In some embodiments, a daily dose of
between 100 mg to 1500 mg of any of the compounds listed in FIG. 1
is administered to humans. In particular, a total daily dose of 100
mg-600 mg of a PKR Activating Compound can be administered to
humans (including, e.g., a dose of 100 mg, 200 mg, 300 mg, 400 mg,
500 mg, or 600 mg, per day, in single or divided doses). In
particular, a total daily dose of 100 mg-600 mg of Compound 1 can
be administered to humans (including, e.g., a dose of 100 mg, 200
mg, 300 mg, 400 mg, 500 mg, or 600 mg, per day, in single or
divided doses). In particular, a total daily dose of 100 mg-600 mg
of any of the compounds listed in FIG. 1 can be administered to
humans (including, e.g., a dose of 100 mg, 200 mg, 300 mg, 400 mg,
500 mg, or 600 mg, per day, in single or divided doses). In some
embodiments, a daily dose of 400 mg (e.g., 400 mg QD or 200 mg BID)
of a PKR Activating Compound is administered to humans. In some
embodiments, a daily dose of 400 mg (e.g., 400 mg QD or 200 mg BID)
of Compound 1, or a pharmaceutically acceptable salt thereof, is
administered to humans. In some embodiments, a daily dose of 400 mg
(e.g., 400 mg QD or 200 mg BID) of any of the compounds listed in
FIG. 1 is administered to humans.
Example 1: Synthesis of Compounds of Formula I
[0064] The PKR Activating Compound 1 was obtained by the method
described herein and the reaction scheme shown in FIG. 4A and/or
FIG. 4B. Compound 1 has a molecular weight of 457.50 Da.
Step 1. 2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride
(3)
[0065] Into a 100 mL round-bottom flask purged and maintained with
an inert atmosphere of nitrogen was placed a solution of n-BuLi in
hexane (2.5 M, 2 mL, 5.0 mmol, 0.54 equiv) and a solution of
n-Bu.sub.2Mg in heptanes (1.0 M, 4.8 mL, 4.8 mmol, 0.53 equiv). The
resulting solution was stirred for 10 min at RT (20.degree. C.).
This was followed by the dropwise addition of a solution of
7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine (2 g, 9.26 mmol, 1.00
equiv) in tetrahydrofuran (16 mL) with stirring at -10.degree. C.
in 10 min. The resulting mixture was stirred for 1 h at -10.degree.
C. The reaction mixture was slowly added to a solution of sulfuryl
chloride (16 mL) at -10.degree. C. The resulting mixture was
stirred for 0.5 h at -10.degree. C. The reaction was then quenched
by the careful addition of 30 mL of saturated ammonium chloride
solution at 0.degree. C. The resulting mixture was extracted with
3.times.50 mL of dichloromethane. The organic layers were combined,
dried over anhydrous sodium sulfate, filtered and concentrated
under vacuum. The residue was purified by silica gel column
chromatography, eluting with ethyl acetate/petroleum ether (1:3).
This provided 1.3 g (60%) of
2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride as a white
solid. LCMS m/z: calculated for C.sub.7H.sub.6ClNO.sub.4S: 235.64;
found: 236 [M+H].sup.+.
Step 2. tert-Butyl
5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrol-
o[3,4-c]pyrrole-2-carboxylate (4)
[0066] Into a 100-mL round-bottom flask was placed
2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride (1.3 g, 5.52
mmol, 1.00 equiv), tert-butyl
1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate (1.16 g, 5.52
mmol), dichloromethane (40 mL), and triethylamine (1.39 g, 13.74
mmol, 2.49 equiv). The solution was stirred for 2 h at 20.degree.
C., then diluted with 40 mL of water. The resulting mixture was
extracted with 3.times.30 mL of dichloromethane. The organic layers
were combined, dried over anhydrous sodium sulfate, filtered and
concentrated under vacuum. The residue was purified by silica gel
column chromatography, eluting with dichloromethane/methanol
(10:1). This provided 1.2 g (53%) of tert-butyl
5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrol-
o[3,4-c]pyrrole-2-carboxylate as a yellow solid. LCMS m/z:
calculated for C.sub.18H.sub.23N.sub.3O.sub.6S: 409.46; found: 410
[M+H].sup.+.
Step 3.
2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-
-pyrrolo[3,4-c]pyrrole (5)
[0067] Into a 100-mL round-bottom flask was placed tert-butyl
5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrol-
o[3,4-c]pyrrole-2-carboxylate (1.2 g, 2.93 mmol, 1.00 equiv),
dichloromethane (30 mL), and trifluoroacetic acid (6 mL). The
solution was stirred for 1 h at 20.degree. C. The resulting mixture
was concentrated under vacuum. The residue was dissolved in 10 mL
of methanol and the pH was adjusted to 8 with sodium bicarbonate (2
mol/L). The resulting solution was extracted with 3.times.10 mL of
dichloromethane. The organic layers were combined, dried over
anhydrous sodium sulfate, filtered and concentrated under vacuum.
The crude product was purified by silica gel column chromatography,
eluting with dichloromethane/methanol (10:1). This provided 650 mg
(72%) of
2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrol-
o[3,4-c]pyrrole as a yellow solid. LCMS m/z: calculated for
C.sub.13H.sub.15N.sub.3O.sub.4S: 309.34; found: 310
[M+H].sup.+.
Step 4.
(S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4-
H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one
(1) and
(R)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-
-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one (2)
[0068] Into a 100 mL round-bottom flask was placed
2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrol-
o[3,4-c]pyrrole (150 mg, 0.48 mmol, 1.00 equiv),
3-hydroxy-2-phenylpropanoic acid (97 mg, 0.58 mmol, 1.20 equiv),
dichloromethane (10 mL), HATU (369 mg, 0.97 mmol, 2.00 equiv) and
DIEA (188 mg, 1.46 mmol, 3.00 equiv). The resulting solution was
stirred overnight at 20.degree. C. The reaction mixture was diluted
with 20 mL of water and was then extracted with 3.times.20 mL of
dichloromethane. The organic layers were combined, dried over
anhydrous sodium sulfate, filtered and concentrated under vacuum.
The residue was purified by prep-TLC eluted with
dichloromethane/methanol (20:1) and further purified by prep-HPLC
(Column: XBridge C18 OBD Prep Column, 100 .ANG., 5 m, 19
mm.times.250 mm; Mobile Phase A: water (10 mmol/L
NH.sub.4HCO.sub.3), Mobile Phase B: MeCN; Gradient: 15% B to 45% B
over 8 min; Flow rate: 20 mL/min; UV Detector: 254 nm). The two
enantiomers were separated by prep-Chiral HPLC (Column, Daicel
CHIRALPAK.RTM. IF, 2.0 cm.times.25 cm, 5 m; mobile phase A: DCM,
phase B: MeOH (hold 60% MeOH over 15 min); Flow rate: 16 mL/min;
Detector, UV 254 & 220 nm). This resulted in peak 1 (2, Rt:
8.47 min) 9.0 mg (4%) of
(R)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-
-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one as a
yellow solid; and peak 2 (1, Rt: 11.83 min) 10.6 mg (5%) of
(S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-
-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one as a
yellow solid.
[0069] (1): .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.13 (d,
J=2.0 Hz, 1H), 7.61 (d, J=2.0 Hz, 1H), 7.31-7.20 (m, 5H), 4.75 (t,
J=5.2 Hz, 1H), 4.50-4.47 (m, 2H), 4.40-4.36 (m, 1H), 4.32-4.29 (m,
2H), 4.11-3.87 (m, 8H), 3.80-3.77 (m, 1H), 3.44-3.41 (m, 1H). LC-MS
(ESI) m/z: calculated for C.sub.22H.sub.23N.sub.3O.sub.6S: 457.13;
found: 458.0 [M+H].sup.+.
[0070] (2): .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.13 (d,
J=2.0 Hz, 1H), 7.60 (d, J=2.0 Hz, 1H), 7.31-7.18 (m, 5H), 4.75 (t,
J=5.2 Hz, 1H), 4.52-4.45 (m, 2H), 4.40-4.36 (m, 1H), 4.34-4.26 (m,
2H), 4.11-3.87 (m, 8H), 3.80-3.78 (m, 1H), 3.44-3.43 (m, 1H). LC-MS
(ESI) m/z: calculated for C.sub.22H.sub.23N.sub.3O.sub.6S: 457.13;
found: 458.0 [M+H].sup.+.
Step 5.
(S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4-
H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one
(1)
[0071] Alternatively, Compound 1 can be synthesized using the
procedure described here as Step 5. A solution of
7-((3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)sulfonyl)-2,3-dihydro-
-[1,4]dioxino[2,3-b]pyridine (130.9 mg, 0.423 mmol) in DMF (2.5 ml)
was cooled on an ice bath, then treated with
(S)-3-hydroxy-2-phenylpropanoic acid (84.8 mg, 0.510 mmol), HATU
(195.5 mg, 0.514 mmol), and DIEA (0.30 mL, 1.718 mmol) and stirred
at ambient temperature overnight. The solution was diluted with
EtOAc (20 mL), washed sequentially with water (20 mL) and brine
(2.times.20 mL), dried (MgSO.sub.4), filtered, treated with silica
gel, and evaporated under reduced pressure. The material was
chromatographed by Biotage MPLC (10 g silica gel column, 0 to 5%
MeOH in DCM) to provide a white, slightly sticky solid. The sample
was readsorbed onto silica gel and chromatographed (10 g silica gel
column, 0 to 100% EtOAc in hexanes) to provide
(2S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6-
H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one (106.5
mg, 0.233 mmol, 55% yield) as a white solid.
Example 2: Biochemical Assay for Identification of PKR Activating
Activity
[0072] PKR Activating Compounds can be identified with the
biochemical Luminescence Assay of Example 2. The PKR activating
activity of a series of chemical compounds was evaluated using the
Luminescence Assay below, including compounds designated Compound
1, Compound 2, and Compounds 6, 7, and 8 below, and the compounds
listed in FIG. 1.
[0073] For each tested compound, the ability to activate PKR was
determined using the following Luminescence Assay. The effect of
phosphorylation of adenosine-5'-diphosphate (ADP) by PKR is
determined by the Kinase Glo Plus Assay (Promega) in the presence
or absence of FBP (D-fructose-1,6-diphosphate; BOC Sciences, CAS:
81028-91-3) as follows. Unless otherwise indicated, all reagents
are purchased from Sigma-Aldrich. All reagents are prepared in
buffer containing 50 mM Tris-HCl, 100 mM KCl, 5 mM MgCl.sub.2, and
0.01% Triton X100, 0.03% BSA, and 1 mM DTT. Enzyme and PEP
(phosphoenolpyruvate) are added at 2.times. to all wells of an
assay-ready plate containing serial dilutions of test compounds or
DMSO vehicle. Final enzyme concentrations for PKR(wt), PKR(R510Q),
and PKR(G332S) are 0.8 nM, 0.8 nM, and 10 nM respectively. Final
PEP concentration is 100 M. The Enzyme/PEP mixture is incubated
with compounds for 30 minutes at RT before the assay is initiated
with the addition of 2.times.ADP and KinaseGloPlus. Final
concentration of ADP is 100 M. Final concentration of KinaseGloPlus
is 12.5%. For assays containing FBP, that reagent is added at 30
.mu.M upon reaction initiation. Reactions are allowed to progress
for 45 minutes at RT until luminescence is recorded by the BMG
PHERAstar FS Multilabel Reader. The compound is tested in
triplicate at concentrations ranging from 42.5 .mu.M to 2.2 nM in
0.83% DMSO. AC.sub.50 measurements were obtained by the standard
four parameter fit algorithm of ActivityBase XE Runner (max, min,
slope and AC.sub.50). The AC.sub.50 value for a compound is the
concentration (.mu.M) at which the activity along the four
parameter logistic curve fit is halfway between minimum and maximum
activity.
[0074] As set forth in Tables 2 and 3 below and in FIG. 1,
AC.sub.50 values are defined as follows: .ltoreq.0.1 .mu.M (+++);
>0.1 .mu.M and .ltoreq.1.0 .mu.M (++); >1.0 .mu.M and
.ltoreq.40 .mu.M (+); >40 .mu.M (0).
TABLE-US-00002 TABLE 2 Luminescence Assay Data AC.sub.50 AC.sub.50
AC.sub.50 Compound (PKRG332S) (PKRR510Q) (WT) 1 ++ +++ +++ 2 + +
+
TABLE-US-00003 TABLE 3 Additional Luminescence Assay Data AC.sub.50
AC.sub.50 Compound Structure (PKRG332S) (PKRR510Q) 6 ##STR00023##
++ + 7 ##STR00024## 0 0 8 ##STR00025## 0 0
[0075] Compounds and compositions described herein are activators
of wild type PKR and certain PKR mutants having lower activities
compared to the wild type. Such mutations in PKR can affect enzyme
activity (catalytic efficiency), regulatory properties, and/or
thermostability of the enzyme. One example of a PKR mutation is
G332S. Another example of a PKR mutation is R510Q.
Example 3: Enzyme Assays of a PKR Activating Compound
[0076] The effect of 2 .mu.M Compound 1 on maximum velocity
(V.sub.max) and PEP K.sub.m (Michaelis-Menten constant, i.e., the
concentration of PEP at which v=1/2v.sub.max) was evaluated for
wt-PKR and PKR-R510Q. Tests were conducted in the presence and
absence of fructose-1,6-bisphosphate (FBP), a known allosteric
activator of PKR. Assessments were made up to 60 min at RT, and
V.sub.max and PEP K.sub.m were calculated. The effect of Compound 1
on V.sub.max ranged from no effect to a modest increase (see FIG. 5
for a representative curve). Compound 1 consistently reduced the
PEP K.sub.m, typically by .about.2 fold, for wt-PKR and PKR-R510Q
in the presence or absence of FBP (Table 4), demonstrating that
Compound 1 can enhance the rate of PKR at physiological
concentrations of PEP.
TABLE-US-00004 TABLE 4 Effect of Compound 1 on PKR Enzyme Kinetic
Parameters No FBP 30 .mu.M FBP Kinetic 2 .mu.M 2 .mu.M Enzyme
Parameter.sup.a DMSO Compound 1 DMSO Compound 1 WT- V.sub.max 1.00
1.14 1.19 1.16 PKR PEP K.sub.m 4.84 2.44 1.98 1.00 PKR V.sub.max
1.54 1.56 1.00 1.29 R510Q PEP K.sub.m 6.20 1.70 2.01 1.00 .sup.aAll
values in Table 4 are normalized to 1.00, relative to the other
values in the same row.
[0077] Activation of wt-PKR and PKR-R510Q by different
concentrations of Compound 1 was evaluated for PEP concentrations
at or below K.sub.m. Compound 1 increased the rate of ATP
formation, with AC.sub.50 values ranging from <0.05 to <0.10
.mu.M and a range of <2.0 to <3.0 maximum-fold activation
(i.e., <200% to <300%) (Table 5). Representative data from
PKR-R510Q showed that the effect was concentration dependent (FIG.
6).
TABLE-US-00005 TABLE 5 Activation of PKR Wild and Mutant Types by
Compound 1 PK Enzyme Maximum-fold Activation AC.sub.50 (.mu.M)
WT-PKR <2.0 <0.05 PKR R510Q <3.0 <0.10
Example 4: Cell Assays of a PKR Activating Compound
[0078] The activation of wt-PKR by Compound 1 in mature human
erythrocytes ex vivo was evaluated in purified RBCs purchased from
Research Blood Components. Cells treated with Compound 1 for 3 hr
in glucose-containing media were washed, lysed, and assayed using a
Biovision Pyruvate Kinase Assay (K709-100). The assay was repeated
multiple times to account for donor-to-donor variability and the
relatively narrow dynamic range. Mean maximum activation increase
(Max-Min) was <100% and mean 50% effective concentration
(EC.sub.50) was <125 nM (Table 6). wt-PKR was activated in a
concentration-dependent manner (FIG. 7).
TABLE-US-00006 TABLE 6 Wild Type PKR Activation in Human Red Blood
Cells Treated with Compound 1 Replicate Max-Min (%) EC.sub.50 (nM)
1 <125 <250 2 <150 <150 3 <100 <50 4 <50
<50 Mean <100 <125
[0079] Mouse RBCs were isolated fresh from whole blood using a
Ficoll gradient and assayed with methods similar to those used in
the human RBCs assays. Maximum activation increase, and EC.sub.50
values were comparable to the effects in human RBCs (Table 7).
TABLE-US-00007 TABLE 7 Effect of Compound 1 on PKR Activation in
Mouse Red Blood Cells Replicate Max-Min (%) EC.sub.50 (nM) 1 <50
<125 2 <100 <125 Mean <100 <125
Example 5: Pharmacokinetic/Pharmacodynamic Studies of Compound 1 in
Wild Type Mice
[0080] Two pharmacokinetic (PK)/phamacodynamic (PD) studies were
conducted in Balb/c mice that were administered Compound 1 once
daily by oral gavage (formulated in 10% Cremophor EL/10% PG/80% DI
water) for 7 days (QD.times.7) at doses of 0 (vehicle), 3.75, 7.5,
15, 30, 60 mg/kg (Study 1); 0 (vehicle), 7.5, 15, 30, 60, 120, or
240 mg/kg (Study 2). On the 7th day, whole blood was collected 24
hours after dosing and snap frozen. Samples were later thawed and
analyzed by LC/MS for 2,3-DPG and ATP levels. In both studies,
Compound 1 was well tolerated. No adverse clinical signs were
observed and there were no differences in body weight change
compared with the vehicle group.
[0081] The levels of 2,3-DPG decreased with Compound 1 treatment
(FIGS. 8A and 8B (Studies 1 and 2) and FIG. 9 (Study 2)). In
general, reductions were >20% at .gtoreq.15 mg/kg Compound 1,
and >30% for 120 and 240 mg/kg Compound 1. Together, the results
from the highest doses provide in vivo evidence that 2,3-DPG
decreases with PKR activation.
[0082] Evaluation of ATP levels in these studies showed that
treatment with Compound 1 increased levels of ATP. In Study 1, ATP
increased 21% and 79% with 30 and 60 mg/kg Compound 1,
respectively, compared to vehicle, and in Study 2, ATP levels
increased with exposure with doses up to 120 mg/kg Compound 1 with
a maximum increase of .about.110% compared to vehicle (FIG. 10A and
FIG. 10B). At the highest dose, 240 mg/kg Compound 1, ATP levels
increased by 45%. Levels of ATP correlated with Compound 1 exposure
in a manner similar across both studies.
Example 6: A SAD/MAD Study to Assess the Safety, Pharmacokinetics,
and Pharmacodynamics of Compound 1 in Healthy Volunteers and Sickle
Cell Disease Patients
[0083] Compound 1 will be evaluated in a randomized,
placebo-controlled, double blind, single ascending and multiple
ascending dose study to assess the safety, pharmacokinetics, and
pharmacodynamics of Compound 1 in healthy volunteers and sickle
cell disease patients. The use of Compound 1 is disclosed herein
for treatment of sickle cell disease in humans.
[0084] Compound 1 is an oral small-molecule agonist of pyruvate
kinase red blood cell isozyme (PKR) being developed for the
treatment of hemolytic anemias. This human clinical trial study
will characterize the safety, tolerability and the
pharmacokinetics/pharmacodynamics (PK/PD) of a single ascending
dose and multiple ascending doses of Compound 1 in the context of
phase 1 studies in healthy volunteers and sickle cell disease
patients. The effects of food on the absorption of Compound 1 will
also be evaluated, in healthy volunteers.
[0085] The objectives of the study include the following: [0086] 1.
To evaluate the safety and tolerability of a single ascending dose
and multiple ascending doses of Compound 1 in healthy volunteers
and sickle cell disease (SCD) patients. [0087] 2. To characterize
the pharmacokinetics (PK) of Compound 1. [0088] 3. To evaluate the
levels of 2,3-diphosphoglycerate (DPG) and adenosine triphosphate
(ATP) in the red blood cells (RBCs) of healthy volunteers and SCD
patients after single and multiple doses of Compound 1. [0089] 4.
To evaluate the relationship between Compound 1 plasma
concentration and potential effects on the QT interval in healthy
volunteers. [0090] 5. To evaluate the effect of single ascending
doses of Compound 1 on other electrocardiogram (ECG) parameters
(heart rate, PR and QRS interval and T-wave morphology) in healthy
volunteers. [0091] 6. To explore food effects on the PK of Compound
1 in healthy volunteers. [0092] 7. To explore the association of
Compound 1 exposure and response variables (such as safety,
pharmacodynamics (PD), hematologic parameters as appropriate).
[0093] 8. To explore effects of Compound 1 after single and
multiple doses on RBC function. [0094] 9. To explore effects of
Compound 1 after multiple doses in SCD patients on RBC metabolism,
inflammation and coagulation.
[0095] This is a first-in-human (FIH), Phase 1 study of Compound 1
that will characterize the safety, PK, and PD of Compound 1 after a
single dose and after repeated dosing first in healthy adult
volunteers and then in adolescents or adults with sickle cell
disease. The study arms and assigned interventions to be employed
in the study are summarized in Table 8. Initially, a dose range of
Compound 1 in single ascending dose (SAD) escalation cohorts will
be explored in healthy subjects. Enrollment of healthy subjects
into 2-week multiple ascending dose (MAD) escalation cohorts will
be initiated once the safety and PK from at least two SAD cohorts
is available to inform the doses for the 2-week MAD portion of the
study. The MAD cohorts will then run in parallel to the single dose
cohorts. A single dose cohort is planned to understand food effects
(FE) on the PK of Compound 1. After the SAD and FE studies in
healthy subjects are completed, the safety, PK and PD of a single
dose of Compound 1 that was found to be safe in healthy subjects
will then be evaluated in sickle cell disease (SCD) subjects.
Multiple dose studies in SCD subjects will then be initiated upon
completion of MAD studies in healthy volunteers. Compound 1 will be
administered in 25 mg and 100 mg tablets delivered orally.
TABLE-US-00008 TABLE 8 Arms Assigned Interventions Experimental:
Single ascending dose cohorts Drug: Compound 1/Placebo in healthy
subjects Healthy volunteer subjects will receive Healthy volunteer
subject cohorts Compound 1/placebo and be monitored randomized 6:2
receiving a single dose of for side effects while undergoing
Compound 1 or placebo. The first cohort pharmacokinetics and
pharmacodynamic will receive 200 mg of Compound 1 or studies
placebo. Dose escalation will occur if Compound 1 or placebo is
tolerated. The maximum dose of Compound 1 or placebo will be 1500
mg. Planned doses for the SAD cohorts are listed in Table 9.
Experimental: Multiple ascending dose Drug: Compound 1/Placebo
cohorts in healthy subjects Healthy volunteer subjects will receive
Healthy volunteer subject cohorts Compound 1/placebo and be
monitored randomized 9:3 to receive Compound 1 or for side effects
while undergoing placebo for 14 days continuous dosing.
pharmacokinetics and pharmacodynamic The first cohort will receive
100 mg of studies Compound 1 or placebo daily X 14 days.
Alternatively, the first cohort will receive 200 mg (e.g., 100 mg
BID or 200 mg QD) of Compound 1 or placebo daily X 14 days. The
maximum dose of Compound 1/placebo will be 600 mg Compound
1/placebo daily for 14 days. Planned doses for the MAD cohorts are
listed in Table 10. Experimental: Food Effect Cohort in healthy
Drug: Compound 1 subjects Healthy subjects will receive Compound
Healthy Volunteer subject cohort of 10 1 with or without food and
undergo subjects who will receive a single dose of pharmacokinetic
studies Compound 1 with food and without food. Dose will be
administered per the protocol defined dose. Healthy Volunteer
subject cohort of 10 subjects who will receive a single dose of
Compound 1 with food and without food. Dose will be 500 mg of
Compound 1, but is subject to change based on the pharmacokinetic
profile of Compound 1 observed in the initial SAD cohorts and the
safety profile of Compound 1 observed in prior SAD and MAD cohorts.
Experimental: Single ascending dose cohorts Drug: Compound
1/Placebo in SCD subjects SCD subjects will receive Compound Sickle
cell disease subject cohort 1/placebo and be monitored for side
randomized 6:2 receiving a single dose of effects while undergoing
Compound 1 or placebo. The dose of pharmacokinetic and
pharmacodynamics Compound 1/placebo administered will be studies a
dose that was found to be safe in healthy subjects. The dose of
Compound 1/placebo administered also will be a dose that was found
to be pharmacodynamically active (e.g., results in a reduction in
2,3-DPG) in healthy subjects. Experimental: Multiple ascending dose
Drug: Compound 1/Placebo cohorts in SCD subjects SCD subjects will
receive Compound Sickle cell disease subject cohorts 1/placebo and
be monitored for side randomized 9:3 to receive Compound 1 or
effects while undergoing placebo for 14 days continuous dosing.
pharmacokinetic and pharmacodynamics The dose of Compound 1/placebo
studies administered will be a dose less than maximum tolerable
dose evaluated in MAD healthy volunteers. The dose of Compound
1/placebo also will be a dose that was found to be
pharmacodynamically active (e.g., results in a reduction in RBC
2,3-DPG and increase in RBC ATP) in MAD healthy volunteers.
TABLE-US-00009 TABLE 9 Dose Level/Cohort Dose Tablet Strength
(#/day) SAD 1 200 mg 100 mg (2/day) SAD 2 400 mg 100 mg (4/day) SAD
3 700 mg 100 mg (7/day) SAD 4 1100 mg 100 mg (11/day) SAD 5 1500 mg
100 mg (15/day)
TABLE-US-00010 TABLE 10 Dose Level/Cohort Dose Tablet Strength
(#/day) MAD 1 100 mg 100 mg (1/day) or 25 mg (4/day) MAD 2 200 mg
100 mg (2/day) MAD 3 400 mg 100 mg (4/day) MAD 4 600 mg 100 mg
(6/day)
[0096] Outcome Measures
[0097] Primary Outcome Measures.
1. Incidence, frequency, and severity of adverse events (AEs) per
CTCAE v5.0 of a single ascending dose and multiple ascending doses
of Compound 1 in adult healthy volunteers and SCD patients.
[0098] [Time Frame: Up to 3 weeks of monitoring]
2. Maximum observed plasma concentration (Cmax)
[0099] [Time Frame: Up to 3 weeks of testing]
3. Time to maximum observed plasma concentration (Tmax)
[0100] [Time Frame: Up to 3 weeks of testing]
4. Area under the plasma concentration-time curve from time zero
until the 24-hour time point (AUC0-24)
[0101] [Time Frame: Up to 3 weeks of testing]
5. Area under the plasma concentration-time curve from time zero
until last quantifiable time point (AUC0-last)
[0102] [Time Frame: Up to 3 weeks of testing]
6. Area under the plasma concentration-time curve from time zero to
infinity (AUC0-inf)
[0103] [Time Frame: Up to 3 weeks of testing]
7. Terminal elimination half-life (t1/2)
[0104] [Time Frame: Up to 3 weeks of testing]
8. Apparent clearance (CL/F)
[0105] [Time Frame: Up to 3 weeks of testing]
9. Apparent volume of distribution (Vd/F)
[0106] [Time Frame: Up to 3 weeks of testing]
10. Terminal disposition rate constant (Lz)
[0107] [Time Frame: Up to 3 weeks of testing]
11. Renal clearance (CIR)
[0108] [Time Frame: Up to 3 weeks of testing]
[0109] Secondary Outcome Measures:
12. Change from baseline in the levels of 2,3-diphosphoglycerate
(DPG) and adenosine triphosphate (ATP) in the red blood cells
(RBCs) of healthy volunteers and SCD patients after single and
multiple doses of Compound 1.
[0110] [Time Frame: Up to 3 weeks of testing]
13. Model-based estimate of change from baseline QT interval
corrected using Fridericia's correction formula (QTcF) and 90%
confidence interval at the estimated Cmax after a single dose of
Compound 1 in healthy volunteers.
[0111] [Time Frame: up to 7 days]
14. Change from baseline heart rate after a single dose of Compound
1 in healthy volunteers
[0112] [Time Frame: up to 7 days]
15. Change from baseline PR after a single dose of Compound 1 in
healthy volunteers
[0113] [Time Frame: up to 7 days]
16. Change from baseline QRS after a single dose of Compound 1 in
healthy volunteers
[0114] [Time Frame: up to 7 days]
17. Change from baseline T-wave morphology after a single dose of
Compound 1 in healthy volunteers
[0115] [Time Frame: up to 7 days]
[0116] Exploratory Outcome Measures:
18. Effect of food on C.sub.max, AUC.sub.0-24/AUC.sub.last 19.
Effect of AUC.sub.last/AUC.sub.0-24, C.sub.max, minimum plasma
concentration (C.sub.min), peak-to trough ratio, dose linearity,
accumulation ratio on safety, PD, and hematologic parameters of
interest, as assessed by exposure-response analyses 20. Effect of
chronic Compound 1 dosing on SCD RBC response to oxidative stress
in SCD Patients (including evaluation of glutathione, glutathione
peroxidase and superoxide dismutase levels) 21. Effect of chronic
Compound 1 dosing on measurable markers of inflammation in SCD
Patients (C-reactive protein, ferritin, interleukin [IL]-113, IL-6,
IL-8, and tumor necrosis factor-.alpha.) 22. Effects of chronic
Compound 1 dosing on measurable markers of hypercoagulation in SCD
patients (D-dimer, prothrombin 1.2, and thrombin-antithrombin [TAT]
complexes)
[0117] Eligibility
[0118] Minimum age: 18 Years (healthy volunteers); 12 Years (SCD
subjects)
[0119] Maximum age: 60 Years
[0120] Sex: All
[0121] Gender Based: No
[0122] Accepts Healthy Volunteers: Yes
[0123] Inclusion Criteria: [0124] Healthy volunteer: subjects must
be between 18 and 60 years of age; SCD: subjects must be between 12
and 50 years of age [0125] Subjects must have the ability to
understand and sign written informed consent, which must be
obtained prior to any study-related procedures being completed.
[0126] Subjects must be in general good health, based upon the
results of medical history, a physical examination, vital signs,
laboratory profile, and a 12-lead ECG. [0127] Subjects must have a
body mass index (BMI) within the range of 18 kg/m2 to 33 kg/m.sup.2
(inclusive) and a minimum body weight of 50 kg (healthy volunteer
subjects) or 40 kg (SCD subjects) [0128] For SCD subjects, sickle
cell disease previously confirmed by hemoglobin electrophoresis or
genotyping indicating one of the following hemoglobin genotypes:
Hgb SS, Hgb S.beta..sup.+-thalassemia, Hgb
S.beta..sup.0-thalassemia, or Hgb SC [0129] All males and females
of child bearing potential must agree to use medically accepted
contraceptive regimen during study participation and up to 90 days
after. [0130] Subjects must be willing to abide by all study
requirements and restrictions.
[0131] Exclusion Criteria. [0132] Evidence of clinically
significant medical condition or other condition that might
significantly interfere with the absorption, distribution,
metabolism, or excretion of study drug, or place the subject at an
unacceptable risk as a participant in this study [0133] History of
clinically significant cardiac diseases including condition
disturbances [0134] Abnormal hematologic, renal and liver function
studies [0135] History of drug or alcohol abuse
[0136] Results (Healthy Volunteers)
[0137] Four healthy SAD cohorts were evaluated at doses of 200,
400, 700, and 1000 mg, and four healthy MAD cohorts received 200 to
600 mg total daily doses for 14 days at QD or BID dosing (100 mg
BID, 200 mg BID, 300 mg BID, and 400 mg QD). In the food effect
(FE) cohort, 10 healthy subjects received 200 mg of Compound 1 QD
with and without food.
[0138] No serious adverse events (SAEs) or AEs leading to
withdrawal were reported in the SAD and MAD cohorts of healthy
volunteers. In PK assessments, Compound 1 was rapidly absorbed with
a median T.sub.max of 1 hr postdose. Single dose exposure increased
in greater than dose-proportional manner at doses .gtoreq.700 mg.
In multiple-doses delivered BID or QD, linear PK was observed
across all dose levels (100-300 mg BID, 400 mg QD), and exposure
remained steady up to day 14, without cumulative effect. Compound 1
exposure under fed/fasted conditions was similar.
[0139] PD activity was demonstrated at all dose levels evaluated in
Compound 1-treated subjects (Table 11). Table 11 reports the mean
maximum percentage change in 2,3-DPG and ATP across all doses and
timepoints in the SAD and MAD cohorts. As shown in Table 11, a mean
decrease in 2,3-DPG, and a mean increase in ATP, relative to
baseline, was observed in both the SAD and MAD cohorts. Within 24
hr of a single dose of Compound 1, a decrease in 2,3-DPG was
observed. After 14 days of Compound 1 dosing these PD effects were
maintained along with an increase in ATP over baseline.
Accordingly, the mean maximum reduction in the concentration of
2,3-DPG was at least about 40% in patients receiving Compound 1 in
the SAD study and at least about 50% in patients receiving Compound
1 in the MAD study.
TABLE-US-00011 TABLE 11 Summary of Mean Maximum Percent Change in
Key PD Measures from Baseline SAD MAD Placebo Compound 1 Placebo
Compound 1 PD Marker Statistics (N = 8) (N = 24) (N = 12 (N = 36)
2,3-DPG Mean -19.5 -46.8 -17.0 -56.3 (95% CI) (-25.0, -14.0)
(-50.3, -43.2) (-22.9, -11.1) (-58.9, -53.7) P-value <0.0001
<0.0001 ATP Mean 9.2 24.4 7.2 68.5 (95% CI) (0.5, 18.0) (18.4,
30.3) (-0.3, 14.7) (63.6, 73.3) P-value 0.0094 <0.0001
[0140] In the SAD cohorts, the subjects' blood 2,3-DPG levels were
measured periodically after dosing by a qualified LC-MS/MS method
for the quantitation of 2,3-DPG in blood. Decreased 2,3-DPG blood
levels were observed 6 hours following a single dose of Compound 1
at all dose levels (earlier timepoints were not collected). Maximum
decreases in 2,3-DPG levels generally occurred .about.24 hours
after the first dose with the reduction sustained .about.48-72 hr
postdose. Table 12 reports the median percentage change in 2,3-DPG
blood levels, relative to baseline, measured over time in healthy
volunteers after a single dose of Compound 1 (200 mg, 400 mg, 700
mg, or 1000 mg) or placebo. Accordingly, the median reduction in
the concentration of 2,3-DPG, relative to baseline, was at least
about 30% at all dose levels tested 24 hours after administration
of the single dose.
TABLE-US-00012 TABLE 12 Median Percentage Change in 2,3-DPG Levels
Time After Dose Dose Placebo 200 mg 400 mg 700 mg 1000 mg 0 0.0 0.0
0.0 0.0 0.0 6 -7.8 -18 -23 -29 -20 8 -7.6 -17 -29 -28 -31 12 -4.0
-25 -40 -41 -44 16 -6.0 -33 -35 -46 -50 24 -2.0 -31 -39 -49 -48 36
-6.9 -33 -38 -46 -47 48 -15 -29 -31 -48 -47 72 -6.9 -18 -30 -33
-21
[0141] FIG. 11 is a graph of the blood 2,3-DPG levels measured over
time in healthy volunteers who received a single dose of Compound 1
(200 mg, 400 mg, 700 mg, or 1000 mg) or placebo. As shown in FIG.
11, healthy volunteers who received Compound 1 experienced a
decrease in blood 2,3-DPG levels, relative subjects who received
the placebo. FIG. 12 is a graph of the blood 2,3-DPG levels
measured 24 hours post-dose in healthy volunteers who received a
single dose of Compound 1 (200 mg, 400 mg, 700 mg, or 1000 mg) or
placebo. As shown in FIG. 12, healthy volunteers who received
Compound 1 experienced a decrease in blood 2,3-DPG levels at 24
hours post-dose, relative to subjects who received the placebo.
[0142] In the MAD cohorts, the subjects' blood 2,3-DPG levels were
measured periodically after dosing by a qualified LC-MS/MS method
for the quantitation of 2,3-DPG in blood. The maximum decrease in
2,3-DPG on Day 14 was 55% from baseline (median). 2,3-DPG levels
reached a nadir and plateaued on Day 1 and had not returned to
baseline levels 72 hours after the final dose on Day 14. Table 13
reports the median percentage change in 2,3-DPG blood levels,
relative to baseline, measured over time after the first dose on
days 1 and 14 in healthy volunteers who received daily doses of
Compound 1 (100 mg BID, 200 mg BID, or 300 mg BID) or placebo for
14 days. Accordingly, the median reduction in the concentration of
2,3-DPG, relative to baseline, was at least about 25% at all dose
levels tested 24 hours after administration of the first dose on
day 1 and at least about 40% at all dose levels tested 24 hours
after administration of the first dose on day 14.
TABLE-US-00013 TABLE 13 Median Percentage Change in 2,3-DPG Levels
(Days 1 and 14) Time After Dose First 100 mg BID 200 mg BID 300 mg
BID Placebo Daily Day Day Day Day Dose 1 14 1 14 1 14 1 14 0 0.0
-42.0 0.0 -48.2 0.0 -59.4 0.0 -7.6 6 -16.1 -44.3 -13.1 -48.5 -18.8
-53.0 -2.9 -10.9 8 -12.1 -44.7 -22.3 -44.3 -23.8 -54.2 -0.6 -1.6 12
-18.1 -43.6 -23.1 -42.2 -31.6 -55.3 -7.1 -1.6 16 -18.4 -43.9 -33.9
-42.9 -40.7 -52.4 -6.7 -5.3 24 -27.8 -44.1 -43.5 -44.3 -50.8 -52.1
1.1 -10.7 48 -34.7 -38.7 -44.5 -1.0 72 -20.2 -20.2 -32.9 -7.0
[0143] FIG. 13 is a graph of the blood 2,3-DPG levels measured over
time in healthy volunteers who received daily doses of Compound 1
(100 mg BID, 200 mg BID, 300 mg BID, or 400 mg QD) or placebo for
14 days. As shown in FIG. 13, healthy volunteers who received
Compound 1 experienced a decrease in blood 2,3-DPG levels, relative
subjects who received the placebo. FIG. 14 is a graph of the blood
2,3-DPG levels measured on day 14 in healthy volunteers who
received daily doses of Compound 1 (100 mg BID, 200 mg BID, 300 mg
BID, or 400 mg QD) or placebo for 14 days. As shown in FIG. 14,
healthy volunteers who received Compound 1 experienced a decrease
in blood 2,3-DPG levels, relative to subjects who received the
placebo.
[0144] In the MAD cohorts, the subjects' blood ATP levels were
measured on day 14 by a qualified LC-MS/MS method for the
quantitation of ATP in blood. ATP levels were elevated, relative to
baseline, on day 14, and remained elevated 60 hours after the last
dose. Table 14 reports the median percentage change in blood ATP
levels, relative to baseline, measured over time after the first
dose on day 14 in healthy volunteers who received daily doses of
Compound 1 (100 mg BID, or 200 mg BID) or placebo for 14 days.
TABLE-US-00014 TABLE 14 Median Percentage Change in ATP Levels (Day
14) Time After First Dose Daily Dose 100 mg BID 200 mg BID Placebo
0 41.5 55.3 -0.5 6 43.8 48.1 2.8 8 47.8 58.4 -4.1 12 45.4 56.2 2.3
16 44.8 57.0 -6.8 24 55.0 64.0 2.9 48 52.2 58.9 4.7 72 49.2 54.0
2.2
[0145] FIG. 15 is a graph of the blood ATP levels measured on day
14 in healthy volunteers who received daily doses of Compound 1
(100 mg BID, 200 mg BID, 300 mg BID, or 400 mg QD) or placebo for
14 days. As shown in FIG. 15, healthy volunteers who received
Compound 1 experienced an increase in blood ATP levels, relative to
subjects who received the placebo.
[0146] FIG. 16 is a graph plotting the blood concentration of
Compound 1 (ng/mL) measured in healthy volunteer (HV) patients on a
first (left) axis and the concentration of 2,3-DPG (micrograms/mL)
measured in these HV patients on a second (right) axis after
administration of a single dose of Compound 1 (400 mg). Solid
symbols represent geometric means and Standard errors of the
observed Compound 1 plasma and 2,3 DPG concentrations. As shown in
the figure, the observed 2,3 DPG modulation does not track directly
plasma pharmacokinetics (blood concentration of Compound 1) where
the pharmacodynamic maximum (i.e., the minimum of the 2,3-DPG
concentration, at time .about.24 h) occurred nearly 24 h after the
pharmacokinetic maximum (i.e., maximum of the PK curve, at time
.about.1-2 h). The observed pharmacodynamic response in HVs was
durable, with a calculated PD half-life of .about.20 h, where
2,3-DPG depression was observed long after plasma levels were
undetectable. Taken together, this suggests that identifying the
pharmacologically active dose cannot be adequately performed using
pharmacokinetic parameters (C.sub.max/C.sub.min/AUC) in isolation,
but rather support an approach that includes integrating the
temporal pharmacokinetic/pharmacodynamic relationship to provide
the platform of evidence that QD dosing may be feasible in sickle
cell disease patients.
Example 7: Analysis of ATP and 2,3 DPG in K2EDTA Whole Blood by
LC-MS/MS
[0147] The following procedures are employed for the analysis of
ATP and 2,3-DPG in human whole blood K2EDTA using a protein
precipitation extraction procedure and analysis by LC-MS/MS.
[0148] This bioanalytical method applies to the parameters
described below:
TABLE-US-00015 Assay Range 25,000-1,500,000 ng/mL Extraction Volume
15.0 uL Species/Matrix/ Water as a surrogate for Human
Anticoagulant Whole Blood K2EDTA Extraction type Protein
Precipitation Sample Storage 80.degree. C. Mass Spectrometer
API-5500 Acquisition software Analyst/ Aria System
[0149] The following precautions are followed:
[0150] 1. Standard and QC samples are prepared on ice and stored in
plastic containers.
[0151] 2. Study samples and QC samples are thawed on ice.
[0152] 3. Extraction is performed on ice.
[0153] The following definitions and abbreviations are
employed:
TABLE-US-00016 CRB Carryover remediation blanks FT Freeze-thaw MPA
Mobile phase A MPB Mobile phase B NA Not applicable NR Needle rinse
RT Retention time SIP Stability in progress TBD To be
determined
[0154] The following chemicals, matrix, and reagents are used:
TABLE-US-00017 K2EDTA Human Whole Blood, BioreclamationIVT or
equivalent (Note: BioReclamationIVT and BioIVT are considered
equivalent) Acetonitrile (ACN), HPLC Grade or better Ammonium
Acetate (NH.sub.4OAc), HPLC grade or equivalent Ammonium Hydroxide
(NH.sub.4OH, 28-30%), ACS grade or better Dimethylsulfoxide (DMSO),
ACS grade or better Formic Acid (FA), 88% ACS grade Isopropanol
(IPA), HPLC Grade or better Methanol (MeOH), HPLC Grade or better
Water (H.sub.2O), Milli-Q or HPLC Grade ATP - Analyte, Sponsor or
supplier ATP-IS- IS, Sponsor or supplier 2,3-DPG - Analyte, Sponsor
or supplier 2,3-DPG-IS- IS, Sponsor or supplier
[0155] The following procedures are used for reagent preparation.
Any applicable weights and volumes listed are nominal and may be
proportionally adjusted as long as the targeted composition is
achieved:
TABLE-US-00018 Nominal Volumes Final Solution for Solution Storage
Solution Composition Preparation Conditions Mobile Phase A 10 mM
Weigh Ambient (MPA) Ammoniumn approximately Temperature Acetate in
770.8 mg of water pH 8.5 Ammonium Acetate; add to a bottle with
1000 mL of water. Adjust pH to 8.3-8.7 using Ammonium Hydroxide.
Mobile Phase B 5:95 MPA:ACN Add 50.0 mL of Ambient (MPB) MPA to 950
mL of Temperature CAN. Mix. Needle Rinse 1 25:25:25:25:0.1 Add 500
mL of (NR1) (v:v:v:v:v) MeOH, 500 mL of Ambient MeOH:ACN: ACN, 500
mL of H2O:IPA:NH.sub.4OH H.sub.2O, 500 mL of Temperature IPA, and 2
mL of NH.sub.4OH. Mix. Needle Rings 2 90:10:0.1 (v:v:v) Add 2 mL of
FA to Ambient (NR2) H.sub.20:MeOH:FA 200 mL of MeOH Temperature and
1800 mL of H.sub.20. Mix.
[0156] Calibration standards are prepared using water as the matrix
according to the table presented below. The indicated standard is
prepared by diluting the indicated spiking volume of stock solution
with the indicated matrix volume.
TABLE-US-00019 Stock Spiking Matrix Final Final Calibration Stock
Conc. Vol. Vol. Vol. Conc. Standard Solution (ng/mL) (mL) (mL) (mL)
(ng/mL) STD-6 ATP Stock 60,000,000 0.0100 0.380 0.400 1,500,000
2,3-DPG Stock 60,000,000 0.0100 STD-5 STD-6 1,500,000 0.100 0.200
0.300 500,000 STD-4 STD-6 1,500,000 0.0500 0.325 0.375 200,000
STD-3 STD-6 1,500,000 0.0250 0.350 0.375 100,000 STD-2 STD-5
500,000 0.0500 0.450 0.500 50,000 STD-1 STD-5 500,000 0.0250 0.475
0.500 25,000 Cond. STD-5 500,000 0.0250 0.975 1.00 12,500
[0157] Quality control standards are prepared using water as the
matrix according to the table presented below. The indicated
quality control standard is prepared by diluting the indicated
spiking volume of stock solution with the indicated matrix
volume.
TABLE-US-00020 Quality Stock Spiking Matrix Final Final Control
Stock Conc. Vol. Vol. Vol. Conc. Standard Solution (ng/mL) (mL)
(mL) (mL) (ng/mL) QC-High ATP Stock 60,000,000 0.160 7.68 8.00
1,200,000 2,3-DPG 60,000,000 0.160 Stock QC-Mid QC-High 1,200,000
1.50 4.50 6.00 300,000 QC-Low QC-Mid 300,000 1.50 4.50 6.00
75,000
[0158] An internal standard spiking solution is prepared with a
final concentration of 12,500 ng/mL ATP and 2,3-DPG by diluting
stock solutions of ATP and 2,3-DPG at concentrations of 1,000,000
ng/mL with water. 0.200 mL each of the ATP and 2,3-DPG stock
solutions are diluted with 15.6 mL of water to produce a final
volume of 16.0 mL at a final concentration of 12,500 ng/mL of ATP
and 2,3-DPG.
[0159] The following procedures are used for sample extraction
prior to analysis via LC-MS/MS. 15.0 .mu.L of the calibration
standards, quality controls, matrix blanks, and samples are
aliquoted into a 96-well plate. 50.0 .mu.L of the internal standard
spiking solution is added to all samples on the plate, with the
exception of the matrix blank samples; 50.0 .mu.L of water is added
to the matrix blank samples. Subsequently, 150 .mu.L of water is
added to all samples on the plate. The plate is then covered and
agitated by vortex at high speed for ten minutes, after which 750
.mu.L of methanol is added to all samples on the plate. The plate
is covered and agitated by vortex for approximately 1 minute. The
plate is then centrifuged at approximately 3500 RPM at
approximately 4.degree. C. for five minutes. After centrifugation,
a liquid handler is used to transfer 50 L of each sample to a new
96-well plate, and 200 .mu.L of acetonitrile is added to all
samples on the plate. The newly prepared plate is covered and
agitated by vortex for approximately 1 minute. The plate is then
centrifuged at approximately 3500 RPM at approximately 4.degree. C.
for 2 minutes.
[0160] The following LC parameters and gradient conditions are used
for analysis of the extracted samples:
TABLE-US-00021 LC Parameters Analytical Column Vendor: SeQuant
Description: ZIC-pHILIC Dimensions: 50 mm .times. 2.1 mm Column
Heater Temperature: 40.degree. C. Plate Rack Position: Cold Stack
Cold Stack Set Point: 5.degree. C. Mobile Phase Mobile Phase A 10
mM Ammoniumn (MPA) Acetate in water pH 8.5 Mobile Phase B 5:95
MPA:ACN (MPB) Injection Volume 5 .mu.L
TABLE-US-00022 LC Gradient Time Flow Gradient Step (s) (mL/min)
Setting % MPB 1 50 0.400 Step 5 2 30 0.400 Ramp 95 3 70 0.400 Step
5
Data is collected starting at 0.08 min and is collected over a data
window length of 0.70 min.
[0161] The following MS parameters are used for analysis of the
extracted samples using an API-5500 Mass Spectrometer:
TABLE-US-00023 Interface: Turbo Ion Spray Ionization, positive-ion
mode Scan Mode: Multiple Reaction Monitoring (MRM) Scan Parameters:
Parent/Product: Dwell Time (ms): 506.0/159.0 50 521.0/159.0 25
265.0/166.8 50 268.0/169.8 25 Source Temperature: 400.degree.
C.
[0162] The collected MS data is analyzed and sample concentrations
are quantified using peak area ratios with a linear 1/x.sup.2
regression type.
* * * * *