Discovery of GBT440, an Orally Bioavailable R-State Stabilizer of Sickle Cell Hemoglobin

Article abstract of DOI:10.1021/acsmedchemlett.6b00491

We report the discovery of a new potent allosteric effector of sickle cell hemoglobin, GBT440 (36), that increases the affinity of hemoglobin for oxygen and consequently inhibits its polymerization when subjected to hypoxic conditions. Unlike earlier allosteric activators that bind covalently to hemoglobin in a 2:1 stoichiometry, 36 binds with a 1:1 stoichiometry. Compound 36 is orally bioavailable and partitions highly and favorably into the red blood cell with a RBC/plasma ratio of ～150. This partitioning onto the target protein is anticipated to allow therapeutic concentrations to be achieved in the red blood cell at low plasma concentrations. GBT440 (36) is in Phase 3 clinical trials for the treatment of sickle cell disease (NCT03036813).

Full text of DOI:10.1021/acsmedchemlett.6b00491

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Letter
pubs.acs.org/acsmedchemlett
Discovery of GBT440, an Orally Bioavailable R‑State Stabilizer of
Sickle Cell Hemoglobin
Brian Metcalf,† Chihyuan Chuang,‡ Kobina Dufu,† Mira P. Patel,† Abel Silva-Garcia,† Carl Johnson,†
Qing Lu,‡ James R. Partridge,† Larysa Patskovska,∥ Yury Patskovsky,∥ Steven C. Almo,∥
Matthew P. Jacobson,¶ Lan Hua,¶ Qing Xu,† Stephen L. Gwaltney, II,† Calvin Yee,† Jason Harris,†
Bradley P. Morgan,‡ Joyce James,‡ Donghong Xu,‡ Athiwat Hutchaleelaha,† Kumar Paulvannan,§
Donna Oksenberg,† and Zhe Li*,†
†Global Blood Therapeutics, Inc., South San Francisco, California 94080, United States
‡Cytokinetics, Inc., South San Francisco, California 94080, United States
∥Albert Einstein College of Medicine, Bronx, New York 10461, United States
¶Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United States
§Tandem Sciences, Inc., Menlo Park, California 94025, United States
S
* Supporting Information
ABSTRACT: We report the discovery of a new potent
allosteric effector of sickle cell hemoglobin, GBT440 (36), that
increases the affinity of hemoglobin for oxygen and
consequently inhibits its polymerization when subjected to
hypoxic conditions. Unlike earlier allosteric activators that bind
covalently to hemoglobin in a 2:1 stoichiometry, 36 binds with
a 1:1 stoichiometry. Compound 36 is orally bioavailable and
partitions highly and favorably into the red blood cell with a
RBC/plasma ratio of ∼150. This partitioning onto the target protein is anticipated to allow therapeutic concentrations to be
achieved in the red blood cell at low plasma concentrations. GBT440 (36) is in Phase 3 clinical trials for the treatment of sickle
cell disease (NCT03036813).
KEYWORDS: Sickle cell disease, sickle cell hemoglobin, allosteric modulator, aldehyde, Schiff-base formation, oxygen affinity,
red blood cell partitioning
polymerization by inducing an allosteric conformational change
ickle cell disease (SCD) is caused by a single mutation in the
Sβ chain of hemoglobin (Hb) where a hydrophilic βGlu6 has that increases the oxygen affinity of the HbS. It was suggested
that 1 preferentially stabilizes the R2 state,3 which has higher
been exchanged for a hydrophobic βVal6. Under low oxygen
affinity for oxygen,4 thus preventing polymerization under
conditions the mutant hemoglobin (HbS) polymerizes via the
hypoxic conditions. More potent synthetic aldehyde analogues
of 5HMF such as BW12C795,6 and Tucaresol7 (Figure 1a) were
developed earlier and shown clinically to reduce hemolysis when
20−30% of the HbS is modified. Neither BW12C79 (2) nor
Tucaresol (3) was developed further although 5HMF (1) is
reported to be in a clinical trial.8 Tucaresol was terminated
apparently due to immunotoxicity issues. That a positive
pharmacodynamic effect is observed at 20−30% modification
of HbS is a key point as it implies a target modification to be
attained for a desirable result. This proposed modification target
results from studies in individuals with compound heterozygosity
for HbS and pancellular hereditary persistence of fetal
hemoglobin (S/HPFH) infers that the presence of 10−30%
HbF provides clinical benefits with patients having few, if any,
mutated βVal6 from one Hb tetramer and a hydrophobic cavity
formed by β1Ala70, β1Phe85, and β1Leu88 from a laterally
located Hb tetramer. These polymers result in the red blood cell
(RBC) losing its deformability properties and taking on a sickle-
like shape. These sickle cells are unable to pass through narrow
capillaries resulting in painful vaso-occlusive crises.1 The sickle
cells undergo hemolysis leading to anemia and a shortened
lifespan. The only approved therapy is a cytotoxic drug,
hydroxyurea,2 that works via an unknown mechanism but
involves inducing the expression of fetal hemoglobin, which is
protective against sickling. There is variable patient response and
compliance to hydroxyurea.2
An approach to therapy would be to maintain the HbS in the
oxygenated state, as polymerization occurs only in the
deoxygenated state under hypoxic conditions. The natural
product, 5-hydroxymethylfurfural (5HMF, 1, Figure 1a), was
shown3 to bind to HbS via a reversible Schiff-base linkage to the-
N-terminal valine of the α chain, and thereby prevent
Received: December 2, 2016
Accepted: January 23, 2017
Published: January 23, 2017
© XXXX American Chemical Society
A
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high concentration of Hb in the RBC would act as a sink for the
aldehyde thus protecting it from being rapidly metabolized.
In our initial assays, allosteric modulation of purified HbS was
determined using a Hemox Analyzer (TCS) to generate Oxygen
Equilibrium Curves (OEC) and measuring the change in p50.
The assay therefore is a measure of the test compound’s ability to
maintain the oxygenated or R state of Hb. Data in Table 1 are
Table 1. B-Ring SARs on Oxygen Affinity with Fused Bicyclics
a
Data expressed as Δp50%: the % change from baseline of pO2 at
which HbS (at 25 μM) is 50% saturated with O2 in the presence of test
compound at 30 μM. Please see Supporting Information for standard
deviation and number of tests.
Figure 1. (a) Literature Hb binding aldehydes. (b) Structures of
compounds 4, 5, and 6.
reported as the % change in p50 from baseline (Δp50) when
compounds were tested at 30 μM against purified Hb (a mixture
of HbA and HbS) at 25 μM. Here, compound 4 had a Δp50 (%)
of 34% at 30 μM, while 5 with a bicyclic A-ring was similarly
active with a Δp50 (%) = 33%. However, in a time course study
where the onset of oxygenation was measured as a surrogate for
on-rate, 5 impacted the oxygenation with a much faster rate than
4, which in turn was faster than 5HMF (Figure 2). Thus, the
maximum effect of 5 was seen several minutes after the first
measurement was taken, while 1 and 4 took almost 1 h to do so.
sickle cell-related events.9 As the concentration of Hb in the RBC
is estimated at 5 mM, a positive allosteric modulator that binds in
a reversible covalent manner would need to reach a minimum
concentration of 1 mM to allow 20% occupancy in the RBC in
order to be effective. This requirement poses a significant
challenge with respect to preventing off-target effects while
attaining such high concentrations in the target RBC.
The X-ray structure of 5HMF in coordination with HbS is
available.3 Furthermore, compound 4 (Figure 1b) and other
synthetic analogues have been shown to have potency in
hemoximetry assays.10 The increase in oxygen affinity of Hb is
illustrated by the ability of the test compound to facilitate 50%
oxygen saturation at lower oxygen pressure than the normal
control, hence the term, left shifter. The X-ray structure of 4 with
Hb has also been described.11 Analogously to 5HMF (1),
compound 4 was proposed to bind in Schiff-base linkage to the
N-terminal valines of each α chain of HbS with the aromatic rings
of 4 protruding into the intradomain cavity and appearing to
interact with one another. We proposed by molecular modeling
that 5 (Figure 1b) and other fused bicyclic analogues would fill
this cavity more efficiently than 4 by providing stronger
interactions between the aromatic rings themselves, perhaps
via increasing dipole interactions.
From the outset we were aware of the propensity for aldehydes
to be metabolized rapidly. While we were also concerned with
the possibility that Schiff-base linkages could be formed with
exposed amines on any protein, the N-terminal valine on the α
chain in Hb, having a pKa = 6.912 is primarily unprotonated at
physiological pH and hence is more nucleophilic than
protonated amines, such as the ω amino group of exposed
lysine which has a pKa = 10.5 in polypeptides.13 This differential
pKa of the N-terminal valine offers an in-built possibility for
selectivity. We also hoped that the rapid reversibility of these
Schiff-base linkages would result in the aldehyde moving from
protein to protein until it arrived at the most stable situation, that
being reversibly bound to Hb. This would require that we build in
high affinity for Hb, compared to other proteins. In addition, the
Figure 2. Time-dependent change in hemoglobin-oxygen affinity for 1,
4, and 5. Measurement was taken after a 20 min oxygenation/
deoxygenation cycle in the Hemox Analyzer.
Consequently, as we anticipated that a fast on-rate would be an
advantage, we elected to study further analogues bearing a
bicyclic aromatic B-ring. In addition, with a concern that the para
relationship of phenyl ethers, as in 4 and 5, may be a metabolic
liability we prepared the methoxypyridine 6 (Figure 1b) that, to
our surprise, elicited a Δp50 = 44%. This methoxypyridine A-ring
was maintained through much of our early SAR program, while
we concentrated on optimizing the aromatic B-ring that resides
near the opening of the cavity. The X-ray crystal structure of 6
with HbS (Figure 3, PDB code 5UFJ) confirmed our molecular
B
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Table 3. B-Ring SARs on Oxygen Affinity with Purified HbS
and Whole Blood and on the Change in Delay Time to the
Onset of Polymerization
a
Data expressed as Δp50%: the % change from baseline of pO2 at
which HbS (at 25 μM) is 50% saturated with O2 in the presence of test
compound at 30 μM. Data expressed as Δp50%: the % change from
baseline of pO2 at which whole blood (at 20% hematocrit, 1 mM) is
b
Figure 3. Cocrystal structure of 6 (in stick) with CO-ligand HbS.
50% saturated with O2 in the presence of test compound at 1 mM.
c
Data expressed as ΔT%: the % change from baseline of delay time of
modeling hypothesis that 6 appears to form a Schiff-base linkage
with the N-terminal valine of each α subunit, and the aromatic
rings appear to interact with each other via π stacking. A
representative set of the bicyclic B-ring analogues that we
prepared is shown in Tables 1 and 2.
d
HbS at 50 μM in the presence of test compound at 75 μM. Not
determined; please see Supporting Information for standard deviation
and number of tests.
the Δp50 (%) in both the purified Hb and whole blood OEC.
Compound 31 was outstanding as it maintained strong activity in
the whole blood assay.
Table 2. B-Ring SARs on Oxygen Affinity with Fused Bicyclic
with Substituent
As our hypothesis was that compounds that improved the
affinity of HbS for oxygen should prevent polymerization when
HbS is subjected to hypoxic conditions, we developed a
polymerization assay to confirm activity of the most potent
compounds identified in the whole blood assay.14,15 Here
purified HbS in a high concentration of KH2PO4 as a crowding
agent induces polymerization by a temperature jump from 4 to
37 °C, and the impact of test compound on delay time to onset of
polymerization is measured. Only key compounds from Table 3
were tested. The %Δ in the delay time (measured as the delay
time in the presence of compound relative to the delay time of
control) is presented in Table 3. At this stage, compound 31
remained the compound of most interest, as it was clearly
superior in the critical whole blood OEC and confirmed by
increasing the delay time in the polymerization assay.
Having arrived at an optimal B-ring substitution pattern as in
31, we began a brief survey of alternate A-ring aldehydes while
maintaining the B-ring of 31 constant. Representative structures
are shown in Table 4. We intended to survey the impact of
changing the electronics of the aldehyde and hence the strength
of the Schiff-base linkage (37, 38, 41, 43), and the consequences
of a potential H bond from the phenolic OH to the imine of the
Schiff-base (36 vs 44, 42 vs 43).16 Underlining the importance of
the aldehyde itself, des-formyl compound 46, which lacks the
aldehyde, is inactive.
a
Data expressed as Δp50%: the % change from baseline of pO2 at
which HbS (at 25 μM) is 50% saturated with O2 in the presence of test
compound at 30 μM. Please see Supporting Information for standard
deviation and number of tests.
We hypothesized from our modeling that a heterocyclic ring
attached to the bicyclic B-ring might benefit from further
interactions with Hb protein; however, our initial efforts in this
direction did not reveal compounds of greater potency (Table 2)
despite greater MW and surface area, and we discontinued
studies toward these tricyclic compounds.
At this stage we elected to study the most potent compounds
for their ability to increase Hb-oxygen affinity in whole blood
from transfused SCD subjects, compared to the earlier protocol
that used HbS/HbA protein purified from sickle disease blood.
The impact of plasma protein binding became immediately
apparent with even the most potent compounds described in
Tables 1 and 2 losing a significant amount of their activity in
whole blood, even at mM concentrations. However, biaryl
structures, where one ring would be twisted with respect to the
other, were found to be beneficial as shown by the compounds in
Table 3. In particular, the combination of N at the 3-position and
a pyrazole substituent at 2 with the pyrazole 1N-substituted with
an isopropyl group proved among the most potent, increasing
Several compounds from Table 4 were subjected to PK
studies; however, compound 36 is clearly the superior compound
when considering the hemox results with purified Hb and whole
blood, polymerization assays, and initial PK screening. The
impact of the phenolic hydroxyl in 36 is evidenced by the
comparison in T1/2 in rat for 36 (19.1 h in blood) versus T1/2 for
C
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Table 4. A-Ring SARs on Oxygen Affinity with Purified HbS
and Whole Blood and on the Change in Delay Time to the
Onset of Polymerization, and PK Properties in Rats
Table 5. In Vitro CYP Inhibition with 36
Control Inhibitor (IC50
Cmpd 36 IC50
Enzyme
Substrate
(μM))
(μM)
CYP1A2
CYP2C8
CYP2C9
Phenacetin
Paclitaxel
Furafylline (6.29)
Quercetin (1.20)
58.6
7.9
Tolbutamide
Sulfaphenazole (0.205)
8.5
CYP2C19 S-Mephentoin Tranylcypromine (10.3)
20.0
148
81.9
12.5
CYP2D6
CYP3A4
CYP3A4
Bufuralol
Quinidine (0.035)
Midazolam
Testosterone
Ketoconazole (0.018)
Ketoconazole (0.015)
We followed the SAR with X-ray structures of key compounds.
The fused bicyclic compounds as in Table 1 all presented similar
poses to compound 6 with a 2:1 stoichiometry of allosteric
modulator/HbS. However, the substituted pyridines such as
compound 31 and 36 demonstrated a 1:1 stoichiometry (Figure
4 for 31, PDB code 5U3I; X-ray structure of 36 was published
previously17).
a
Data expressed as Δp50%: the % change from baseline of pO2 at
which HbS (at 25 μM) is 50% saturated with O2 in the presence of test
compound at 30 μM. Data expressed as Δp50%: the % change from
baseline of pO2 at which whole blood (at 20% hematocrit, 1 mM) is
b
50% saturated with O2 in the presence of test compound at 1 mM.
c
Data expressed as ΔT%: the % change from baseline of delay time of
d
HbS at 50 μM in the presence of test compound at 75 μM. Not
e
f
determined. Tested at 500 μM. AUC(0-∞) value normalized by PO
dose [(ng·h/mL)/(mg/kg)]. Please see Supporting Information for
standard deviation and number of tests.
the des-hydroxy compound 44 (2.1 h). This difference may result
from a stronger intramolecular H bond in the resultant Schiff-
base of 36 with Hb compared to that from 44. These results
prompted us to study 36 further in pharmacokinetic assays, to
assess its oral bioavailability.
The pharmacokinetics of 36 was assessed in rats, dogs, and
monkeys following i.v. and p.o. administration and has been
noted in our earlier publication.17 Compound 36 has favorable
oral bioavailability of 60, 37, and 36% in rats, dogs, and monkeys,
respectively, with similar blood and plasma half-lives of
approximately 20 h each. Of particular note is RBC/plasma
ratio of ∼150 compared to ∼38 for 31. This high RBC/plasma
ratio demonstrates that 36 is rapidly sequestered into RBCs in
preference to plasma and implies that therapeutically effective
blood concentrations in the target cell can be achieved at
relatively low plasma concentrations. Thus, the possibility of off-
target effects is diminished. T1/2 value of 36 in all animal species
was significantly shorter than the T1/2 of red blood cells (∼20
days), which supports that binding of 36 to hemoglobin is a
reversible process.
The ability of 36 to inhibit the major human liver CYP
isozymes (CYP 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4) was
evaluated using pooled human liver microsomes and isozyme-
specific probe substrates. While 36 inhibited these isozymes with
IC50 ranging from 7.9 to 148 μM (Table 5), we feel that this level
of inhibition is mitigated by the low plasma concentrations that
are anticipated to allow therapeutic concentrations in the RBC as
a consequence of the partitioning into the RBC. This high
partitioning into RBC should protect against metabolism and
against possible drug−drug interactions. There was no significant
time-dependent inhibition of 36 against CYP2C19 and 3A4.
Compound 36 is not a substrate for either P-gp or BCRP
transporters.
Figure 4. Zoomed image of the binding pocket for 31. The H-bond with
Ser131 from the neighboring α chain appears to be a key interaction for
maintaining the R-state of HbS.
A driving force for this pose and the 1:1 stoichiometry
appeared to be the H bond from the pyrazole N to Ser131 on the
second α chain, with the aldehyde forming the usual Schiff-base
linkage to the N-valine of the first α chain (Figure 5). In this way
31 and other analogues filled the space normally taken up by two
fused analogues or by two molecules of 5HMF (1). Not
coincidentally those compounds that complexed Hb with a 1:1
stoichiometry performed better than compounds with a 2:1
stoichiometry in the whole blood assay and in the polymerization
assay. As demonstrated in Figure 5, compounds 31 and 36 have
similar orientations when bound to HbS. Compound 36 has
favorable PK properties, and this difference is not immediately
revealed by simply comparing how the compounds interact with
HbS. We surmise that the PK of 36 is superior to that of 31 owing
to an intramolecular H bond to the Schiff-base in the case of 36.
The synthesis of this series of compounds is exemplified by the
convergent synthesis of 36, shown in Scheme 1. The key
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treatment. The second key reactant, the chloride 54, was
prepared via a Suzuki coupling reaction between the pyrazole
boronate ester 51 and the chloropyridine 52 to give the alcohol
53, which was chlorinated by thionyl chloride. Other aldehydes
were prepared by varying the boronate ester and the phenol
coupling agents (Supporting Information).
In summary, we have developed a series of compounds that
increase the oxygen affinity of HbS, both on the isolated protein
and in whole blood from sickle cell patients. Key compounds
increase the delay time to the onset of polymerization of HbS.
These “left shifters” bind in a Schiff-base linkage to the amine of
the N-terminal valine of the α chain of HbS. In comparison to
those earlier reported such as 5HMF (1) and BW12C (2) that
bind in a 2:1 stoichiometry, X-ray analysis reveals that key
compounds such as 31 and GBT440 (36)17 bind to a single α
chain in a 1:1 stoichiometry to the HbS tetramer. Compound 36
demonstrates favorable pharmacokinetics in the rat, dog, and
monkey. As noted in the rat, oral bioavailability is 60%, the T1/2 is
19.1 h, and there is dramatic partitioning into blood with a RBC/
plasma ratio of ∼150. It is anticipated that this unique
partitioning into the target cell should allow therapeutic
concentrations to be attained in the RBC at comparatively low
plasma concentrations and thereby reduce the likelihood of off-
target effects. Compound 36 (GBT440) is currently in Phase 3
clinical trials (NCT03036813) in SCD patients.
Figure 5. Overlay of compounds 6, 31, and 36. All compounds bind with
Val1 (green). Compound 6 binds 2:1 per Hb tetramer, and both
compounds 31 and 36 bind 1:1 per tetramer. This difference in
stoichiometry is further demonstrated with the omit maps included in
Figures S1 and S2.
ASSOCIATED CONTENT
* Supporting Information
■
S
a
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00491.
Scheme 1. Synthesis of 36
All experimental procedures and compounds character-
ization, material and methods about Hemox and polymer-
ization assays, as well as DMPK and in vivo experiment
protocols (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: zli@globalbloodtx.com.
■
ORCID
Matthew P. Jacobson: 0000-0001-6262-655X
Zhe Li: 0000-0002-3780-0760
Notes
The authors declare the following competing financial
interest(s): B.M., K.D., M.P., A.S., C.J., J.P., Q.X., S.G., C.Y.,
J.H., A.H., D.O., and Z.L. are employees of Global Blood
Therapeutics, which has a commercial interest in hemoglobin
modulators; M.P.J. owns GBT stock.
Carl Johnson: chagroth@stanford.edu. Lan Hua: hualan@nibs.
ac.cn. Jason Harris: Jharris@jrhbiopharma.com. Joyce James:
joycjames@gmail.com. Larysa Patskovska: larysa.patskovska@
accentbiosystems.com. Stephen L. Gwaltney: sgwaltney@
chrysalistherapeutics.com. Yury Patskovsky: yury.patskovsky@
accentbiosystems.com.
a
Reagents and conditions: (a) MOMCl, DIEPA, DCM, 0 °C to rt 2 h,
90%; (b) nBuLi, DMF, THF, −78 to 0 °C, 94%; (c) 12 N HCl, THF,
rt, 1.5 h, 81%; (d) Pd(dppf)Cl2, NaHCO3, H2O/dioxane, 100 °C, 12
h, 40%; (e) SOCl2, DCM, rt, 100%; (f) Na2CO3, DMF, 65 °C, 1.5 h,
81%; (g) 12 N HCl, THF, rt, 3 h, 96%.
coupling step of phenol 50 with the benzylic chloride 54 is
affected using sodium carbonate as base in DMF at 65 °C for 1.5
h. Compound 55 was isolated in 81% yield by pouring the
reaction mixture into ice water and filtering the solid. The
protecting MOM group was removed using HCl (12 N) in THF.
On neutralization the 36 free base was isolated by filtration. The
key phenolic reactant, 50, was prepared from 2-bromoresorcinol
(47) first by MOM protection of both hydroxyls, then lithiation
via butyl lithium followed by quenching with DMF and HCl
ACKNOWLEDGMENTS
■
The authors thank Dr. Peter Rademacher for running HRMS for
selected compounds. We wish to acknowledge the Hemoglobin-
opathy Center at the Children’s Hospital Oakland Research
Institute (CHRCO, Oakland, CA) for providing blood from
sickle cell patients. We thank J. Holton, G. Meigs, and ALS staff
E
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Letter
T.; Abraham, D. J.; Venitz, J.; Safo, M. K. Crystallographic analysis of
human hemoglobin elucidates the structural basis of the potent and dual
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Sect. D: Biol. Crystallogr. 2011, D67, 920−928.
of beamline 8.3.1 for data collection and helpful discussions.
Beamline 8.3.1 at the Advanced Light Source is operated by the
University of California Office of the President, Multicampus
Research Programs and Initiatives grant MR-15-328599 and
Program for Breakthrough Biomedical Research, which is
partially funded by the Sandler Foundation. Additional support
comes from National Institutes of Health (GM105404,
GM073210, GM082250, GM094625), National Science Foun-
dation (1330685), Plexxikon Inc., and the M.D. Anderson
Cancer Center. The Advanced Light Source (Berkeley, CA), a
national user facility operated by Lawrence Berkeley National
Laboratory on behalf of the US Department of Energy under
contract number DE-AC02-05CH11231, Office of Basic Energy
Sciences, through the Integrated Diffraction Analysis Technol-
ogies program, supported by the US Department of Energy
Office of Biological and Environmental Research.
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ABBREVIATIONS
■
5HMF, 5-hydroxymethylfurfural; Cmax, the maximum concen-
tration; DIPEA, N,N-diisopropylethylamine; Hb, hemoglobin;
HbA, adult hemoglobin; HbS, sickle cell hemoglobin; MOMCl,
methoxymethyl chloride; OEC, oxygen equilibrium curve;
Pd(dppf)Cl2, [1,1′-Bis(diphenylphosphino)ferrocene]-
palladium(II) dichloride; RBC, red blood cell; R and R2 states,
relaxed or oxygenated states of hemoglobin; SCD, sickle cell
disease; T1/2, half-life, the time required for Cmax to drop in half
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DOI: 10.1021/acsmedchemlett.6b00491
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX