Water-soluble progesterone analogues are effective, injectable treatments in animal models of traumatic brain injury

Article abstract of DOI:10.1021/ml200303r

After more than 30 years of research and 30 failed clinical trials with as many different treatments, progesterone is the first agent to demonstrate robust clinical efficacy as a treatment for traumatic brain injuries. It is currently being investigated in two, independent phase III clinical trials in hospital settings; however, it presents a formidable solubility challenge that has so far prevented the identification of a formulation that would be suitable for emergency field response use or battlefield situations. Accordingly, we have designed and tested a novel series of water-soluble analogues that address this critical need. We report here the synthesis of C-20 oxime conjugates of progesterone as therapeutic agents for traumatic brain injuries with comparable efficacy in animal models of traumatic brain injury and improved solubility and pharmacokinetic profiles. Pharmacodynamic analysis reveals that a nonprogesterone steroidal analogue may be primarily responsible for the observed activity.

Full text of DOI:10.1021/ml200303r

Letter
pubs.acs.org/acsmedchemlett
Water-Soluble Progesterone Analogues Are Effective, Injectable
Treatments in Animal Models of Traumatic Brain Injury
David B. Guthrie,† Donald G. Stein,‡ Dennis C. Liotta,†,§ Mark A. Lockwood,† Iqbal Sayeed,‡ Fahim Atif,‡
Richard F. Arrendale,† G. Prabhakar Reddy,† Taylor J. Evers,† Jose R. Marengo,† Randy B. Howard,†
Deborah G. Culver,† and Michael G. Natchus*,†
†Emory Institute for Drug Discovery (EIDD), Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
‡Emory University School of Medicine, Department of Emergency Medicine, Emory University, Atlanta, Georgia 30322, United
States
§Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
S
* Supporting Information
ABSTRACT: After more than 30 years of research and 30 failed clinical trials with as many different
treatments, progesterone is the first agent to demonstrate robust clinical efficacy as a treatment for traumatic
brain injuries. It is currently being investigated in two, independent phase III clinical trials in hospital settings;
however, it presents a formidable solubility challenge that has so far prevented the identification of a
formulation that would be suitable for emergency field response use or battlefield situations. Accordingly, we
have designed and tested a novel series of water-soluble analogues that address this critical need. We report
here the synthesis of C-20 oxime conjugates of progesterone as therapeutic agents for traumatic brain injuries with comparable
efficacy in animal models of traumatic brain injury and improved solubility and pharmacokinetic profiles. Pharmacodynamic
analysis reveals that a nonprogesterone steroidal analogue may be primarily responsible for the observed activity.
KEYWORDS: Traumatic brain injury, traumatic brain injury, progesterone, neurosteroid, progesterone analogues
espite decades of effort, scientists have not found a
pharmacological agent that consistently improves func-
multisite phase III clinical trials are currently being conducted
throughout the United States and Europe.22,23
D
tional outcomes after traumatic brain injury (TBI). After more
than 30 failed clinical trials,1 there is still no safe and effective
neuroprotective treatment for TBI, and most major pharma-
ceutical companies have suspended research and development
in this field. However, an innovative, safe, and effective
approach to TBI treatment is now providing the promise of a
breakthrough.
TBI produces a complex succession of molecular events in
addition to the immediate loss of nervous tissue caused by
concussions, contusions, and ballistic injuries. The “brain injury
cascade” initiates rapidly after the initial trauma and unfolds
over days, weeks, and even months. Therefore, a key tenet of
brain injury treatment is that the sooner one can treat/prevent
edema, inflammation, and neuronal loss, the better the
functional outcome will be.18 Unfortunately, current clinical
protocols for the use of PROG require patients to be
transported to a hospital setting, thus losing valuable time
before the treatment can be administered.
PROG is insoluble in aqueous-based formulations, so it must
be delivered in a freshly prepared lipid formulation which
requires a fairly complicated and time-consuming preparation.
Furthermore, the plasma half-life of PROG is only 25 min,24 so
treatment requires continuous iv drip or multiple injections
with an oil-based formulation that delays release to the systemic
circulation. A treatment for TBI that can be administered easily
and rapidly in the field remains elusive.
Preclinical and clinical research demonstrates that the
hormone progesterone (PROG) is a potent neurosteroid
which, when acutely administered, can dramatically reduce
cerebral edema, inflammation, tissue necrosis, and programmed
cell death, while at the same time providing and stimulating
trophic (growth and survival) support to damaged nerve
cells.2−11 Unlike many other potential neuroprotective agents,
PROG is pleiotropic; that is, it acts simultaneously at a number
of different receptor sites in both neurons and glial cells.11 It
also plays a protective role in reducing inflammation and
cellular damage in other body tissues directly affected by TBI,
thus addressing the multiple organ damage that typically occurs
after severe traumatic injuries.12−19 A recently completed phase
II, single-center, double-blind, randomized, controlled trial in
100 moderate-to-severe TBI patients showed that 3 days of
PROG, administered by continuous intravenous (iv) infusion,
reduced mortality by over 60% and significantly improved
functional outcomes at 30 days postinjury.20 A second,
independent clinical trial has recently replicated these
findings.21 On the basis of these positive outcomes, two
As a potential approach toward delivering a field-ready
treatment for TBI, we have adopted a strategy of preparing
novel water-soluble PROG prodrugs and analogues. We
previously reported an initial set of such compounds with
Received: December 19, 2011
Accepted: March 15, 2012
Published: March 15, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
modifications at the C3 center of PROG that allowed
incorporation of water solubilizing groups.25 After surveying
numerous chemotypes, analogue P1-185 (1, Figure 1) was
may also contribute intrinsic therapeutic activity. It also raised
the question of whether PROG arises directly by metabolism of
1 or in a stepwise pathway via the free oxime 2.
While C-3 oxime conjugates such as 1 meet our initial goals
of improved water solubility and therapeutic efficacy, their
syntheses are somewhat challenging. Installation of the oxime
moiety at the C-3 position requires a protection/deprotection
at the C-20 position, and the oxime itself forms in very low E/Z
selectivity, necessitating a tedious chromatographic separation,
which results in low yields of the desired geometric isomer. To
avoid these problems, we designed a second generation of C-20
oxime conjugates (Figure 1) that applies a similar design
principle to 1 but utilizes the more sterically biased C-20
ketone as a synthetic handle to stereoselectively attach a
solubilizing group onto the oxime linker.
The C-20 oxime analogues were synthesized by the general
methods shown in Scheme 1. Starting from readily available
a
Scheme 1. General Synthesis of C-20 PROG Analogues
Figure 1. Progesterone and water-soluble analogues.
identified as our first generation lead compound. P1-185
possesses an improved solubility profile and shows activity
similar to that of PROG in in vitro and in vivo models of brain
injury TBI. The structure of 1 was designed to allow
introduction of a solubilizing group that is susceptible to
hydrolysis in the bloodstream. This was then attached to the
PROG skeleton by a oxime linker, which can undergo formal
hydrolysis in vivo.26 A pharmacokinetic experiment was carried
out in rats using the iv route of administration to follow the
disappearance of 1 and the potential appearance of its related
free oxime (2) and/or PROG. The graph in Figure 2A shows
that 1 is hydrolyzed rapidly to provide large concentrations of
the free oxime 2 and smaller concentrations of PROG. While
this study provided a possible explanation for the observed in
vivo efficacy, it left open the question of whether the free oxime
a
Methods and reagents: (a) NH2OR−HCl, pyridine, Δ; (b) N-
methylpiperidone, Al(OiPr)3, PhMe, Δ; (c) NH2OH−HCl, pyridine,
Δ; (d) RCO2H, iPr2NEt, EDCI, cat. DMAP, CH2Cl2, rt; (e)
deprotection and salt formation (TFA then HCl) or methylation
(MeI, acetone) if necessary.
pregnenolone, condensation with an appropriate O-substituted
hydroxylamine, followed by an Oppenauer oxidation, furnished
C-20 O-alkyl oximes stereoselectively in 53−66% overall yields
in only two steps. Oxime esters were synthesized through the
common intermediate 3, which was performed with mod-
ifications to known procedures on up to 50 mmol scale in 75%
overall yield over two steps. EDCI-promoted coupling of 3 with
the appropriate carboxylic acid, followed by deprotection or
methylation of any amino groups as needed, resulted in oxime
esters 6−22. These routes allowed us to construct multiple
analogues in a rapid fashion (Table 1).
Analogues with adequate aqueous solubility were screened in
our first-pass in vitro assay, which measures the analogues’
capacities to reduce neuronal cell death after a glutamate
challenge. Most of the soluble oxime-ester conjugates tested
had a maximum efficacy of 20−27% reduction in cell death at a
5 μM dose. These values compared favorably with our first-
generation lead 1. In contrast, PROG requires a 20 μM dose to
surpass this efficacy and is much less potent than many of our
compounds at a 5 μM dose. Because the oxime analogues are
not metabolized in this assay, these values suggest that the
compounds likely possess inherent neuroprotective abilities.
To assess the relative properties of the C-20 and C-3 oxime
series, the valine conjugate at C-20 (7) was prepared as a direct
Figure 2. Pharmacokinetic and metabolic profiles of 1 and 7 when
dosed iv at 10 mg/kg in rats. (See Supporting Information for error
analysis of the data.)
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Table 1. Aqueous Solubilities, Stabilities, and In Vitro Efficacy of Analogues
a
b
c
d
e
As determined by laser nephelometry. Hydrolysis of the C−O bond to give 3. Details about in vitro. nd = not determined. s = stable (t1/2 > 10
f
days). ns = not soluble; could not be determined.
comparison to the C-3 oxime 1. These compounds were
compared head to head for their ability to reduce cerebral
edema in rats following bilateral cortical contusion as described
previously.24 The two compounds produced similar levels of
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Letter
therapeutic benefit, both of which compared favorably with that
of natural PROG (Figure 3A). PROG reduced brain edema
Figure 4. Plasma levels of free oxime in rats after im injection of parent
compounds.
Figure 3. In vivo efficacy of PROG and various analogues in a rat
model of injury induced brain edema.
Cmax and AUC than the relative C-3 oxime 1. The more
chemically stable oxime ester at C-20, 13, produced levels of
free oxime that were similar to those from 1, thus suggesting
that other properly designed C-20 oxime esters could
potentially generate similar concentration profiles.
after 24 h by 62% compared to that in untreated rats, while 1
and 7 reduced brain edema by 59% and 56%, respectively.
Subsequently, we examined the pharmacokinetic profile of 7 in
rats after a single iv dose at 10 mg/kg. Again, we observed low
levels of the parent compound 7, high levels of the relative free
oxime 3, and therapeutic levels of PROG (Figure 2B). The
levels of PROG that were produced were slightly superior to
those generated by 1, so the C-20 oxime series proved to be a
good scaffold on which to examine the effect of modifications
on the water-solubilizing group.
We hypothesize that the observed difference in pharmaco-
kinetic profiles between iv and im administration may be due to
differences in the metabolic stability of the free oximes relative
to their relative oxime esters. We have shown that both 7 and 1
are readily cleaved in both rat and human plasma (see
Supporting Information), which explains the appearance of the
free oxime after either mode of administration. By contrast, the
oxime esters behaved differently than the free oximes when
exposed to rat S9 liver fractions. Oxime ester 7 generated
measurable quantities of the free oxime 3, as well as PROG.
However, when 3 was exposed directly to S9 fractions, it was
relatively stable and no measurable quantities of PROG were
generated. We therefore hypothesize that the oxime esters are
metabolized in the liver to give PROG directly, without the
involvement of the free oxime as an intermediate in the process.
It appears that when the esters are administered iv, a substantial
fraction reaches the liver before being hydrolyzed, resulting in
the production of PROG. However, when the agents are
administered im, they are hydrolyzed to their corresponding
oximes before they reach general circulation, thereby avoiding
conversion to PROG in the liver.
Given that our intended route of administration for these
compounds is im, it became critical to determine whether free
oximes 2 and 3 are intrinsically active. Accordingly, we
administered the C-3 oxime 2 iv at 4 mg/kg and showed that
it was cleared from plasma with a half-life of 2.7 h without the
formation of measurable concentrations of PROG (see Supporting
Information for data). We then demonstrated that when 2 was
administered iv at 4 mg/kg to rats 1 h after a controlled cortical
impact, it reduced brain edema by 60% (Figure 3B). We
therefore conclude that oxime 2 is intrinsically active.
Experiments to determine the levels of the intrinsic neuro-
protective activity of 3 are currently in progress.
We anticipate that a field-ready therapy for TBI will likely
need to be delivered via an intramuscular (im) injection in an
aqueous formulation to allow for rapid release into circulation.
We therefore measured the aqueous solubility of all new
analogues using nephelometry at pH 7 and 4. We also evaluated
their chemical stability in neutral media. These results are
summarized in Table 1. The valine conjugate 7 was more
soluble (146 μM) than PROG (≪25 μM) in neutral aqueous
buffer, but it exhibited markedly increased solubility in a
cyclodextrin formulation of at least 60 mM. Interestingly, 7
hydrolyzed in phosphate buffer (pH = 7.0) with a half-life of
11.4 h, while it was indefinitely stable at pH < 5. Proline
conjugate 8 was also highly unstable, with a half-life of only 52
min. At neutral pH, α-amino acid esters have been reported to
undergo general base catalyzed hydrolysis, assisted by complex-
ing to buffer components,27 and this behavior proved to be
operative for these analogous oxime esters as well. This
aqueous instability could have skewed solubility results at
neutral pH, as the common byproduct of hydrolysis was the
highly insoluble 3 in each case. To address the chemical
stability issue, we generated a series of analogues with altered
water-solubilizing groups. Restricting a β- or γ-amino nitrogen
inside a nipecotate or isonipecotate moiety, respectively, led to
much higher stability, with half-lives ranging from 1.5 to 4 days,
while increasing solubility. Tertiary substitution on N,N-
dimethylglycine ester 10 led to a half-life of only 6.2 h, but
including the α-nitrogen in a piperazine ring as in 13 increased
the half-life to nearly 3 days. Analogues with an alkyl C−O
bond, such as 5, were indefinitely stable but had very low
biological activity (vide infra).
In conclusion, we have developed a set of water-soluble
analogues of PROG with substantial efficacy in animal models
of TBI, which compare favorably to native PROG. These
analogues possess intrinsic neuroprotective activity and are well
suited for field administration via im injection. A set of these
analogues has been optimized to maximize their aqueous
solubility and stability. We are now in the process of
characterizing key compounds from this series with a view
toward identifying a clinical candidate and developing it as a
field-ready treatment for TBI. These new agents should be
rapidly and easily administered in the field to reduce the time
Having resolved the chemical stability issue in aqueous
media, we next evaluated the pharmacokinetic profile of these
compounds after im injection in rats. Again, we looked at both
1 and 7 as analogous representatives of the C-3 and C-20 oxime
families. Interestingly, as shown in Figure 4, both compounds
generated measurable concentrations of only the corresponding
oximes without evidence of either the parent prodrug or
PROG. Also, the C-20 oxime 7 generated a significantly higher
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between injury and treatment to improve overall patient
outcomes.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic experimental details, analytical data of compounds,
and biological assay protocols. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mnatchu@emory.edu.
Author Contributions
The manuscript was written through contributions of D.B.G.,
D.G.S., M.A.L., M.G.N., and D.C.L. All authors have given
approval to the final version of the manuscript.
(16) Ishrat, T.; Sayeed, I.; Atif, F.; Hua, F.; Stein, D. G. Progesterone
and allopregnanolone attenuate blood-brain barrier dysfunction
following permanent focal ischemia by regulating the expression of
matrix metalloproteinases. Exp. Neurol. 2010, 226 (1), 183−90.
(17) Stein, D. G. The case for progesterone. Ann. N. Y. Acad. Sci.
2005, 1052, 152−69.
Funding
We gratefully acknowledge financial support from The Emory
Institute for Drug Discovery, Emory University.
Notes
(18) Stein, D. G. Progesterone in the treatment of acute traumatic
brain injury: a clinical perspective and update. Neuroscience 2011, 191,
101−6.
The authors declare no competing financial interest.
ABBREVIATIONS
■
(19) Stein, D. G.; Wright, D. W. Progesterone in the clinical
treatment of acute traumatic brain injury. Expert Opin. Invest. Drugs
2010, 19 (7), 847−857.
PROG, progesterone; TBI, traumatic brain injury; EDCI, N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
DMAP, 4-dimethylaminopyridine; TFA, trifluoroacetic acid
(20) Wright, D. W.; Kellermann, A. L.; Hertzberg, V. S.; Clark, P. L.;
Frankel, M.; Goldstein, F. C.; Salomone, J. P.; Dent, L. L.; Harris, O.
A.; Ander, D. S.; Lowery, D. W.; Patel, M. M.; Denson, D. D.; Gordon,
A. B.; Wald, M. M.; Gupta, S.; Hoffman, S. W.; Stein, D. G. ProTECT:
A Randomized Clinical Trial of Progesterone for Acute Traumatic
Brain Injury. Ann. Emerg. Med. 2007, 49 (4), 391−402.
(21) Xiao, G. M.; Wei, J.; Yan, W. Q.; Wang, W. M.; Lu, Z. H.
Improved outcomes from the administration of progesterone for
patients with acute severe traumatic brain injury: a randomized
controlled trial. Crit. Care 2008, 12 (2), R61.
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