Download Development of Rapidly Metabolized and Ultra-Short

Development of Rapidly Metabolized and
Ultra-Short-Acting Ketamine Analogs
Martyn Harvey, MD, FACEM,* Jamie Sleigh, MD,* Logan Voss, PhD,* Jiney Jose, PhD,†
Swarna Gamage, PhD,† Frederik Pruijn, PhD,† Sarath Liyanage, PhD,† and William Denny, PhD†
BACKGROUND: Ketamine is a well-established, rapidly acting dissociative anesthetic. Clinical
use is limited by prolonged psychotomimetic phenomena on emergence, often requiring the
coadministration of additional hypnotic drugs. We hypothesized that the development of ketamine ester analogs with ultrashort offset times might markedly reduce the dysphoric emergence phenomena and, hence, increase the utility of a ketamine-like hypnotic and analgesic.
Here, we describe the results of studies that seek to define the pharmacology of 5 esters of
((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)pentanoate hydrochloride, the first ketamine analogs
designed to be susceptible to ultrarapid metabolism.
METHODS: Five norketamine ester analogs (R1–R5) were compared by ability to produce loss of
righting and nociceptive blunting in rats. Toxicity testing was performed for 2 analogs (R1, R5) with
50% lethal dose (LD50) estimation in rats. In vitro metabolic stability was tested in rabbit plasma
and whole blood by high-performance liquid chromatography. Behavioral and hemodynamic effects
were observed in rabbits. We estimated the pharmacokinetics of these analogs in rabbits.
RESULTS: All 5 norketamine esters produced rapid loss of righting reflex and diminished pedal
withdrawal with ultrarapid offset in the models studied (return of righting reflex 87 seconds
[interquartile range (IQR) 78–131] R1 vs 996 seconds [IQR 840–1304] ketamine in rats; P <
0.01). The LD50 was comparable to that of ketamine (LD50 R1 50.2 mg/kg [95% confidence
interval, 30–63]). For all analogs, hydrolysis to sole carboxylic acid derivatives was most rapid in
vivo (clearance 1.61 L/kg/min R1 [IQR 0.40–2.42]), followed by whole blood and then plasma.
Analog R5 demonstrated relatively greater nociceptive blunting than hypnotic effect (P < 0.001;
pedal withdrawal score comparison with R1).
CONCLUSIONS: The 5 norketamine ester analogs retain the hypnotic characteristics of the parent
compound, yet display rapid offset due to ultrarapid metabolism. (Anesth Analg 2015;XXX:00–00)
K
etamine [racemic (2-(2-chlorophenyl)-2-(methylamino)
cyclohexanone] is a dissociative anesthetic with
profound analgesic and sympathomimetic properties. It has major advantages over opioids: minimal respiratory depression, preservation of protective airway reflexes,
no hyperalgesic effects, a reduction in analgesic tolerance,
and efficacy in opioid-resistant pain.1,2 Analgesic effects are
achieved at approximately 400 ng/mL.3 A dissociative anesthetic state develops when the plasma concentration exceeds
2000 ng/mL. The most important adverse effects of ketamine
are its psychotomimetic properties, which occur at drug
concentrations of just 100 ng/mL.4 Because ketamine has an
elimination half-life of 2 to 3 hours,2,5,6 patients may experience prolonged (20–120 minutes) psychic disturbances after
anesthesia. This limits the clinical utility of ketamine in adults.
Ketamine analogs with very rapid clearance might
limit the psychomimetic period to only a few minutes.
From the *Waikato Clinical School, University of Auckland, Hamilton, New
Zealand; and †Auckland Cancer Society Research Center, School of Medical
Sciences, Auckland, New Zealand.
Accepted for publication January 12, 2015.
Funding: Biopharma funding Grant No 76650.
Conflict of Interest: See Disclosures at the end of the article.
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF versions of
this article on the journal’s website (www.anesthesia-analgesia.org).
Reprints will not be available from the authors.
Address correspondence to Martyn Harvey, MD, FACEM, Emergency Department, Waikato Hospital, Pembroke St., Hamilton, New Zealand. Address
e-mail to [email protected].
Copyright © 2015 International Anesthesia Research Society
DOI: 10.1213/ANE.0000000000000719
XXX 2015 ‫ ڇ‬Volume XXX ‫ ڇ‬Number XXX
This would reduce the need for adjunctive hypnotics
and extend the utility of such ketamine-like compounds,
particularly in remote locations where analgesia is
important, such as the radiological suite, the emergency department, or an out-of-hospital accident scene.
We pursued a strategy similar to “soft” designer drugs
(remifentanil,7 remimazolam,8,9 methoxycarbonyl-etomidate10,11), to create pharmacologically active esters that
would be rapidly inactivated via hydrolysis to inactive
metabolites.
Our initial experience with development and testing of
these agents has been described elsewhere.12 From these
experiments in rodents, 5 norketamine ester analogs were
identified with ketamine-like sedative effects but with
significantly reduced duration of action. In this study, we
determined the behavioral effects in rats and rabbits. We
also characterized the in vivo toxicity in rats and pharmacokinetic characteristics in rabbits.
METHODS
Animals
Animal studies were conducted at the Ruakura Animal
Research Center, Hamilton, New Zealand. All experimental protocols were reviewed and approved by the Ruakura
Animal Ethics Committee. Adult female Sprague-Dawley
rats (350–400 g) were obtained from breeding stock from
the Ruakura site. Adult male New Zealand white rabbits
(2910–3340 g) were purchased from a credentialed supplier
and quarantined at the Ruakura site for 3 weeks before
use. All animals were housed in gender-specific enclosures
with no chance of pregnancy. Standard, 12-hour day–night
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Ketamine Analog Development
lighting was maintained, and free access to feed and water
was allowed until the day of the animal study.
Synthesis of N-Alkylated Norketamine
Esters R1 to R5
We previously reported our initial synthesis and screening of 24 norketamine analog test compounds12 and identified 5 of these compounds as having the most desirable
characteristics of potent sedative effects with rapid offset.
In this report, the following alkylated norketamine esters
were interrogated for behavioral effects and toxicity in rats,
and behavioral effects and pharmacokinetic analysis in
rabbits: SN35210 = racemic-methyl 4-((1-(2-chlorophenyl)2-oxocyclohexyl)amino)pentanoate hydrochloride (R1);
SN35371 = (S)-methyl 4-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)pentanoate hydrochloride (R2); SN35476 = racemic ethyl 3-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)
propanoate hydrochloride (R3); SN35486 = racemic isopropyl 4-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)butanoate hydrochloride (R4); and SN35563 = racemic isopropyl
3-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)propanoate
hydrochloride (R5).
The compounds of interest were synthesized from norketamine, prepared from commercially available (2-chlorophenyl)(cyclopentyl)methanone.13 Esters were then
prepared by treatment of norketamine with the appropriate alkyl halides Br(CH2)nCO2(CH2)R, and converted to
the hydrochloride salts with HCl gas (Fig. 1). Purity was
determined by high-performance liquid chromatography
(HPLC) monitoring at 272 nm and was ≥95% for all compounds. Enantiomeric purity was analyzed by chiral HPLC
(Chiralcel OJ-H column, 0.46 × 45 cm) (Chiral Technologies,
Illkirch, France). The mobile phase was 85% hexane/15%
EtOH (v/v) with a flow rate of 0.6 mL/min. The purity for
R2 was determined by monitoring at 254 and 280 nm and
was ≥95%. All compounds were solubilized in 0.9% saline
solution before administration.
Synthesis of N-Alkylated Norketamine
Carboxylic Acids M1 to M5
N-Alkylated norketamine esters (R1–R5) and sodium hydroxide solution were dissolved in MeOH/EtOH. The reaction
mixture was stirred at 50°C overnight. After completion of
reaction, the solvent was evaporated and the residue obtained
was acidified with 2 N HCl in ether. EtOAc was added to the
above solution and stirred for 5 minutes. The solution was filtered, and the filtrate was evaporated to obtain a white solid
that was triturated with EtOAc and filtered, and then dried to
obtain the product as white solid. The purity of the carboxylic
acids (M1–M5) was determined by monitoring at 254 and 280
nm and was ≥95% for all compounds.
All compounds were synthesized by the laboratory of
the Auckland Cancer Society Research Centre, Auckland,
New Zealand.
Calibration Standards/Analysis
Final products were analyzed by reverse-phase HPLC
(Alltima C18 5-μm column, 150 × 3.2 mm; Alltech
Associated, Inc., Deerfield, IL) using an Agilent 1100 LC
equipped with a diode array detector. The mobile phase
was 80% CH3CN/20% H2O (v/v) in 45 mM HCO2NH4 at
pH 3.5 and 0.5 mL/min. Calibration for each compound
was performed using 100% fetal calf serum spiked with a
range of ester/carboxylic acid concentrations (1–3, 10, 30,
60, and 100 μM). Concentrations of norketamine esters and
carboxylic acids for subsequent in vitro and in vivo experiments were determined by HPLC analysis using the above
parameters. After thawing, all experimental samples were
Figure 1. Synthesis of study drugs (R1–
R5) from norketamine (1). Structure of
ketamine provided for comparison. All
analogs are racemic save R2, which is
the S-enantiomer of R1. Me = methyl;
Et = ethyl; iPr = isopropyl.
2
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completely dried in a vacuum evaporator, and 75 μL of 1:3
acetonitrile formate buffer (0.45 M, pH 4.5) was added, vortexed, and centrifuged again; 50 μL was injected to HPLC.
Standard curves were prepared from known stock solutions of the compounds to determine HPLC area peaks for
each for the 5 compounds and for the 4 carboxylic acids.
Standard curves were estimated by linear regression. The
regression curves were used to determine the relevant concentrations of test compounds via correlation with measures of HPLC peaks.
The lowest stock concentration for each analyte was variably 1to 3 μM. Concentrations below this were extrapolated
to 0. The lowest recorded values for the esters were: R1 0.1
μM, R2 0.21 μM, R3 0.31 μM, R4 2.29 μM, and R5 0.86 μM.
The lowest recorded carboxylic acid concentrations were
M1 4.95 μM, M2 5.49 μM, M3/M5 6.29 μM, and M4 3.65 μM.
Each test sample was assayed only once for ester and
carboxylic acid. The method does not readily provide for
determination of variability coefficients.
Behavioral Effects in Rats
Norketamine esters were evaluated for effect on responsiveness (hypnosis/analgesia) in rats. Sedative activity (demonstrated primarily by loss of righting reflex [LORR]) and
nociceptive blunting (demonstrated by attenuation of pedal
withdrawal score [PWS], a graduated response to standardized digital pressure [Supplemental Digital Content
1, http://links.lww.com/AA/B94, Table 1]) served as key
outcome measures. Using a crossover experimental design,
each norketamine analog was compared with ketamine in
the same 3 animals (total 15 rats). An interval of 24 hours
was allowed between agent evaluation.
Rats were restrained in a 3-inch-diameter, 9-inch-long
acrylic chamber and underwent venous cannulation of a lateral tail vein. Mini-bore extension tubing was attached, and the
animal was released to a solitary enclosure. Infusion of study
agents (R1–R5) at 10 mg/mL was begun at 20 mg/kg/min
and maintained until LORR (defined as inability of the animal
to spontaneously right from a position of dorsal recumbency
to that of sternal recumbency). Thereafter, the infusion was
continued at identical rate until the animal exhibited a PWS
of 1.1 On reaching a PWS of 1, the infusion was decreased
to 6.7 mg/kg/min and titrated in an up-and-down fashion (increment or decrement of 1 mg/kg/min at 30-second
intervals) in an attempt to maintain a goal level of sedation
defined as constant LORR and stable PWS of 1. Infusions
were continued for a total of 10 minutes before cessation.
Righting reflex status and PWS were recorded at 1-minute intervals from commencement until these parameters
returned to baseline. Sedation index (defined as weightadjusted mean drug dose [mg] per minute of righting reflex
loss) and nociceptive index (defined as weight-adjusted
mean analog dose [mg] per unit decrease in PWS score from
baseline) were computed to enable comparison of in vivo
potency. Time from cessation of infusion to return of righting reflex was recorded.
Toxicity: IV LD50 Determination
Determination of 50% lethal dose (LD50) by the IV route
for norketamine esters R1 and R5 was performed in 14
female Sprague-Dawley rats weighing 280 to 320 g. After
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catheterization of the lateral tail vein, study drugs were
injected for 30 seconds. Initial drug dosing was at 9.5 mg/kg
with subsequent dosing in accord with the AOT 425 statistical programa using an up-and-down methodology for
LD50 determination. All drugs were administered in 1 mL
of 0.9% saline solution regardless of dosage. Limb lead electrocardiogram and visual inspection of respiratory effort
was monitored throughout. Death was confirmed by the
absence of respiratory and cardiac activity.
Metabolic Stability in Plasma and Whole Blood
The metabolic stability of the norketamine esters was tested
in rabbit plasma and whole blood. For each compound, 20
mL of whole blood was drawn from arterial puncture of the
central ear artery from one male New Zealand white rabbit.
Animals were then returned to enclosures and later used for
study no less than 2 weeks later. Blood was anticoagulated
(100 IU heparin) and divided. A volume of 10 mL of whole
blood was centrifuged at 2300g for 3 minutes, with plasma
then pipetted from underlying red call mass and buffy coat.
At time 0, study drugs were added to plasma/whole
blood to a concentration of 0.0335 mg/mL (approximately
100 μM). Plasma/whole blood was then continuously vortexed at 37°C. At time equals 1, 3, 5, 10, and 20 minutes,
0.3-mL samples were withdrawn and immediately added to
equal volumes of ice-cold acetonitrile (Sigma-Aldrich Co, St
Louis, MO) to arrest metabolic activity. Samples were manually agitated before undergoing centrifugation at 9000g for
4 minutes, before aliquoting, and freezing at −80°C before
analysis. Norketamine ester concentrations (R1–R5) and
corresponding norketamine carboxylic acid (M1–M5) concentrations were determined by HPLC analysis according
to the method described above.
Behavioral Effects and Population
Pharmacokinetic Evaluation in Rabbits
Preliminary pharmacokinetic analysis of the norketamine
eaters R1 to R5 was conducted in 5 male adult New Zealand
white rabbits (average weight of 3.07 kg; range 2.91–3.34 kg)
after rapid loading and subsequent maintenance infusions.
Norketamine esters and ketamine were evaluated in the
same animals at an interval of at least 3 days to minimize
intersubject variation in behavioral and pharmacokinetic
metrics. Venous cannulation of the marginal ear vein was
undertaken for the purpose of study drug administration.
Arterial cannulation of both ears was performed to enable
continuous invasive monitoring of hemodynamic variables
and arterial blood sampling.
Ketamine or norketamine esters at 10 mg/mL were infused
at the rate of 20 mg/kg/min to LORR (nominally time 0).
Thereafter, infusion rates were reduced to 2 mg/kg/min and
continued to 10 minutes before cessation. Assessment
of behavioral parameters (LORR, PWS, anesthetic score
[a 6-point score describing levels of rabbit anesthesia14;
Supplemental Digital Content 2, http://links.lww.com/
AA/B95, Table 2]), and hemodynamic parameters were
obtained at 1-minute intervals. During and after the infusion, blood was sampled via arterial catheter at 1, 3, 5, 10, 11,
Available at: http://www.epa.gov/oppfead1/harmonization/. Accessed
December 2013.
a
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Ketamine Analog Development
Table 1. Norketamine Ester In Vivo Potency and Speed of Offset in Rats
Dose to LORR
(mg/kg)
Dose to PWS =1
(mg/kg)
SI
NI
Time to righting from
cessation infusion (sec)
R1
29.4
(23.9–47.5)
45.1
(27.3–60)
11.3
(10.1–11.3)
6.4
(6.3–7.2)
87*
(78–131)
R2
38.4
(30.5–38.9)
41.8
(40.5–48.6)
19.7
(17.9–25.5)
10.6
(9.0–15.3)
110*
(60–115)
R3
25.6
(24.6–94.1)
31.6
(27.8–105.9)
14.8
(10.9–32.5)
6.6
(4.8–19.5)
133*
(118–280)
R4
43.8
(25.6–55.3)
73.6
(34.4–91.6)
18.1
(13.1–18.4)
11.3
(9.2–24.1)
20*
(10–80)
R5
32.6
(23.4–43.2)
39.7
(25.7–44.6)
14.7
(9.9–24.5)
4.7
(3.9–5.1)
68*
(60–120)
Ketamine
20.3
(17.2–26.1)
25.8
(20.8–30.5)
4.3
(3.6–4.6)
1.6
(1.5–2.1)
996
(840–1304)
All data = median (IQR).
LORR = loss of righting reflex; PWS = pedal withdrawal score; SI = sedation index; NI = nocioceptive index.
*P < 0.01, comparison with ketamine
13, 15, 20, and 40 minutes. Whole blood samples of 0.3 mL
were immediately spiked with 0.3 mL ice-cold acetonitrile
and manually agitated to effect complete precipitation of
proteins. Samples were than centrifuged at 9000g for 4 minutes and the supernatant aliquotted before freezing at −80
°C until analysis by HPLC for study agent and acid metabolite according to the method described above.
Basic pharmacokinetic parameters, clearance (CL), volume of distribution (V), and elimination half-life (t½), were
calculated from the arterial concentrations of norketamine
analogs R1 to R5. A 1-compartment population (i.e., all-in)
pharmacokinetic model was used (FOCE-L-B algorithm)
with the NLME7 engine in Phoenix NMLE 1.1 (Pharsight
Corporation, St Louis, MO). Because the data were well
described by a 1-compartment model, we did not undertake evaluation using further compartmental analysis. The
model parameter and associated error estimates (theta values) reported all returned relatively small error estimates.
In addition, visual inspection of the fitted data and the
numerous residual plots all confirmed good and statistically
sound model fits. Preliminary modeling of pharmacokinetic
(PK)– pharmacodynamic (PD) parameters was furthermore
performed using a sigmoid Emax model. Simulations of the
plasma concentrations of norketamine esters R1 to R5 based
on population PK metrics were constructed and linked to
PD models of PWS and anesthetic scores.
Statistical Analysis
Statistical analyses were conducted using GraphPad Prism
5.0 (GraphPad Software Inc., La Jolla, CA). All data are
reported as median (interquartile range) unless otherwise
stated. Comparison of time metrics was conducted using the
Mann-Whitney statistic. Continuous metrics were compared
over time using 2-way repeated measures analysis of variance. A P value of <0.05 was deemed statistically significant.
RESULTS
Responsiveness in Rats with Norketamine
Ester Infusion
All agents (R1–R5) demonstrated lesser in vivo potencies with
a 1.5- to 2-fold increase in dosing required to reach LORR and
PWS end points compared with ketamine. Consistent with
hypothesized rapid metabolism, all analogs demonstrated
lightening of sedation after cessation of the initial rapid infusion, therefore requiring upward titration of subsequent
maintenance infusion rates. Recovery, as demonstrated by
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return of righting reflex, was approximately 10 times faster
than ketamine for all the norketamine esters. Dose to LORR
and dose to a PWS of 1, sedation and nociceptive indices,
and time to regaining righting reflex for all agents are presented in Table 1. The quality of sedation achieved with ketamine analog infusion differed between agents. In particular,
R1 exhibited the most ketamine-like effect with consistent
LORR and depressed PWS, whereas R5 exhibited less consistent LORR yet more profound PWS blunting (Supplemental
Digital Content 3, http://links.lww.com/AA/B96, Fig. 1).
Toxicity
Norketamine ester R1 was administered to 7 rats at doses of
9.5, 30, 95, 30, 95, 30, and 95 mg/kg. Death was observed to
occur in animals 3, 5, and 7 after administration of 95, 63, and
73 mg/kg, respectively (death occurring before administration
of the full dose in the latter 2 animals). Therefore, the final score
is OOOOXXX and LD50 50.2 mg/kg (95% confidence interval
[CI], 30–63 mg/kg). R5 was administered to 7 rats at dose 9.5,
30, 95, 30, 95, 30, and 95 mg/kg. Death was observed in animals 3, 5, and 7 after administration of 95 mg/kg. Therefore,
the final score is OOOOXXX and LD50 56.0 mg/kg (95% CI 30–
95 mg/kg). IV LD50 for ketamine in rats is previously reported
at 58.9 mg/kg (Pfizer ketamine material safety datasheet.b
Metabolic Stability in Plasma and Whole Blood
Initial assessment of the metabolic stability of compounds
R1 to R5 was performed in rabbit plasma and rabbit whole
blood. Consistent with our hypothesis of degradation by
hydrolysis of the distal ester moiety, fragmentation to the
appropriate carboxylic acid and corresponding alcohol was
proposed (Fig. 2).
Plots of norketamine ester (R1–R5) and associated carboxylic acid metabolite (M1–M5) concentration in rabbit plasma
and whole blood are presented in Figure 3. Each data point
represents the mean of 2 separate experiments. Maximal
variation in values was 11% and 21% for drug and metabolite, respectively. Percentage decrease in analog concentration
from time 1 minute (assumed complete mixing) to 20 minutes
in plasma was greatest for R1 (42%), followed by R4 (26%), R5
(20%), and R3 (−33%). Percentage decrease in analog concentration from time 1 minute (assumed complete mixing) to 20
minutes in whole blood was greatest for R2 (83%), followed
Available at: http://www.pfizer.com/files/products/material_safety_
data/526.pdf. Accessed December 22, 2013.
b
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by R3 (76%), R1 (70%), R4 (63%), and R5 (−1.5%). For each test
agent, HPLC analysis showed the presence of only one major
metabolite. On the basis of these results, we conclude that the
rapid metabolism of norketamine esters R1 to R5 occurs exclusively via the proposed pathway outlined for R1 in Figure 2.
Responsiveness in Rabbits and
Pharmacokinetic Evaluation
Hypnotic effect was then evaluated in whole rabbits with
loss of righting and strength of pedal withdrawal serving
Figure 2. Proposed fragmentation pathway for compound racemicmethyl 4-((1-(2-chlorophenyl)-2-oxocyclohexyl)amino)pentanoate hydrochloride (R1) to its carboxylic acid derivative (M1) and methanol.
as primary outcome measures. All agents exhibited rapid
induction of ketamine-like sedation characterized by
lost righting reflex and blunted nociceptive responses.
Hemodynamic parameters and behavioral metrics during
sedation with norketamine analogs and ketamine are presented in Figure 4. All norketamine ester analogs displayed
relative stability of heart rate and mean arterial pressure
throughout the monitoring period. Myotonic activity was
observed early in the course of drug administration with
agent R3, and to a lesser extent with agents R4 and R5.
Offset of sedation was most rapid in agent R3, with animals righting 3 minutes into the maintenance infusion and
PWS normalization before cessation of maintenance infusion (Table 2, Fig. 4). Analog R1 exhibited the most consistent LORR and PWS reduction, yet still maintained rapid
offset. Consistent with the pattern of sedation previously
observed in our rat experiments, the analog R5 exhibited
more profound, and prolonged, nociceptive attenuation
(Supplemental Digital Content 4, http://links.lww.com/
Figure 3. Metabolic stability of norketamine esters in rabbit plasma and rabbit whole blood. Test agents are represented with closed icons
(R1–R5). Carboxylic acid metabolites are represented with open icons (M1–M5). The metabolites of R3 and R5 (M3 and M5, respectively) are
identical compounds; R2 was not analyzed in rabbit plasma due to technical difficulty.
Figure 4. Hemodynamic parameters and behavioral effects of analog R1–R5 and ketamine infusion in rabbits. Shaded portion represents
initial loading infusion. Dotted lines at 0 and 10 minutes represent initiation and cessation of maintenance infusion, respectively. Closed
circles (● ) represent heart rate (beats/min); closed squares (◼) represent mean arterial pressure (mm Hg); upright triangles (▲) represent
pedal withdrawal score; inverted triangles (▼) represent anesthetic score; grey bars represent fraction righting. All data mean (SD).
XXX 2015 ‫ ڇ‬Volume XXX ‫ ڇ‬Number XXX
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Ketamine Analog Development
Table 2. Norketamine Ester Doses, Pharmacokinetic Parameters, and Behavioral Effects Summary in Rabbits
Dose to LORR
(mg/kg)
Total dose
(mg/kg)
CL (L/kg/min)
V (L/kg)
t½ (min)
Time to righting
(sec)
Time to anesthetic
score normalization (sec)
Time to PWS
normalization (sec)
R1
26.5
(25.7–27.6)
46.4
(45.7–48.0)
0.72
(0.137)
0.65
(0.182)
0.65
(0.162)
42†
(−42.5 to 58.5)
180*
(120–210)
0
(−240 to 60)
R2
23.3
(21.8–28.5)
42.7
(40–47.4)
0.69
(0.100)
3.39
(0.694)
3.3
(1.07)
−122†
(−187 to 0)
120*
(0–270)
−120
(−270 to 30)
R3
23.2
(22.7–25.2)
44.2
(42.5–44.8)
0.35
(0.066)
1.68
(0.186)
3.3
(0.73)
−450†
(−479 to −330)
60*
(−240 to 120)
−300*
(−390 to −120)
R4
25.6
(23.0–26.0)
47.2
(42.8–50.5)
0.19
(0.017)
1.01
(0.104)
3.7
(0.51)
192†
(94 to 280)
600*
(240–600)
180
(90–240)
R5
27.5
(26.5–34.1)
47.1
(46.3–54.2)
0.21
(0.026)
1.02
(0.126)
3.4
(0.68)
80†
(−155 to 174)
1800
(1350–1800)
1800 †
(1650–1800)
Ketamine
10.3
(9.7–12.8)
28.6
(26.6–29.9)
NA
NA
NA
1320
(960 to 1560)
1500
(1350–1800)
300
(−150 to 750)
All times from cessation infusion (t=10minutes). Data = median (IQR); population PK data = mean (SD).
LORR = loss of righting reflex; CL = clearance; V = volume of distribution; T½ = half-life; PWS = pedal withdrawal score; NA = ketamine pharmacokinetic analysis
not undertaken.
*P < 0.05.
†P < 0.01, comparisons with ketamine.
AA/B97, Fig. 2). In rabbits, the return to baseline PWS for
R5 occurred very slowly, far outlasting the sedative effects
of the drug (Fig. 4). By this measure, the analgesic effects of
R5 lasted more than twice as long as ketamine, despite animals righting in approximately one-third of the time shown
for the parent compound.
Maximal test substance concentrations were achieved
early in the course of infusion with concentrations declining rapidly after completion of loading infusion, and subsequently after cessation of maintenance infusion (Fig. 5).
Ultrarapid metabolism was observed, which caused the
carboxylic acid metabolite concentrations to exceed those
of study agents at all time points, with maximal concentrations (5- to17-fold study agent concentration) observed at 10
minutes—the time of cessation of infusion of study agents.
The PK parameters obtained from population modeling are
presented in Table 2. These were subsequently linked with
real and simulated PD measures and are presented graphically for R1 in Figure 6, with additional compounds outlined
in Online Supplemental Digital Content 5 (http://links.
lww.com/AA/B98, Fig. 3. While basic, these demonstrate
reasonable agreement between measured and simulated
metrics, suggesting the absence of significant hysteresis
between the plasma drug concentration over time and the
onset and offset of drug effect.
DISCUSSION
In the present study, we report experimental findings of 5
novel norketamine esters designed to exhibit ketamine-like
sedation while providing extremely rapid offset of action secondary to hydrolysis to water-soluble carboxylic acid metabolites. Our synthetic design strategy of including a labile
carboxylate ester moiety joined to the original compound
mirrors that of previous investigators in developing esmolol, remifentanil, methoxycarbonyl-etomidate, and remimazolam, from their respective parents. Such “soft” designer
drugs might have clinical appeal because of their rapid and
predictable metabolism after exerting therapeutic actions,
and hence the ability to readily titrate dosing to clinical effect.
6
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The application of such a rationale to the N-methyl-daspartate antagonist, ketamine, has resulted in 5 ester analogs (R1–R5) with broadly similar PD and PK properties. All
agents, when administered IV, exhibited ketamine-like hypnotic states characterized by LORR and attenuated nociceptive responses. Rapid recovery after cessation of infusions,
or indeed even during slower infusion rates (as seen with
R3 in particular), was apparent for all esters. While congruous across the 2 species under study, offset of sedation was
faster in rats (return of righting reflex was approximately
10-fold less than that of ketamine compared with 3-fold in
rabbits) for all study drugs. This is in keeping with what
is known about the rate of drug metabolism in rodents.10,15
Such ultrarapid metabolism may have contributed to the
wide CIs for measured sedative and nociceptive indices
observed in R3-treated rats in particular.
Although it is difficult to be sure about psychotomimetic
drug effects in animal models, it was apparent in both species receiving norketamine esters that, once normal righting
had been achieved, each animal exhibited normal exploratory and grooming behaviors, which were qualitatively
very different from the prolonged passivity and stereotypical movements seen in animals recovering from ketamine
sedation. Such an observation, although it cannot be quantified, suggests any psychotomimetic phenomena, such as
the emergence reaction often reported by adults undergoing ketamine-based sedation, was attenuated in animals
receiving R1 to R5. This was the goal of our design of rapidly cleared analogs. Clinical studies will be required to
see whether these suggestive animal findings translate into
reduced emergence phenomena in humans.
We did not attempt to identify the site or the enzymatic
system responsible for norketamine analog degradation.
Nevertheless, these preliminary data in rabbits indicate an
appreciable gradient of esterase activity beginning with
plasma, followed by whole blood, then by in vivo metabolism. The huge and rapid increase in metabolites observed
when the ketamine analogs were injected into live animals
suggests that almost all significant esterase activity resides
ANESTHESIA & ANALGESIA
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Figure 5. Arterial concentrations of norketamine esters R1–R5 and carboxylic acid metabolites M1–M5, to 40 minutes in rabbits. Dotted lines
at 0 and 10 minutes represent initiation and cessation of maintenance infusion, respectively.
Figure 6. Simulated plasma PK to PD models of pedal withdrawal
score and anesthetic score for R1 in rabbits. PK = pharmacokinetic;
PD = pharmacodynamic; PWS = pedal withdrawal score.
in the tissues, with breakdown occurring predominantly
as the result of tissue esterases similar to that found for
remimazolam.16 The relatively low potency of the active
ester, combined with rapid buildup of its (slowly excreted
and to date unstudied) metabolites, might prove problematic when administered as an infusion for extended periods.
Nevertheless, ketamine is relatively more potent in humans
than in rodents, and obviously the human dosing regimen
has still to be determined.
Perhaps more intriguing is the spectrum of slightly different actions we observed among the different ketamine
analogs investigated, despite small differences in molecular
structure. At one extreme, we have R1, which has a relatively
smooth hypnotic effect. At the other, we have R5, which has
a relatively weak hypnotic effect and which demonstrated
some degree of myotonus, but which also exerted a potent
and long-acting analgesic action extending after the plasma
drug levels were almost undetectable. Such findings were
reproducible in both animal models under investigation,
and have also been reported in our previous work.12
The origins of the observed differences in behavioral
effects (and those of R5 in particular) are not immediately
apparent. The mechanism of action for ketamine-mediated
XXX 2015 ‫ ڇ‬Volume XXX ‫ ڇ‬Number XXX
analgesia remains to be fully elucidated. Antinociceptive
activity nevertheless likely originates from synergy between
monoamine descending inhibitory system activation,17
NR2B N-methyl-d-aspartate antagonism,18 and direct effects
on opioid receptors.19 On the basis of the present work, we
do not believe this to be an effect of the major metabolite
(M3), because this is the same metabolite produced by R3,
which did not exhibit an excessive prolonged analgesic
effect. Furth ermore, the levels of isopropyl alcohol, the other
significant breakdown product of R5, are nowhere near those
that might produce any systemic analgesia. While mechanistic uncertainty as to the cause of these observed discrepancies remains, their existence raises the interesting possibility
that we might be able to dissociate analgesia from hypnosis,
and perhaps even from psychotomimetic effects, by further
exploration of this group of ketamine-like drugs.
These studies are subject to a number of limitations. The
use of the same animals for sequential PK evaluation of
ketamine analogs, while controlling for interanimal variability in responses, could have introduced bias due to the
effects of study drugs on agent metabolism. Ketamine is
a known cytochrome P-450 inducer,20 and its administration may have enhanced hepatic elimination of analogs
administered later. Similarly, while at present unstudied,
all analogs may exert similar effects on hepatic enzymes.
The serial use of animals for assessment of noxious stimulus may furthermore have introduced bias from potential
adaption of acute pain mechanisms to repeat testing. We
have not sought to quantify the magnitude of any such
phenomenon in the present work.
The designated sampling frequency during PK evaluation furthermore negates more comprehensive analysis
that might have been achieved with greater initial sampling
rates. This is best evidenced in R1 with a calculated half life
less than the minimal sampling interval. Sampling intervals were, however, adopted in line with tolerable limits of
blood loss for rabbits. Given the rapidity of drug degradation, future evaluation in larger animal models will require
far more frequent drug assays to more accurately model
early drug degradation.
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Ketamine Analog Development
We did not undertake PK evaluation of the parent compound ketamine using the method described. Prior investigators investigating ketamine pharmacokinetics in identical
rabbits have reported a plasma half-life of 0.74 ± 0.13 hours21
consistent with the behavioral metrics reported in this work.
Differences in experimental design and the use of multicompartmental modeling techniques, however, render
comparison of additional PK metrics between these studies
impossible. We have not performed in-house LD50 estimation
for ketamine. Our reported values for R1 and R5 are nevertheless in line with those for ketamine in similar rodent models.
From these studies, it is apparent that R1 to R5 exhibit
many characteristics favorable to IV administered hypnotics.
Blood clearance in vivo is rapid, with commensurate reversal
of behavioral effects occurring soon after cessation of infusion. Possible advantages over commonly used GABAergictype hypnotic drugs might include augmented procedural
analgesia, lesser incidence of respiratory depression, and
reduced potential for airway obstruction.22 Demonstration of
hemodynamic stability (in line with the known sympathomimetic effects of the parent drug ketamine) provides additional advantages over short-acting hypnotics now used. The
potential for clinical use may be offset by the seemingly narrower therapeutic indices of these analogs compared with
those of ketamine. Our observed in vivo potencies (approximately 1.5- to 2-fold less than ketamine for LORR and PWS
end points in rats) when combined with similar LD50 metrics suggest a therapeutic index less than that of the parent.
However, the more relevant comparison may be with the
therapeutic index of the IV opioids and hypnotics that these
novel drugs may replace in clinical use.
CONCLUSIONS
Incorporation of rapidly degraded ester moieties into the
parent compound resulted in retention of ketamine-like
hypnotic properties, yet rapid offset due to degradation
to carboxylic acid metabolites, in the 5 norketamine esters
studied. Analgesic activity persisted well beyond that of
any sedative action for one analog (R5). E
DISCLOSURES
Name: Martyn Harvey, MD, FACEM.
Contribution: This author helped design the study, conduct the
study, analyze the data, and write the manuscript.
Attestation: Martyn Harvey has seen the original study data,
reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Conflicts of Interest: Martyn Harvey is a co-inventor on a patent application for test compounds. He, his department, or his
institution could receive royalties relating to the development
of test compounds or their analogs.
Name: Jamie Sleigh, MD.
Contribution: This author helped design the study, conduct the
study, analyze the data, and write the manuscript.
Attestation: Jamie Sleigh has seen the original study data,
reviewed the analysis of the data, and approved the final
manuscript.
Conflicts of Interest: Jamie Sleighis a co-inventor on a patent
application for test compounds. The author or the author’s
department or institution could receive royalties relating to the
development of test compounds or their analogs.
8
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Name: Logan Voss, PhD.
Contribution: This author helped design the study, conduct the
study, analyze the data, and write the manuscript.
Attestation: Logan Voss has seen the original study data,
reviewed the analysis of the data, and approved the final
manuscript.
Conflicts of Interest: Logan Voss is a co-inventor on a patent application for test compounds. The author, the author’s
department or institution could receive royalties relating to the
development of test compounds or their analogs.
Name: Jiney Jose, PhD.
Contribution: This author helped design the study, analyze the
data, and write the manuscript.
Attestation: Jiney Jose has seen the original study data and
approved the final manuscript.
Conflicts of Interest: Jiney Joseis a co-inventor on a patent
application for test compounds. The author, or the author’s
department or institution could receive royalties relating to the
development of test compounds or their analogs.
Name: Swarna Gamage, PhD.
Contribution: This author helped design the study, conduct the
study, and write the manuscript.
Attestation: Swarna Gamage has seen the original study data,
reviewed the analysis of the data, and approved the final
manuscript.
Conflicts of Interest: Swarna Gamage is a co-inventor on a patent application for test compounds. The author, or the author’s
department or institution could receive royalties relating to the
development of test compounds or their analogs.
Name: Frederik Pruijn, PhD.
Contribution: This author helped design the study, conduct the
study, analyze the data, and write the manuscript.
Attestation: Frederik Pruijn has seen the original study data,
reviewed the analysis of the data, and approved the final
manuscript.
Conflicts of Interest: The author declares no conflicts of interest.
Name: Sarath Liyanage, PhD.
Contribution: This author helped analyze the data and write
the manuscript.
Attestation: Sarath Liyanage has seen the original study data,
reviewed the analysis of the data, and approved the final
manuscript.
Conflicts of Interest: The author declares no conflicts of interest.
Name: William Denny, PhD.
Contribution: This author helped design the study, conduct the
study, analyze the data, and write the manuscript.
Attestation: William Denny has seen the original study data,
reviewed the analysis of the data, and approved the final
manuscript.
Conflicts of Interest: William Denny isa co-inventor on a patent application for test compounds. The author, the author’s
department or institution could receive royalties relating to the
development of test compounds or their analogs.
This manuscript was handled by: Steven L. Shafer, MD.
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