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Efficacy of selective mineralocorticoid and glucocorticoid agonists in canine septic shock
Caitlin W. Hicks, BA, Daniel A. Sweeney, MD, Robert L. Danner, MD, Peter Q. Eichacker,
MD, Anthony F. Suffredini, MD, Jing Feng, BS, Junfeng Sun, PhD, Robert Wesley, PhD, Ellen
N. Behrend, VMD, PhD, Steven B. Solomon, PhD, and Charles Natanson, MD
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Supplementary Methods
Details of Study Protocol
During the first 4 h after S. aureus challenge, phenylephrine was titrated to maintain
mean arterial pressure (MAP) >80 mmHg to counter any remaining effects of anesthesia and as
sedation was optimized and sepsis developed. After 4 h, when symptoms of sepsis were fully
developed (based on prior experience with the model), phenylephrine administration was
discontinued and, following a 10 minute washout period, intravascular hemodynamic measures,
an echocardiogram, and blood samples were obtained. Treatment for sepsis was then initiated
based on algorithms to maintain pressures by titrating norepinephrine (NE), oxygenation by
adjusting fractional inspired oxygen concentration (FiO2) and positive end expiratory pressure
(PEEP) levels and acid-base status by adjusting respiratory rate (RR) measured by arterial blood
gas. Preload was maintained with fluid boluses based on scheduled pulmonary artery occlusion
pressure (PAOP) measures (1). Oxacillin (30 mg/kg IV q8 h) was started 4 h after bacterial
inoculation and administered every 8 h thereafter. Conventional intensive care unit support
employed during the ventilation of critically ill large animals was administered as previously
described (1). Animals alive at 96 h were considered survivors and subsequently euthanized
(Beuthanol; 75 mg/kg IV).
Bacterial Preparation and Inoculation
Oxacillin-sensitive S. aureus cultures were prepared and administered into a
subsegmental airspace of the right caudal lobe via a bronchoscope as previously described (1).
The dose of S. aureus (1.5-7.5 x 109 cfu/kg suspended in 10 milliliters) was the same for all
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animals in an individual 96 h experiment and was chosen with the objective of producing 7080% mortality in control animals.
Corticosteroid Dosing
DOC-P: DOC pivalate (2.2 mg/kg SQ) was given in a dose equivalent to that used
clinically to treat adrenal insufficiency in canines (2). This dose provides a maintenance level of
daily mineralocorticoid activity for up to 28 days (3, 4). Animals assigned to receive DOC-P
were given a single injection of DOC pivalate 72 h before bacterial inoculation. Animals
assigned to the placebo group received normal saline in a volume that was equivalent to that
given to the concurrent treatment animal.
DOC-T: Animals assigned to receive DOC-T were given a loading dose of DOC acetate
(0.2 mg/kg dissolved in DMSO 0.45 mL/kg mixed with 250 mL saline given over 45 minutes
IV) immediately after bacterial challenge, followed by daily SQ injections of DOC acetate (0.17
mg/kg dissolved in sesame oil). Animals assigned to the placebo group received equivalent
volumes of DMSO and sesame oil at the same time as the treatment animals. Unlike DOC
pivalate (used in the DOC-P regimen), DOC acetate given by this regimen immediately reaches
therapeutic concentrations in blood. Treated animals in the DOC-T study had blood
concentrations of DOC of 494 ± 91 and 170 ± 5.7 ng/dL at 10 and 24 h after infection,
respectively [measured by a commercial lab (Endocrine Sciences, Calabasas Hills, CA with
normal range 16-46 ng/dL in canines) using previously validated methods (5)], which were
comparable to the levels observed in DOC-P-treated animals at the same time points (395 ± 68
and 163 ± 36 ng/dL at 10 and 24 h after infection, respectively).
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DEX-P: The DEX dosing regimen used to study DEX-P (0.17 mg/kg SQ q12 h starting
48 h before bacterial challenge, followed by 0.014 mg/kg/h IV after challenge) has 10 times
more glucocorticoid activity than the 24 h corticosteroid dose used to treat adrenal insufficiency
in canines (6), and thus is comparable to the stress dose cortisol therapy (hydrocortisone 300
mg/day) used to treat sepsis clinically [i.e. approximately 10 times the daily unstressed
production of cortisone in humans daily for 7 to 10 days (7)]. This DEX-P regimen was designed
to simulate the timing of DOC administration in the DOC-P study. DEX was not started until
48h prior to infection because it was administered intravenously, and thus reaches therapeutic
levels much more rapidly. Thus, the purpose of the asymmetrical design was to produce
therapeutic levels of the two drugs at similar times. Animals assigned to the placebo group
received equivalent volumes of normal saline at the same time as the treatment animals.
DEX-T: The DEX dosing regimen used to study DEX-T was the same as that for DEX-P
(0.014 mg/kg/h IV) except that animals did not receive the SQ doses prior to bacterial challenge.
DEX was given continuously throughout the study so as to mirror the administration of DOC as
closely as possible. Because of its depot preparation, animals treated with SQ DOC injections
were receiving a continuous release of the drug throughout the experiment. Thus DEX was given
as a continuous IV infusion to match this continual release. Animals assigned to the placebo
group received normal saline in a volume that was equivalent to that given to the concurrent
treatment animals.
Fluid and Vasopressor Support
Animals received continuous maintenance fluids (Normasol-M + 27 mEq K+/L, 2
mL/kg/h, IV) beginning at T0. To simulate clinical hemodynamic support and equalize initial
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volume status in all animals, up to three fluid boluses (0.9 % NaCl, 20mL/kg) were administered
at 20 min intervals if PAOP measured at 4 h was < 10 mmHg. For a MAP < 80 mmHg after the
three fluid boluses, a NE infusion was initiated at 0.2 µg/kg/min and adjusted incrementally (0.2
to 0.6 to 1.0, to a maximum of 2.0 µg/kg/min) at 5-min intervals to maintain MAP between 80
and 110 mmHg throughout the 96 h study. At subsequent times (T6, T8, T10, T12, and every 4
hours thereafter), an additional IV fluid bolus (20 mL/kg) was administered if PAOP < 10
mmHg.
Sedation and Analgesia Management
Animals were monitored by a clinician or trained technician at the bedside throughout the
study. Midazolam (0.2 mg/kg loading dose, 50 µg/kg/min infusion IV) sedation and fentanyl (5
µg/kg loading dose, 0.7 µg/kg/min infusion IV) analgesia were titrated based on an algorithm as
previously described (1). Medetomidine infusion (2-5 µg/kg/min) was used to supplement
sedation as needed according to set criteria.
Physiological and Laboratory Measurements
The timing of all physiologic and laboratory measurements is shown in Figure 1.
Hemodynamic parameters [MAP, mean pulmonary artery pressure (PAP), PAOP, central venous
pressure (CVP), heart rate (HR)] measured via a pulmonary artery balloon-tipped thermodilution
catheter placed via the external jugular vein, and airway plateau pressure, measured by an endinspiratory hold maneuver on the ventilator. Arterial and mixed venous blood gases, complete
blood counts (CBC), serum chemistries, and serum cortisol and aldosterone determinations were
measured as previously described (1, 8, 9). Plasma samples for measurement of plasma cytokine
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[interleukin-6 (IL-6) and interleukin-10 (IL-10)] levels were collected and assayed in duplicate
using canine-specific immunoassays according to the manufacturer's instructions (Quantikine
Canine ELISA, R&D Systems, Inc., Minneapolis, MN). Cultures (blood and sputum) were
collected and urinary output was measured as previously described (1).
Statistical Methods
Baseline characteristics within each treatment group were summarized using the mean
and standard error (SEM) and were compared between treatment groups using an F-test. The
effects of DEX and DOC given prophylactically vs. therapeutically on survival were compared
using a Cox proportional hazards model, and survival times between pairs of treatments were
compared using exact log rank tests (StatXact, Cytel Software Corp., Cambridge, MA), using
stratified tests where applicable to account for the potential week effect.
Frequently measured data for components of shock reversal and pulmonary function
scores and central filling pressures were averaged for individual animals across each of 4 time
periods ([0-4 h], (4-12 h], (12-30 h] and (30-96 h]). For less frequently measured variables, we
only compared them at the common time points of 10, 24, 48, 72 and 96h. To evaluate shock
reversal, we standardized MAP and NE using Z-scores and then calculated a “shock reversal”
score based on the difference of the MAP Z-score and NE Z-score. A higher score indicates
improvements in shock reversal. To evaluate pulmonary function, we constructed a “lung injury”
score based on the first principal component of A-aO2, plateau pressure, PAP, SaO2, and RR. To
stabilize the variance and satisfy the normality assumption, all adrenal (aldosterone, cortisol) and
cytokine (IL-6, IL-10) measures were analyzed using log10 transformation. Linear mixed models
(SAS PROC Mixed) were used to compare the effects of DEX and DOC given prophylactically
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vs. therapeutically to account for the actual pairing of animals within each week. Standard
residual diagnostics were used to check model assumptions. Data points with absolute
standardized residuals larger than 3 were considered potential outliers.
To determine the infection probabilities between treatments, we compared the average
probability of a positive test by analyzing culture results using logistic regression, with
Generalized Estimating Equations (GEE; SAS PROC GENMOD) to handle the correlation
induced by multiple tests at each time and multiple times for each animal. Time (0-10 h, 24 h,
48-96 h) was included in the model to account for the possibility of changing infection rates over
time.
SAS version 9.2 (Cary, NC) was used for all analyses except those noted above (i.e.
StatXact). All p-values are two-tailed and considered significant if p ≤ 0.05. We report
interactions based on two-tailed p-values as large as p = 0.06 to limit type II errors.
Supplementary Results
Effects of DOC and DEX Given Prophylactically or Therapeutically on White Blood Cell and
Platelet Counts, Temperature, and Lactate
White Blood Cell and Platelet Counts: DEX-T-treated animals at 10 h and DEX-P-treated
animals at 24 h had higher white blood cell counts compared to controls (4.7  1.4 vs. 1.6  0.3,
p = 0.007; and 7.3  2.9 vs. 1.8  0.6 x103/mm3, p = 0.01, respectively). Also at 10 h after
infection, DOC-P-treated animals had lower mean platelet counts than DOC-T-treated animals,
whereas DEX-P-treated animals had higher mean platelet counts than DEX-P-treated animals (p
= 0.02 for interaction; Table 1). In addition, at 48 h after S. aureus challenge, DEX-T-treated
animals had a higher mean platelet count than both controls and DEX-P-treated animals (282 
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51 vs. 144 27and 119  13 x103/mL; p = 0.03 and 0.01, respectively). Further, DOC-P-treated
animals had a higher mean platelet count at 72 h after infection than controls (233  33 vs. 102 
18 x103/mL; p = 0.02). There were no other significant differences in mean white blood cell or
platelet counts throughout the study (all, p = ns; data not shown).
Lactate: DOC-P-treated animals had minimally higher mean serum lactate concentrations
than DOC-T at 10 h, whereas DEX-P-treated animals had more markedly increased mean serum
lactate levels compared to DEX-T at this time point (p = 0.03 for quantitative interaction; see
Table 1). DOC-P-treated animals also had lower mean lactate concentration compared to
controls at 24 h (0.73 0.11 vs. 1.31  0.19 mmol/L, p = 0.03). There were no other significant
differences in mean serum lactate concentrations throughout the study (all, p = ns; data not
shown).
Body Temperature: At 10 h after S. aureus challenge DEX-P-treated animals had higher
mean body temperatures compared to both controls and DEX-T-treated animals [37.7  0.2 vs.
37.2  0.1 (p = 0.05) and 37.0  0.3 °C (p = 0.02), respectively]. DEX-P-treated animals also had
higher mean body temperature compared to DEX-T-treated animals at 24 h (38.2 0.4 vs. 37.3 
0.2 °C, p = 0.03). There were no other significant differences in mean body temperature
throughout the study (all, p = ns; data not shown).
Effects on Cytokines (IL-6 and IL-10)
IL-6: DOC-P reduced mean IL-6 concentrations at 10 h after infection compared to
DOC-T, whereas DEX-P increased IL-6 concentrations compared to DEX-T-treated animals at
this time point (p = 0.04 for interaction; Table 1). In addition, DOC-P-treated animals had
significantly lower mean IL-6 concentrations compared to controls 24 h after S. aureus challenge
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(3.42  0.17 vs. 3.96  0.17 pmol/L; p = 0.03). There were no other significant differences in
mean IL-6 concentrations through the study (all, p = ns; data not shown).
IL-10: DEX-T lowered mean IL-10 concentrations compared to DEX-P-treated animals
(0.99  0.32 vs. 1.61  0.10 pmol/L; p = 0.01) 10 h after infectious challenge. At 24 h after
infection both DEX-P- and DEX-T-treated animals had lower mean IL-10 concentrations than
controls (1.00  0.26 and 0.92  0.29 vs. 1.51  0.14 pmol/L, respectively; p = 0.05 for both).
There were no other significant differences in mean IL-10 concentrations throughout the study
(all, p = ns; data not shown).
Supplementary References
1. Minneci PC, Deans KJ, Hansen B, et al: A canine model of septic shock: Balancing animal
welfare and scientific relevance. Am J Physiol Heart Circ Physiol 2007;293:H2487-500
2. Kintzer PP, Peterson ME: Primary and secondary canine hypoadrenocorticism. Vet Clin North
Am Small Anim Pract 1997;27:349-357
3. Lynn RC, Feldman EC, Nelson RW: Efficacy of microcrystalline desoxycorticosterone
pivalate for treatment of hypoadrenocorticism in dogs. DOCP clinical study group. J Am Vet
Med Assoc 1993;202:392-396
4. Van Zyl M, Hyman WB: Desoxycorticosterone pivalate in the management of canine primary
hypoadrenocorticism. J S Afr Vet Assoc 1994;65:125-129
5. Reine NJ, Hohenhaus AE, Peterson ME, et al: Deoxycorticosterone-secreting adrenocortical
carcinoma in a dog. J Vet Intern Med 1999;13:386-390
6. Kintzer PP, Peterson ME: Treatment and long-term follow-up of 205 dogs with
hypoadrenocorticism. J Vet Intern Med 1997;11:43-49
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7. Minneci PC, Deans KJ, Banks SM, et al: Meta-analysis: The effect of steroids on survival and
shock during sepsis depends on the dose. Ann Intern Med 2004;141:47-56
8. Kemppainen RJ, Peterson ME, Sartin JL: Plasma free cortisol concentrations in dogs with
hyperadrenocorticism. Am J Vet Res 1991;52:682-686
9. Behrend EN, Weigand CM, Whitley EM, et al: Corticosterone- and aldosterone-secreting
adrenocortical tumor in a dog. J Am Vet Med Assoc 2005;226:1662-6, 1659