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Research Paper
Arandomized,double-blind,placebo-controlled
study of daily cannabidiol for the treatment of
canine osteoarthritis pain
Chris D. Verrico
a,b
, Shonda Wesson
c
, Vanaja Konduri
d
, Colby J. Hofferek
d
, Jonathan Vazquez-Perez
d
, Emek Blair
e
,
Kenneth Dunner Jr
f
, Pedram Salimpour
g
, William K. Decker
d,h,i
, Matthew M. Halpert
d,
*
Abstract
Over the last 2 decades, affirmative diagnoses of osteoarthritis (OA) in the United States have tripled due to increasing rates of
obesity and an aging population. Hemp-derived cannabidiol (CBD) is the major nontetrahydrocannabinol component of cannabis
and has been promoted as a potential treatment for a wide variety of disparate inflammatory conditions. Here, we evaluated CBD for
its ability to modulate the production of proinflammatory cytokines in vitro and in murine models of induced inflammation and further
validated the ability of a liposomal formulation to increase bioavailability in mice and in humans. Subsequently, the therapeutic
potential of both naked and liposomally encapsulated CBD was explored in a 4-week, randomized placebo-controlled, double-
blinded study in a spontaneous canine model of OA. In vitro and in mouse models, CBD significantly attenuated the production of
proinflammatory cytokines IL-6 and TNF-awhile elevating levels of anti-inflammatory IL-10. In the veterinary study, CBD significantly
decreased pain and increased mobility in a dose-dependent fashion among animals with an affirmative diagnosis of OA. Liposomal
CBD (20 mg/day) was as effective as the highest dose of nonliposomal CBD (50 mg/day) in improving clinical outcomes. Hematocrit,
comprehensive metabolic profile, and clinical chemistry indicated no significant detrimental impact of CBD administration over the 4-
week analysis period. This study supports the safety and therapeutic potential of hemp-derived CBD for relieving arthritic pain and
suggests follow-up investigations in humans are warranted.
Keywords: Osteoarthritis, Cannabidiol, Randomized trial, Liposomal encapsulation, TNF-a, IL-6
1. Background
Arthritis is a leading cause of pain, disfigurement, and disability in
the United States where nearly one-quarter of all adults have
received an affirmative diagnosis.
2
Although the incidence of
rheumatoid arthritis has remained constant, osteoarthritis (OA)
diagnoses have tripled since 2000 due to an aging population,
increasing levels of obesity, and greater physician recognition of
its prevalence. Accordingly, OA is a leading cause of chronic pain
and disability among the elderly.
23
Irrespective of the precipitating
cause, the pathology of joint destruction in arthritis is driven by an
overlapping profile of pathologic inflammatory cytokines including
TNF-a, IL-1b, IL-6, IL-17, and IL-21.
26,46,49
In addition, pain,
inflammation, and joint destruction among both etiologies are
mediated by overlapping subsets of innate cell types, most
prominently neutrophils.
16,45
Treatment of rheumatoid arthritis
consists of both targeted and nonspecific immunosuppressive
drug regimens (disease-modifying antirheumatic drugs), whereas
treatment of OA consists of analgesics, nonsteroidal anti-
inflammatory drugs, glucocorticoids, and joint replacement
supplemented by a weight loss regimen, if applicable. In either
case, pharmacomodulation is not curative and often accompa-
nied by severe side-effects.
6,9,36
Because pain is the pre-
dominant symptom of OA, it is also the primary target of
intervention. Recent reviews comparing the efficacy of pharma-
cotherapies for reducing OA pain conclude opioids are most
effective, however, abuse potential limits utility. Overall, the effect
size across all pharmacotherapies is small (0.39), signaling a need
for additional treatments with novel and complementary mech-
anisms of action.
1,32,54,61
The ubiquitous endocannabinoid system plays a role in many
physiological and pathophysiological processes. Consistent with
this, cannabis and its constituents are increasingly being
recognized as bona fide pharmacologic agents with significant
therapeutic potential. For example, cannabidiol (CBD), the major
nontetrahydrocannabinol (THC) constituent of cannabis, can
exert numerous biological effects through several different
receptors and signaling pathways, including anti-inflammatory
Sponsorships or competing interests that may be relevant to content are disclosed
at the end of this article.
C.D. Verrico, S. Wesson, W.K. Decker, and M.M. Halpert contributed equally to the
preparation of this manuscript.
a
Department of Psychiatry, Baylor College of Medicine, Houston, TX, United
States,
b
Department of Pharmacology, Baylor College of Medicine, Houston, TX,
United States,
c
Sunset Animal Hospital, Houston, TX, United States,
d
Department
of Pathology and Immunology, Center for Cell and Gene Therapy,
e
Valimenta Labs,
Fort Collins, CO, United States,
f
Department of Cancer Biology, University of Texas
MD Anderson Cancer Center, Houston, TX, United States,
g
Boston University
School of Medicine, Boston, MA, United States,
h
Center for Cell and Gene Therapy,
i
Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine,
Houston, TX, United States
*Corresponding author. Address: Tel.: 713-798-1560; fax: 713-798-3700. E-mail
address: halpert@bcm.edu (M.H. Halpert).
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 Web site (www.painjournalonline.com).
PAIN 00 (2020) 1–12
©2020 International Association for the Study of Pain
http://dx.doi.org/10.1097/j.pain.0000000000001896
Month 2020·Volume 00 ·Number 00 www.painjournalonline.com 1
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
effects in both acute and chronic conditions.
10–13,20,25,43,47,51,57
Indeed, preclinical rodent models suggest the therapeutic
potential of CBD in combating the underlying causes of both
rheumatoid arthritis and OA.
15,35,37,48
Although preclinical rodent models have provided evidence of
efficacy for novel compounds to treat pain,
8,19,29
the clinical
efficacy or safety of these compounds in human studies has been
unsatisfying.
58,59
The late-stage failures of promising compounds
in randomized studies have suggested a disconnect between the
preclinical models used to study structural vs symptomatic
aspects of disease.
17
Indeed, the initiating event and many of the
pathological changes in the commonly used, chemically induced
preclinical rodent models of chronic OA pain are not typical of
human OA.
17,56
By contrast, spontaneous models, particularly
domesticated canine models, are more appropriate for assessing
OA pain treatments because they closely mimic the pathophys-
iology and pathogenesis of human OA pain.
17
In the present
work, we determined the in vitro and in vivo effects of CBD on
expression levels of shared, pathologic proinflammatory cyto-
kines, and innate cell subsets in multiple model systems.
Subsequently, the safety and efficacy of CBD were evaluated in
a double-blind, placebo-controlled study in a spontaneous
canine model.
2. Methods
2.1. Cannabidiol
Cannabidiol, provided by MedterraCBD (Irvine, CA), was isolated
solely from hemp grown and extracted under the strict guidelines
of the Kentucky Department of Agricultural Industrial Hemp pilot
program. Subsequent analysis by third party (ProVerde Labora-
tories, Milford, MA) mass spectrometry confirmed the absence of
D9-THC, other cannabinoid derivatives, and contaminants while
further HPLC testing demonstrated CBD isolate purity of 99.9%.
For all assays, CBD was solubilized in fractionated coconut oil.
Liposomal CBD was produced using a sunflower lecithin
(phosphatidylcholine) base. Each liposome was approximately
100 nm, allowing for encapsulation of 10 to 20 mg/mL CBD.
Transmission electron microscopy was used to observe and
confirm the stability of liposomal CBD concentration, size, and
polydispersity after storage at 4˚C for at least 3 months. Briefly,
samples were placed on 150 mesh formvar-coated copper grids
treated with poly-l-lysine for approximately 1 hour, then negatively
stained with filtered aqueous 2% ammonium molybdate 10.02%
BSA, pH 7.0 for 1 minute. Stain was blotted dry from the grids
with filter paper, and samples were allowed to dry. Samples were
then examined in a JEM 1010 transmission electron microscope
(JEOL USA, Peabody, MA) at an accelerating voltage of 80 kV.
Digital images were obtained using the AMT Imaging System
(Advanced Microscopy Techniques Corp, Danvers, MA).
2.2. Cell culture
Mouse RAW267.4 macrophage cells (ATCC, Manasas, VA),
primary mouse splenocytes, human monocytic THP-1 cells
(ATCC), and human PBMC were plated in a single well of a 6-
well plate in 5-mL RPMI (Invitrogen, Carlsbad, CA) medium
supplemented with 10% fetal bovine serum at 5% CO
2
in a 37˚C
humidified incubator for either 2 (lipopolysaccharide [LPS]) or 4
hours (staphylococcal enterotoxin B [SEB]) before addition of
CBD. Lipopolysaccharide and SEB concentrations used were
determined by previous publications and/or empirical testing in
cell culture. TNF-⍺levels in cell culture supernatants were
determined using the TNF Flex Set immunoassay (BD Bioscien-
ces, San Jose, CA) as measured by an LSR II or Canto Violet flow
cytometer (BD Biosciences) and analyzed with FlowJo version
10.0.00003 (Tree Star, Inc, Ashland, OR). All points were assayed
in triplicate with at least 3 independent repetitions unless stated
otherwise.
2.3. Mice
Approximately 342 female, 6- to 10-week-old C57BL/6 J mice
with a weight range of 18 to 27 g were procured from Baylor
College of Medicine or the Jackson Laboratory (Bar Harbor, ME)
and maintained in accordance with the specific IACUC require-
ments of Baylor College of Medicine and in accordance with
animal protocol AN-7942. Mice were housed under controlled
standard conditions (23 61˚C, 55 610% humidity and a 12-hour
light/dark cycle) and provided standard laboratory chow and
autoclaved water ad libitum.
2.4. Croton oil-induced ear inflammation model
All experiments were conducted between 10 AM and 3 PM, to
avoid the influence of circadian variations in corticosteroid levels
in the murine inflammatory response. Croton oil (2.5% in acetone)
was topically applied (100 mL) to the right ear. Two hours after
croton oil was applied, vehicle or 100 mL of 10 mg/mL CBD oil
was topically applied to swollen and control ears. Two hours after
these treatments, ear tissue samples were collected to determine
myeloperoxidase (MPO) activity, and blood samples were
collected by retrobleed to determine circulating TNF-⍺levels.
2.5. Lipopolysaccharide-induced inflammation model
Lipopolysaccharide (200 ng) was administered intraperitoneally.
Two hours after LPS administration, mice were injected in-
traperitoneally with CBD (1, 10, or 100 mg) or administered either
CBD (100 mg) or 18.3% methyl salicylate/16% menthol (Ben-Gay;
Johnson & Johnson, New Brunswick, NJ) topically at the LPS
injection site. Two hours after treatments, blood samples were
collected by retro-orbital bleed to determine cytokine and
neutrophil levels.
2.6. Tissue myeloperoxidase activity
In brief, ear tissue samples (4-mm punch) collected at 1, 2, 3, or 4
hours after croton oil was applied were homogenized in MPO
assay buffer (Abcam, Cambridge, MA) per the manufacturer’s
instructions. Samples and MPO assay buffer were equilibrated to
room temperature before use, and samples were diluted 1:5 in
assay buffer. Groups were assayed in triplicate in individual wells
in 50 mL of reaction mix for 2 hours at room temperature before
addition of 2 mL of stop mixture. Subsequently, 50 mL of TMB
developer substrate was added and incubated for 10 minutes,
and the output was measured by spectrophotometry at
OD
412
nm.
2.7. Cytokine and neutrophil analysis
Mice were bled retro-orbitally at specified intervals. Blood
samples were mixed with 0.5M EDTA to prevent clotting then
pelleted to extract the serum. Red blood cells in the cell pellet
were lysed by suspension in ammonium chloride (Sigma-Aldrich,
St. Louis, MO) per the manufacturer’s instructions. The remaining
white blood cells were then stained for neutrophils by CD45-
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
2C.D. Verrico et al.·00 (2020) 1–12 PAIN
®
APC-Cy7, CD11b-APC, Ly6G-FITC, and CD115-PE (all from
BioLegend, San Diego, CA) before analysis by flow cytometry.
The serum was subsequently analyzed for various cytokines
using the BD flex set (BD Biosciences). In brief, sera were diluted
with supplied buffer per manufacturer’s instructions, incubated
with the appropriate capture antibody/bead for 1 hour at room
temperature, incubated with the detection antibody/bead for
another hour at room temperature, washed, centrifuged,
resuspended in flow buffer, and analyzed by flow cytometry.
2.8. Bioluminescence imaging
Mice were subcutaneously injected with 5 310
5
luc2
1
KRAS
tumor cells near the hindquarters 24 hours before experimenta-
tion. Subsequently, mice were subcutaneously injected with 100
mL either 10-mg/mL naked D-luciferin (Regis Technologies,
Morton Grove, IL) or 10 mg/mL liposomally encapsulated D-
luciferin near the forequarters on the ipsilateral side. Mice were
then analyzed continuously by IVIS imaging (Caliper Life
Sciences, Waltham, MA) for 2 hours.
2.9. Human subjects bioavailability trial design
A longitudinal crossover study to compare the bioavailability of
liposomal vs naked CBD was approved and performed under an
IRB-approved protocol under the auspices and guidance of the
Institute for Regenerative and Cellular Medicine (IRCM, Santa
Monica, CA). After provision of informed consent, subjects were
randomized regarding the order of which to receive an isolate of
either naked CBD or liposomally encapsulated CBD. At first study
visit, peripheral blood was drawn after overnight fasting to
measure the baseline CBD blood levels. Subjects then orally
ingested an amount of isolate equivalent to 10 mg CBD in either
naked or liposomally encapsulated form. One hour after the
product was ingested, a second blood draw was taken to
determine circulating levels of CBD. Two weeks later at the
second study visit, the same procedure was followed with the
exception that the study subject was administered the converse
form of delivery not received at the first study visit. Subjects were
eligible for inclusion if (1) between the ages of 25 and 70, (2) able
to read and sign the informed consent and stay compliant with
study requirements and schedule, (3) not taking any other CBD
product concurrently, and (4) in good general health. Patients
with terminal illnesses were prohibited from study participation.
Bioavailability ratio of liposomally encapsulated CBD to naked
CBD administration was calculated using an LOQ value of 0.05
ng/mL (limit of detection) if naked CBD administration produced
undetectable levels of circulating CBD.
2.10. Osteoarthritis veterinary trial design
Canine veterinary studies were performed with oversight as
stipulated by Baylor College of Medicine IACUC protocol AN-
7705. The study population consisted of client-owned dogs
presenting to Sunset Animal Hospital (Houston, TX) for evaluation
and treatment of lameness due to OA. Owners completed a brief
questionnaire to define the affected limb(s), duration of lameness,
and duration of analgesic or other medications taken. Dogs were
considered for inclusion in the study if they (1) received an
affirmative diagnosed of OA by a veterinarian and (2) demon-
strated signs of pain according to assessment by their owners,
detectable lameness on visual gait assessment, and painful
joint(s) upon palpation. Complete blood count (CBC) and serum
chemistry were performed at presentation to rule out other
underlying disease. Dogs were excluded by the attending study
veterinarian if they exhibited evidence of uncontrolled renal,
endocrine, neurologic, or neoplastic disease or were undergoing
physical therapy. No cases of OA were related to trauma, and no
animals with end-stage disease were enrolled. All other medi-
cations were discontinued at least 2 weeks before enrollment,
and dogs were not allowed to receive any medications during the
4-week study period except the study medication. Large (.20
kg, mean 41 615 kg) domestic canines were enrolled in the 4-
week, randomized placebo-controlled trial in which both owner
and veterinarian were blinded. After provision of informed owner
consent, 20 study subjects were randomly assigned 1:1:1:1 to 1
of 4 groups: placebo, 20 mg/day (0.5 mg/kg) naked CBD, 50 mg/
day (1.2 mg/kg) naked CBD, or 20 mg/day liposomal CBD.
Simple randomization was achieved by providing the blinded
study drug regimens to the veterinary investigator in a randomized
numerical order labeled 1 to 20 as assigned by the rolling of a die.
After randomization, aggregate average weight of each study
group remained within one SD of all other study groups. Blood
was collected for CBC and clinical chemistry at initiation and at
day 30 of treatment. Before treatment initiation and at day 30,
each dog was evaluated by the study veterinarian who assessed
locomotion because it related to walking, running, and assuming
a standing position from both a sitting and lying down position on
a 5-point scale (1 5best) during physical examination. Owners
also evaluated dogs before treatment and at weeks 4 and 6 using
the Helsinki Chronic Pain Index, a validated, 11-item assessment
of treatment response in dogs with OA pain scored ordinally on
a scale from 0 to 4.
18
2.11. Statistical analysis
Data are expressed as the mean 6SD unless otherwise
specified. Student’s t-test was used for pairwise comparisons,
and one-way analysis of variance followed by post hoc Tukey–
Kramer was used for analysis of multiple comparisons. Normality
of data was determined by Q-Q plot. Statistical significance was
defined as P,0.05 unless stated otherwise. Sample sizes for
mouse, canine, and human experiments were based on power
analysis indicating that a difference in mean value (Dm) as small as
0.25-fold could be detected with a power of 0.8 and type I error
rate (a) of 0.05 with a sample size of 4 subjects assuming a SD (s)
of 0.33. Given this calculation, we chose a sample size of 5
subjects for all experimental groups to permit even greater
statistical discernment power (,Dm of 25%) and/or to accom-
modate greater variance (s.1/3 SD) between groups.
3. Results
3.1. Cannabidiol reduces proinflammatory TNF-asecretion
in vitro
It has been widely reported that CBD possesses significant anti-
inflammatory properties in a variety of different experimental
systems.
27
To validate that the CBD used for these studies might
potentiate anti-inflammatory effects relevant to arthritis, 2
different inflammatory stimuli were applied to 4 different relevant
cell populations including a mouse monocyte cell line, a human
monocyte cell line, primary mouse PBMC, and primary human
PBMC. As illustrated in Figure 1, both LPS and SEB induced log-
fold elevations in TNF-asecretion in comparison with untreated
or CBD-only treated controls from RAW267.4 mouse cells,
primary mouse PBMC, THP-1 human cells, and primary human
PBMC. However, concurrent application of 100 ng/mL CBD in
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
Month 2020·Volume 00 ·Number 00 www.painjournalonline.com 3
conjunction with LPS treatment induced a 42% (primary human
PBMC) to 97% (human THP-1 cells) reduction in TNF-a
secretion. Similarly, concurrent application of 100 ng/mL CBD
in conjunction with SEB treatment induced a 55% (RAW267.4
mouse cells) to 63% (human THP-1 cells) reduction in TNF-a
secretion (Figs. 1A–D,*P,0.05 or **P,0.01).
3.2. Cannabidiol induces broad anti-inflammatory effects
in vivo
Encouraged by the in vitro data, we next used 2 different mouse
inflammatory models to analyze the impact of CBD on local and
systemic inflammation in vivo. We first used the croton oil model in
which topical administration of croton oil to the ear of a mouse
induces an inflammatory reaction that includes edema, erythema,
neutrophil influx, and the production of proinflammatory TNF-⍺.
28
Two hours after application of 2.5% croton oil 6topical
application of 1 mg CBD, local MPO activity (a proxy for neutrophil
influx) was measured. As indicated in Figure 1E, MPO activity in
the treated ear was reduced over 80% (*P,0.05) with
concurrent application of CBD. Four hours after croton oil
application, levels of circulating TNF-awere assessed. As shown
in Figure 1F, circulating TNF-awas decreased by 50% among
mice to which croton oil 1CBD had been applied in comparison
with croton oil alone (*P,0.05). Cannabidiol treatment also
significantly reduced the development of edema.
Figure 1. CBD reduces hallmarks of arthritis-related inflammation in vitro. A total of 5 310
6
cells of the specified type were plated in triplicate in 6-well plates in 5-
mL RPMI 110% FBS followed by addition of either 1-ng/mL LPS for 4 hours or 100-ng/mL SEB for 6 hours with or without the addition of 100-ng/mL CBD after 2
hours. After the incubation period, the media were analyzed using the BD TNF-⍺Flex set. (A) TNF-⍺levels in murine RAW267.4 macrophage cell line. (B) TNF-⍺
levels in primary mouse splenocytes. (C) TNF-⍺levels in human THP-1 monocyte cell line. (D) TNF-⍺levels in primary human PBMC. Representative experiment of
3 shown. Error bars 6SD. *P,0.05, **,0.01 by Student’s two-tailed ttest for all (A2D). Cohorts of female mice were also treated on one ear with 100-mL 2%
croton oil-acetone, and ear edema was allowed to occur for 1 to 4 hours. At 2 hours, mice were treated on the swollen ear with either 100 mL of vehicle or 100 mLof
10-mg/mL CBD oil. In addition, a group of untreated mice also received 100-mL CBD oil (E). At each time point indicated, 4-mm biopsies from the most central
portion of swelling were obtained, homogenized, and measured for myeloperoxidase (MPO) activity by ELISA. (F) After 4 hours, each cohort was retro-orbitally bled
for analysis of circulating TNF-⍺concentrations using the BD TNF⍺Flex set. Each cohort consisted of n 55 mice. Representative experiment of 3 shown. Error
bars 6SD. *P,0.05 by Student’s two-tailed ttest. CBD, cannabidiol; LPS, lipopolysaccharide.
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
4C.D. Verrico et al.·00 (2020) 1–12 PAIN
®
When administered intraperitoneally, LPS induces an inflam-
matory response that includes increased expression of proin-
flammatory TNF-aand IL-6, 2 cytokines relevant to the
pathogenesis of arthritis. In this model system, 200 ng of LPS
was administered intraperitoneally. Two hours later, mice were
then treated intraperitoneally with increasing doses of CBD (1, 10,
or 100 mg) or topically with a single CBD dose of 100 mg. After an
additional 2 hours, the impact of CBD treatment on circulating
cytokine levels was assessed. As indicated, intraperitoneal
administration of CBD reduced circulating TNF-aand IL-6 levels
in a dose-responsive fashion, and 100 mg of topically applied
CBD generated an anti-inflammatory effect similar to that of 100
mg injected intraperitoneally (Fig. 2A/B). Interestingly, systemic
administration of CBD alone increased levels of anti-inflammatory
IL-10 in the absence of inflammatory stimulus, an effect that was
significantly potentiated in the presence of LPS (Fig. 2C).
Application of 18.3% methyl salicylate/16% menthol (Ben-Gay)
made no significant impact on any of these cytokine
concentrations. In contrast to alterations in proinflammatory
and anti-inflammatory cytokine levels, significant changes to
circulating neutrophil chemoattractants CXCL1 (KC) and CXCL2
(MIP-2) were not observed (Fig. 2D/E). Nonetheless, circulating
neutrophil levels were reduced up to 60% among LPS-treated
mice to which CBD had been administered (Fig. 2F).
3.3. Liposomal packaging of cannabidiol enhances
bioavailability in vivo
Although CBD clearly displayed a role in regulating inflammation
in both in vitro and in vivo murine models, the relatively low
bioavailability of this hydrophobic molecule when administered
orally may reduce its effectiveness. To potentially improve
absorption of hydrophobic CBD isolate, we packaged it within
liposomes, a vehicle delivery system previously shown to improve
uptake of other hydrophobic compounds.
30
Using sunflower
lecithin as a base, phosphatidylcholine liposomes approximately
Figure 2. Intraperitoneal CBD administration reduces inflammatory cytokines and circulating neutrophils in an in vivo LPS inflammatory model. Cohorts of female
mice were treated intraperitoneally with 200-ng LPS for 2 hours and subsequently administered intraperitoneal CBD, intraperitoneal PBS control, topical CBD, or
topical Ben-Gay control as indicated. After an additional 2 hours, mice were retro-orbitally bled and circulating cytokines were analyzed with the appropriate BD
Flex set. (A) TNF-a. (B) IL-6. (C) IL-10. (D) CXCL1. (E) CXCL2. (F) Flow cytometry analysis of the cellular portion was performed each hour to determine relative
number of neutrophils (CD115
neg
CD11b
1
Ly6G
1
) in circulation. Representative experiment shown. Error bars 6SD. *P,0.05, **,0.01 by one-way ANOVA.
CBD, cannabidiol; LPS, lipopolysaccharide.
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
Month 2020·Volume 00 ·Number 00 www.painjournalonline.com 5
100 nm in diameter and loaded with 10 to 20 mg/mL CBD were
produced. Electron microscopy demonstrated that liposomal
CBD was stable at both room temperature and 4˚C and between
pH 5 to 9 for a period of 3 months (Fig. 3A).
To compare the bioavailability of molecules encapsulated
within this liposomal containment system to that of naked
molecules, we developed a proof-of-principle system using
liposomally encapsulated D-luciferin and a luciferase-
expressing tumor cell line. In this assay system, 5 310
5
luc2
1
tumor cells were implanted subcutaneously near the hindquarters
of C57BL/6 mice. Twenty-four hours later, D-luciferin (100 mL@
10 mg/mL) or liposomally encapsulated D-luciferin (100 mL @ 10
mg/mL) were administered subcutaneously near the forequar-
ters. Luminescence was then monitored for a continuous 2-hour
period by IVIS with post hoc photon measurement at the target
serving as a proxy for substrate absorption into circulation and
bioavailability. As shown in Figures 3B and C, liposomal
packaging of D-luciferin significantly enhanced both the speed
and magnitude at which this substrate was able to reach the
tumor site and induce photon emission, resulting in a full log-fold
enhancement of peak emissions at 60 minutes after D-luciferin
administration (*P,0.05, **P,0.01 for time points indicated).
Next, using the LPS acute inflammatory model, 200-ng LPS was
administered intraperitoneally, and circulating TNF-⍺was
assayed every 30 minutes and plotted as a percentage of
preadministration TNF-⍺. Two hours after introduction of
LPS, mice were orally gavaged with 100-mL 10-mg/mL CBD,
10-mg/mL liposomal CBD, 10-mg/mL liposomal D-luciferin, or
PBS. As shown in Figure 3D, orally administered liposomal CBD
began to significantly reduce rising TNF-alevels within an hour of
administration, whereas an additional hour was required before
orally administered naked CBD significantly reduced rising TNF-a
levels in comparison with negative controls. Moreover, although
both naked and liposomally encapsulated CBD administered
orally significantly reduced relative levels of circulating TNF-⍺
below those of the negative controls, liposomally encapsulated
CBD made a significantly greater impact on such levels (*P,
0.05, **P,0.01 at 4 hours after CBD administration).
Encouraged by these data, we sought to validate enhanced
bioavailability of liposomally encapsulated CBD in healthy human
volunteers under the auspices of an IRB-approved and monitored
human crossover study. In brief, after provision of informed
consent, healthy human volunteers were randomized to receive
10-mg oral CBD in either a naked or liposomally encapsulated
formulation. Circulating CBD levels were determined from
preadministration and 1-hour postadministration blood draws.
At a second study visit, this procedure was repeated in the same
volunteer using the converse delivery method (ie, naked vs
liposomally encapsulated), and a bioavailability ratio was calcu-
lated. For instances in which naked CBD administration pro-
duced undetectable levels of circulating CBD, the bioavailability
ratio was calculated using an LOQ value of 0.05 ng/mL (limit of
detection). Among the 5 study volunteers for whom data were
available, the bioavailability of liposomally encapsulated CBD was
17.1 616-fold greater than that of naked CBD at 1-hour
postadministration (*P,0.05). Furthermore, although 2 of 5
Figure 3. Liposomal encapsulation of small molecules enhances bioavailability. Sunflower lecithin (phosphatidylcholine) was used as a base to make liposomes
approximately 100 nm in size that could encapsulate small molecules at a concentration of 10 to 20 mg/mL and retain pol ydispersity and size for at least 3 months
at 4˚C. (A) Stable size and polydispersity observed by transmission electron microscopy (TEM). (B) Cohorts of mice were implanted subcutaneously injected with
500,000 luc2
1
cells near the hindquarters. (B/C) Twenty-four hours later, D-luciferin (100 mL, 10 mg/mL) or D-luciferin liposomes (100 mL, 10 mg/mL) were applied
subcutaneously near the forequarters, and animals were continually imaged by IVIS for 2 hours with subsequent photon measurement at the target serving as
a proxy for absorption and bioavailability. (D) The ability of liposomal CBD to reduce TNF-⍺production relative to controls and naked CBD was determined. For B
and C, n 55 mice per cohort. Representative experiment of 3 shown. For D, n 58 mice per cohort. Representative experiment of 2 shown. Error bars 6SD. *P,
0.05, **P,0.01 by Student’s two-tailed ttest. CBD, cannabidiol.
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
6C.D. Verrico et al.·00 (2020) 1–12 PAIN
®
subjects exhibited undetectable circulating CBD levels after oral
administration of naked CBD isolate, all 5 subjects exhibited
detectable levels of circulating CBD levels after oral administration
of liposomally encapsulated CBD (Table 1).
3.4. Short-term administration of cannabidiol to domestic
canines diagnosed with osteoarthritis is safe and improves
quality of life
Although there exists a variety of different preclinical mouse
models of arthritis, as noted, these model chemical and
pathologic features of the disease have been poorly predictive
in determining symptomatic or therapeutic responses.
17
In an
effort to better model treatment efficacy, we conducted
a randomized, double-blind, placebo-controlled trial among
large (.20 kg; mean 541 615 kg) outbred canines with an
affirmative veterinary diagnosis of OA and experiencing de-
creased mobility and quality of life. After diagnosis and provision
of owner informed consent, animals were enrolled and randomly
provided with identical medication bottles which contained one
of 4 treatments including 10-mg/mL naked CBD, 25-mg/mL
naked CBD, 10-mg/mL liposomal CBD, or a placebo consisting
only of fractionated coconut oil. Baseline and day 30 CBC and
metabolic panel as well as alanine aminotransferase (ALT) and
alkaline phosphatase (ALKP) were also determined. Sympto-
mology was assessed by the attending study veterinarian
through clinical examination on days 0 and 30 and by each
animal’s owner on study days 0, 30, and 45 using the Helsinki
Chronic Pain Index assessment.
18
Characteristics of each
enrolled animal are provided in Supplementary Table 1 (available
at http://links.lww.com/PAIN/B3). As shown in Figure 4A/B,
owner assessment of animal symptomology was not signifi-
cantly altered by administration of placebo or 20-mg/day naked
CBD; however, administration of 50-mg/day naked CBD or 20-
mg/day liposomal CBD generated statistically significant reduc-
tions in pain symptomology (**P,0.01), an effect that remained
statistically significant (*P,0.05) for at least 15 days after
cessation of therapy. With some variability, veterinarian clinical
examination largely matched that of the owner’s assessment
with generally no improvements observed among animals
administered placebo or 20-mg/day naked CBD, and significant
improvements noted among all 4 assessment categories (sitting
to standing, lying to standing, walking, and running) among
dogs who received 50-mg/day naked CBD and 20-mg/day
liposomal CBD as evidenced by group compilation raw
assessment scores (Figs. 5A–D)orasecondaryanalysis(Fig.
6) that considered only whether symptomology in a given study
participant worsened, remained the same, or improved over the
course of therapy (*P,0.05, **P,0.01, ****P,0.001). No sex
differences with regard to treatment efficacy were observed,
and there were no significant alterations to CBC, metabolic
panel, or ALT/ALKP values over the course of the study in any
group (Figs. 7A–C and Table 2).
4. Discussion
Arthritis is a painful degenerative condition that impacts the lives
of almost a quarter of all Americans, with OA in particular
accounting for 60% of all-cause arthritis diagnoses.
2,23
Be-
cause current treatment regimens are not curative and can be
accompanied by significant comorbidities,
6,9,36
the present
studies were undertaken to validate whether the recently
legalized supplement CBD might positively impact the sympto-
mology of this degenerative condition. We first validated the
widely reported anti-inflammatory effects of CBD administration
both in vitro and in vivo, demonstrating substantial impact on
inflammatory cytokines and innate immune cell subsets relevant
to the pathophysiology of arthritis. After additional experimen-
tation that established greater bioavailability of liposomally
encapsulated vs naked CBD in both mice and humans, we
demonstrated the short-term clinical efficacy of CBD in
a double-blind, placebo-controlled veterinary study in which
neither owner nor veterinarian knew the content of the study
medications. In this study, neither animals given placebo nor
animals given a low daily dose of naked CBD responded to
therapy in any significant fashion. Conversely, animals given
a high dose of naked CBD or a low dose of liposomally
encapsulated CBD experienced significant improvements in
quality of life scores as documented by both owner and
veterinarian assessments. In this setting, administration of
CBD was not associated with any significant alterations to
circulating lymphocyte subsets, clinical chemistry values, or
assessed metabolic parameters.
In vitro and in vivo studies focused on important pathologic
mechanisms applicable to a wide variety of arthritis etiologies.
We found that CBD significantly reduced LPS- and SEB-
induced production of TNF-⍺in human and mouse cell lines
and PBMC, consistent with the results of previous stud-
ies.
3,51,60
Similarly, in a croton oil-induced murine model of
inflammation, we found that topical administration of CBD
significantly reduced TNF-⍺production and MPO activity, the
latter of which is consistent with previous reports of systemic
CBD administration in mice.
4,50
Consistent with previous in
vivo studies, we demonstrated that CBD also significantly
reduced LPS-induced proinflammatory cytokine
33,37
and
neutrophil production,
40
while increasing anti-inflammatory
IL-10 production in a dose-responsive fashion.
33
Given the
wide variety of grades, formulations, and suppliers of
commercially available CBD, it was important to validate and
characterize the functional activity of the CBD isolate planned
for use in subsequent veterinary studies. In those studies, the
finding that 50 mg/day of naked CBD-improved treatment
outcomes is consistent with a previous study in dogs with
OA
14
; however, this is the first report of a randomized, double-
blind, placebo-controlled study that uses a spontaneous
model for assessing the potential therapeutic effects of CBD
for treating OA pain and increasing quality of life. As in humans,
the pathogenesis of canine OA involves changes in all tissues
of the synovial joint.
5,24,31,34,38
The dominant symptom of OA
for both humans and dogs is pain, and the current therapeutic
goal for both species is management of that pain and
Table 1
Naked vs liposomally encapsulated circulating CBD levels in
healthy volunteers.
Subject Naked CBD Liposomal CBD Ratio
Pre
(ng/mL)
Post
(ng/mL)
Pre
(ng/mL)
Post
(ng/mL)
1 0.00 0.87 0.00 5.90 6.8
2 0.00 0.00 0.00 0.87 17.4
3 0.00 0.14 0.10 2.00 13.6
4 0.00 0.00 0.19 2.40 44.2
5 0.00 0.45 0.00 1.60 3.6
Averages 0.29 60.37 2.55 61.95 17.1 616.1
CBD, cannabidiol.
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
Month 2020·Volume 00 ·Number 00 www.painjournalonline.com 7
associated movement deficits.
7
Thus, an extrapolation of
these findings suggest that CBD could be useful for treating
pain and improving quality of life in humans with an affirmative
diagnosis of OA and/or other inflammatory conditions that
might be ameliorated by a reduction in proinflammatory
cytokines and pathologic neutrophil activity.
The absorption of CBD administered by smoking, vapor-
ization, buccal spray, or oral ingestion is highly variable and
results in extremely inconsistent pharmacokinetic profiles
when investigated.
21,39,42,44,52
Cannabidiol also shows limited
oral bioavailability due to poor aqueous solubility and extensive
first-pass metabolism.
22,41,55
Although the current study did
not assess pharmacokinetic parameters among canine study
participants, the effect of liposomal CBD on LPS-induced
TNF-aproduction in mice provides an objective measure of its
pharmacodynamic drug action and suggests a greater
Figure 4. Daily administration of CBD for 30 days improves owner-perspective quality of life scores among large dogs with affirmative diagnosis of osteoarthritis.
Twenty large domestic canines with affirmative diagnosis of osteoarthritis were enrolled in a double-blind, placebo-controlled randomized study.Animalswere
administered coconut oil placebo, 20-mg/day naked CBD, 50-mg/day naked CBD, or 20-mg/day liposomal CBD. Owners assessed their animals by means of the
Helsinki Chronic Pain Index (HPCI) on days 0, 30, and 45. (A) Individual HPCI values were plotted for each study cohort on days 0 and 30. (B) Cohort HPCI values
were plotted on days 0, 30, and 45. Error bars 6SD. *P,0.05, **P,0.01 by Student’s two-tailed ttest. CBD, cannabidiol.
Figure 5. Daily administration of CBD for 30 days improves veterinarian-perspective subset quality of life scores among large dogs with affirmative diagnosisof
osteoarthritis. Study enrolled canine subjects were scored by the (blinded) study veterinarian on days 0 and 30 using a scale of 1 (best) to 5 (worst) for 4 different
movements consisting of sitting to standing (A), lying to standing (B), walking (C), and running (D). Subset scale data comparing day 0 and day 30 scores for each
task are shown by cohort. Error bars 6SEM. *P,0.05, **P,0.01 by Student’s two-tailed ttest. CBD, cannabidiol.
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
8C.D. Verrico et al.·00 (2020) 1–12 PAIN
®
bioavailability than naked CBD. Although previous studies
regarding the bioavailability of liposomal CBD are not found in
the literature, a single study of D
9
-THC, the main psychoactive
constituent in cannabis, reported that liposomal encapsulation
improved bioavailability in rats in comparison with
administration of the naked molecule.
53
Based on these
animal studies, we performed an IRB-approved crossover
study in healthy human volunteers to validate approved
bioavailability of CBD after liposomal encapsulation. The data
demonstrated a 17-fold increase in bioavailable circulating
Figure 6. Daily administration of CBD for 30 days improves veterinarian-perspective overall quality of life scores among large dogs with affirmative diagnosis of
osteoarthritis. Study-enrolled canine subjects were scored by the (blinded) study veterinarian on days 0 and 30 using a scale of 1 (best) to 5 (worst) for 4 different
movements consisting of sitting to standing, lying to standing, walking, and running. Data are represented as pie charts indicating percent of each cohort that
showed improvement, worsening, or no change in condition for the animals enrolled in each study group. **P,0.01, ****P,0.001 by Pearson’s x
2
.CBD,
cannabidiol.
Figure 7. Daily administration of CBD for 30 days does not alter alanine aminotransferase (ALT) or alkaline phosphatase (ALKP) levels. Blood was drawn from
animals we enrolled in the clinical study on days 0 and 30, and Chem10 analysis was performed. (A) Relative changes in circulating ALT and ALKP values over the
30-day period. (B, C) Specific changes in circulating ALT and ALKP values over the 30-day period. Dark horizontal lines outline normal range. Error bars 6SD. No
statistically significant changes were observed. CBD, cannabidiol.
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
Month 2020·Volume 00 ·Number 00 www.painjournalonline.com 9
Table 2
Veterinary study CBC, metabolic panel, and clinical chemistry values.
Day 0 Day 30 Day 0 Day 30 Day 0 Day 30 Day 0 Day 30 Day 0 Day 30
WBC (K/uL) WBC (K/uL) Lymphocytes
(K/uL)
Lymphocytes
(K/uL)
Neutrophils
(K/uL)
Neutrophils
(K/uL)
Basophils
(K/uL)
Basophils
(K/uL)
Eosinophils
(K/uL)
Eosinophils
(K/uL)
Placebo 8.84 (62.52) 9.1 (62.01) 1.34 (60.51) 1.6 (60.68) 6.45 (62.25) 6.2 (61.75) 0.015 (60.007) 0.017 (60.009) 0.535 (60.38) 0.596 (60.24)
20 mg/day 9.496 (61.11) 10.168 (63.01) 1.486 (60.50) 1.628 (60.68) 6.954 (60.81) 7.446 (62.36) 0.014 (60.008) 0.024 (60.005) 0.4 (60.2) 0.564 (60.3)
20 mg lips/day 9.77 (61.71) 8.89 (61.35) 1.68 (60.73) 1.95 (60.82) 6.74 (61.75) 5.875 (61.31) 0.0225 (60.005) 0.0175 (60.003) 0.67 (60.45) 0.452 (60.25)
50 mg/day 8.39 (61.07) 9.25 (61.74) 1.72 (60.78) 1.91 (60.82) 5.44 (60.95) 5.99 (61.66) 0.0125 (60.003) 0.03 (60.01) 0.495 (60.19) 0.46 (60.38)
Day 0 Day 30 Day 0 Day 30 Day 0 Day 30 Day 0 Day 30
RBC (K/uL) RBC (K/uL) HCT (%) HCT (%) HGB (g/dL) HGB (g/dL) Platelets (K/uL) Platelets (K/uL)
Placebo 6.34 (61.01) 7.1 (61.15) 47.8 (66.25) 44.3 (69.1) 15.2 (62.34) 15.8 (62.1) 242.0 (647.2) 248.5 (642.2)
20 mg/day 7.71 (60.41) 7.79 (60.48) 50.36 (63.29) 50.46 (64.84) 17.2 (61.12) 17.1 (61.36) 253.4 (645.8) 259.8 (668.8)
20 mg lips/day 7.38 (60.88) 7.41 (60.73) 50.82 (64.2) 49.82 (65.12) 16.97 (61.76) 17.25 (61.43) 261.0 (642.1) 272.0 (623.4)
50 mg/day 7.08 (60.79) 6.74 (61.35) 48.25 (67.11) 44.0 (67.45) 16.32 (62.27) 14.75 (62.45) 387.5 (642.48) 427.0 (653.35)
Day 0 Day 30 Day 0 Day 30 Day 0 Day 30
Glucose
(mg/dL)
Glucose
(mg/dL)
Creatinine
(mg/dL)
Creatinine
(mg/dL)
BUN (mg/dL) BUN (mg/dL)
Placebo 98.0 (67.53) 105.5 (69.25) 1.25 (60.19) 1.28 (60.22) 13.0 (64.24) 14.5 (64.1)
20 mg/day 103.8 (614.24) 102.6 (67.89) 1.34 (60.38) 1.34 (60.34) 18.6 (66.22) 18.6 (64.39)
20 mg lips/day 119.5 (632.1) 102.25 (68.31) 1.125 (60.17) 1.175 (60.377) 15.20 (66.16) 15.0 (68.32)
50 mg/day 107.25 (66.75) 114.0 (610.23) 1.375 (60.35) 1.5 (60.42) 21.25 (64.5) 16.5 (60.7)
Day 0 Day 30 Day 0 Day 30 Day 0 Day 30
Albumin (G/dL) Albumin (G/dL) Globulin (G/dL) Globulin (G/dL) A:G ratio A:G ratio
Placebo 3.225 (60.41) 3.4 (60.38) 3.62 (60.52) 3.65 (60.42) 0.9 (60.12) 0.93 (60.1)
20 mg/day 3.08 (60.14) 3.3 (60.24) 3.94 (60.16) 3.7 (60.44) 0.82 (60.18) 0.92 (60.16)
20 mg lips/day 3.52 (60.34) 3.52 (60.18) 3.575 (60.36) 3.425 (60.46) 1.0 (60.14) 1.05 (60.13)
50 mg/day 3.375 (60.377) 3.5 (60.075) 4.05 (60.33) 4.05 (60.22) 0.825 (60.15) 0.8 (60.1)
Day 0 Day 30 Day 0 Day 30
ALT (U/L) ALT (U/L) ALKP (U/L) ALKP (U/L)
Placebo 52.0 (619.7) 63.25 (615.65) 76.5 (615.2) 81.2 (623.5)
20 mg/day 109.2 (678.7) 85.2 (654.5) 86.4 (659.7) 95.2 (667.8)
20 mg lips/day 128.6 (686.28) 124.6 (690.25) 106.25 (644.9) 147.5 (646.4)
50 mg/day 83.25 (629.57) 79.5 (640.79) 129.9 (621.2) 138.5 (625.4)
ALKP, alkaline phosphatase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; CBC, complete blood count; HCT, hematocrit; RBC, red blood cells; WBC, white blood cells.
Copyright © 2020 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.
10 C.D. Verrico et al.·00 (2020) 1–12 PAIN
®
CBD after oral administration of the liposomal formulation as
compared to the naked isolate.
5. Conclusions
In summary, we demonstrate here that the widely available
supplement CBD exerts robust and quantifiable anti-
inflammatory properties in experimental systems. These exper-
imental results were translatable in a randomized, double-blind,
placebo-controlled trial in a spontaneous canine model of OA. In
this assessment, administration of liposomally encapsulated or
high-dose naked CBD (but not low-dose naked CBD or placebo)
was associated with significant improvements to quality of life as
quantitated by both owner and veterinarian. The results suggest
that clinical studies in humans may be warranted in a variety of
different etiologies and disease stages of arthritis.
Conflict of interest statement
Institutional policy requires W.K. Decker, M.M. Halpert, and V.
Konduri to declare their ownership stakes in Diakonos Research,
Ltd, an unrelated immuno-oncology company. In addition, M.M.
Halpert is a paid scientific advisor for Medterra CBD. The
remaining authors have no conflicts of interest to declare.
Acknowledgements
This study was funded in part by a sponsored research agreement
(to M.M.H.) between Medterra CBD, Inc, and Baylor College of
Medicine. This project was also supported in part by the
Cytometry and Cell Sorting Core at Baylor College of Medicine
with funding from the NIH (AI036211, CA125123, and
RR024574). Flow cytometry analysis was performed with the
expert assistance of Joel M. Sederstrom.
Author contributions: C.D. Verrico wrote the article and analyzed
data. S. Wesson designed and performed experiments, analyzed
data, and contributed critical research tools. V. Konduri analyzed
data. C.J. Hofferek performed experiments. J. Vazquez-Perez
performed experiments. E. Blair designed and performed
experiments, analyzed data, and contributed critical research
tools. K. Dunner analyzed data. P. Salimpour analyzed data and
provided critical research tools. W.K. Decker analyzed data, wrote
the article, and provided critical research tools. M.M. Halpert
designed and performed experiments, analyzed data, wrote the
article, and provided critical research tools.
Appendix A. Supplemental digital content
Supplemental digital content associated with this article can be
found online at http://links.lww.com/PAIN/B3.
Article history:
Received 18 February 2020
Received in revised form 2 April 2020
Accepted 16 April 2020
Available online 24 April 2020
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