This presentation was delivered at a national conference EBCCON2023, SRM medical college, Chennai. The presentation was timed for eight minutes with two minutes of discussion. It describes evaluation of potential analgesic effect of Vitamin D3 in comparison to tramadol and diclofenac using hot plate test and acetic acid induced writhing test. Prior institutes ethics committee permission was taken and CPCSEA guidelines were followed. The study was conducted over a period of 63 days following principle of 5Rs of animal experiment. Animals were reused for two different models.

1. Evaluation of Analgesic and Anti-
inflammatory effects of Four Exploratory
daily doses and a Single High dose of
Vitamin D3 in Two Animal Models of Pain
in Swiss Albino Mice: An Active
controlled, Single-blinded Experimental
Study
Authors:
Nerurkar R.P., Rao K.S., Dhorajiwala S.S. (Presenter)

2. Introduction (1/3)
Pain is a ubiquitous feature, most common symptom compelling an
individual to seek medical attention1
IASP definition of pain: An unpleasant sensory and emotional experience
associated with actual or potential tissue damage, or described in terms of
such damage 2
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Stimuli
types3:
a)
Mechanical
b) Thermal
c)
Chemical

3. Study Rationale
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3
NSAIDs very commonly a/w adverse
effects like:
• acid peptic disease
• salt and water retention
• exacerbation of asthma etc
Opioids known to have severe A/E:
• like respiratory failure,
• postural hypotension, abuse liability
etc.
Many clinical reports4-8 and animal studies9,10 citing vitamin D role in pain
management
There are studies which negates the role of vitamin D in pain management11,12
Above literature review & controversy about the beneficial effects of vitamin
D3 in pain management basis of the study

4. Primary Hypothesis & Research Question (3/3)
Primary hypothesis: Vitamin D3 has pain mitigating
effects
Primary Research question: “Does vitamin D3 given daily
over 21 days have any effect on pain mitigation through
central or peripheral mechanisms in animal models of pain
when compared to tramadol and diclofenac respectively?”
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5. Aim: To evaluate analgesic
effects of vitamin D3 in animal
models of pain:
Objective 1: To compare 3 daily
doses of vitamin D3
(i.e.15/30/60 µg/kg) to the
standard drugs viz tramadol
and diclofenac
Objective 2: To compare a
single high dose of vitamin D3
(i.e. 260 µg/kg) given once in
3 weeks, to standard drugs
tramadol and diclofenac
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6. Methodology (1/3)
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Study design: Prospective, experimenter blinded, Parallel animal experiment
IAEC* permission taken (Project-No.: IAEC/03/19) & CPCSEA* guidelines followed
Place of study: Experimental laboratory of department of Pharmacology, TNMC & Nair Hospital
Forty-two (36+6*) Swiss Albino male mice (20-30 gm), procured from registered breeder
Age: 6-8 weeks
Identification: Cage tagging system
Mice grouping per model: Six groups of six mice each
*Committee for the Purpose of Control and Supervision of Experiments on Animals
Institutional Animal Ethics Committee

7. Parameters of Assessment (2/3)
Hot Plate test Writhing test
I) Comparison of mean latency period (in seconds)
on hot plate at baseline and after 30, 60 and 90
minutes of dosing
a) within groups
b) between group
II) Percent of maximal possible antinociceptive
effect (MPE%) calculated:
MPE%= test latency (s) – control latency (s) x 100
Cut-off time (s) – control latency (s)
I) Between group comparison of:
-mean time for onset of writhing (in seconds)
-total number of writhes in 10 minutes (after
onset)
II) % inhibition calculated using the formula %inhibition
=(avg writhes in control group – avg writhes in treatment group) x 100
- Average writhes in control group
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8. Statistical Analysis (3/3)
Data were described as Mean+SD
Experimental data analysed using:
• One-way ANOVA/ Kruskal Wallis for between group comparison
• RM ANOVA/ Friedman’s test for within group comparison.
• followed by post-hoc Tukey Kramer/ Dunn’s test
Paired t-test
p-value of <0.05 considered significant
GraphPad Instat version 3 statistical software for analysis
Bar diagrams GraphPad Prism 8 & MS-Excel 2016
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9. Results: Hot plate test: day 0(screening) , day 7, 14 & 21:
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Table no.2: Comparison of latency period between groups on screening (Day 0)
Day 0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
latency period 5.33+1.86 5.67+1.37 6.17+2.32× 7.50+1.22× 4.17+1.6 5.17+1.17
p-value
(KW test)
=0.048
Post-hoc test shows statistically significant difference between group 4 and 5 with p-value<0.05.

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14. Results: Model No. 2- Acetic acid induced writhing test
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17. Discussion
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Principle finding of our study: Vitamin D3 exhibited variable analgesic effect
at different doses and at different time interval
Table No. 8-Interpretation of findings
Hot-plate test Writhing test
1. Vitamin D3 at doses of 15, 30, 60 and 260 µ/kg
significantly prolonged mean latency period when
compared to baseline & control more pronounced
on 7th day
2. 15 & 30 µ/kg transient effect, 60 (day 7 & 14) &
260 µ/kg (day 21) long lasting effect outlasted the
effect of tramadol at 60 and 90 minutes
1. Vitamin D3 failed to show consistency in its
analgesic activity
2. Same doses of vitamin D3 produced opposite
responses through different parameters
3. On day 14, 30 µ/kg significantly delayed mean
time of onset of writhe (analgesia), same dose also
increased mean number of writhes,  pain
inducing effect
Likewise, 15 µ/kg on day 21 significantly reduced
mean number of writhes (analgesia) However, with
same dose, mean time of onset of writhing was
abbreviated (pain-inducing effect)

18. What other studies shows?
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Similarities in hot
plate test9
+ve control in both
studies showed
maximum analgesic
effect
60 µ/kg consistently
showed significant
analgesic effect
Positive control-
maximum analgesic
effect
Differences in hot
plate test
Effect of morphine
consistently high
throughout, tramadol
maximum effect at 30
minutes.
Same day hot plate
test repeated at 4
time intervals.-David
et al.
Acute dose study-
David et al.
 Similar studies evaluating analgesic and anti-inflammatory properties by David et. al 9,10 using hot plate, tail flick,
tail pinch and acetic acid induced writhing tests with contrasting and inconclusive findings
Similarities in
writhing test10
Positive control-
maximum
analgesic effect
60 µ/kg:
significant
analgesic effect
Differences
One additional
parameter-
Duration of each
writhe- David et al
Parameters
assessed over 45
minutes of
observation
Morphine tested-
David et al
Other groups -
significant
analgesic effect-
Our study
15 and 30 µ/kg:
pain inducing
effect-- Our study

19. Conclusion & Way forward
We conclude that in comparison to tramadol and diclofenac; 15, 30, 60 & 260 µ/kg of vitamin D3
failed to produce significant analgesic effect when tested on day 7,14 & 21
However, on daily dosing 60 & 260 µ/kg of vitamin D3 showed significant analgesic effect when
compared to baseline readings & vehicle control on day 7, 14 & 21 in hot plate test
Secondly, 15 & 30 µ/kg of vitamin D3, contrary to our expectation, on day 14 & 21 exhibited pain
inducing property in writhing test
Even though our study presents some experimental evidence supporting administration of vitamin D3
in pain management; more detailed, comprehensive studies with composite experimental models,
testing still higher doses are needed to clarify the full extent and mechanism of action of our findings

20. 1.Barret KE. Barman SM. Boitano S. Brooks HL. Ganong’s Review of Medical Physiology. 25th ed. USA: Mcgraw Hill Education; 2016:165.
2. Merskey H. Bogduk N. Classification of Chronic Pain: Descriptions of Chronic Pain Syndromes and Definitions of Pain Terms. 2nd edition. Seattle,Wash,
USA: IASP Press; 1994:199-207.
3. Hall JE. Guyton and Hall Textbook of Medical Physiology. 13th ed. USA: Elsevier’s Saunders; 2016: 621.
4. Gendelman O, Itzhaki D, Makarov S, Bennun M, Amital H. A Randomized double-blind placebo-controlled study adding high dose vitamin D to analgesic
regimens in patients with musculoskeletal pain. Lupus. 2015; 24:483-489
5. Warner AE, Arnspiger SA. Diffuse Musculoskeletal Pain Is Not Associated with Low Vitamin D Levels or Improved by Treatment with Vitamin D. J Clin
Rheumatol. 2008;14: 12–16
6. Gopinath K, Danda D. Supplementation of 1,25 dihydroxy vitamin D3 in patients with treatment naive early rheumatoid arthritis: a randomised controlled
trial. Int J Rheum Dis. 2011; 14: 332–339.
7. Lee P, Chen R. Vitamin D as an analgesic for patients with Type II Diabetes and Neuropathic pain (Research letter). Arch Intern Med. 2008, Apr 14; Vol 168
(No. 7):771-772
8. Buettner C, Reuven RN, Suzanne B, Bernstein C, Schain A, Mittleman MA, Burstein R. Simvastatin and Vitamin D for Migraine Prevention: A Randomized,
Controlled Trial. Ann Neurol. 2015; 78:970–981
9. David A, Goel RK, Patel P, Kunkolol R, Nandal DH. An experimental study to evaluate and compare the analgesic activity of calcitriol with morphine in albino
mice at a tertiary care teaching hospital in Maharashtra, India. International Journal of Research in Medical Sciences.2017, January;Vol 5(No.1):191-195
10. David A, Goel RK, Patel P, Paul P. A Comparative study for antinociceptive potential of vitamin D3 with diclofenac in animal models. International Journal of
Basic & Clinical Pharmacology.2017, Mar;6(3):608-612
11. Power calculation for one-way ANOVA. Available from: https://www.masc.org.au/stats/PowerCalculator/PowerANOVA. Accessed on: 02-12-2019.
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References