extrapolation of invitro data to preclinical data and preclinical data to clinical data screening of immunomodulators

1. EXTRAPOLATION OF INVITRO DATA TO
PRECLINICAL DATA AND PRECLINICAL DATA
TO CLINICAL DATA
&
SCREENING OF IMMUNOMODULATORS
PRESENTED BY
ASWATHY.R.T
FIRST SEMESTER M PHARM
PHARMACOLOGY

2. In Vitro In Vivo Extrapolation
 In vitro (Latin: within the glass) refers to the technique of
performing a given procedure in a controlled environment
outside of a living organism.
 In vivo (Latin for "within the living") refers to
experimentation using a whole, living organism as opposed
to a partial or dead organism.

3. EXTRAPOLATION
Prediction of effects in humans from effects in animal
experiments using toxicokinetic and toxicodynamic datas.
IVIVE
Qualitative and quantitative transposition of experimental
results or observations made invitro to predict phenomena
invivo

4. PURPOSE OF ANIMAL EXPERIMENT
 To test pharmaceuticals
 To investigate animal behavior
 For educational purpose
 For medical purpose
 others

5. Extrapolation of drug dose or pharmacokinetic parameter to
a species of interest is called scaling
 ISOMETRIC SCALING – dose for one species
is applied to all
 ALLOMETRIC SCALING – correlating mass of
an organism with physiological parameters

6. Initial dose in human is calculated by
 Dose by factor
 Similar drug approach
 Pharmacokinetically guided approach
 Comparative approach

7. DOSE CONVERSION BETWEEN ANIMAL AND HUMAN
Human
dose
Apply safety
factors
Select appropriate
animal species
Convert NOAEL to human
equivalent dose
Determine NOAEL in animal species

8. DOSE BY FACTOR
 Empirical approach and use the no observed
adverse effect levels (NOAEL) of drug from
preclinical toxicological studies to estimate
human equivalent dose (HED).
 Here, the dose selection is based on minimum risk
of toxicity, instead of choosing one with minimum
pharmacologic activity in humans

9. Example
 The most sensitive species for a particular drug is the rat,
with a NOAEL of 15 mg·kg-1·day-1. To calculate the HED
according to the FDA guidelines:
 HED = animal dose mg kg-1 ×( animal weight kg/ human
weight kg)0.33
= 15 × (0.35 ×60)0.33 = 3.4mg⋅kg-1⋅day−1
 Assuming the human weight to be 60 kg, the HED is 206
mg·day-1. Applying a safety factor of 10, the starting dose
in humans is 20.6 mg, so a dose of 20 mg·day-1 would be
selected.

10.  The correction factor (Km) is estimated by dividing the
average body weight (kg) of species to its body surface
area (m2).
 HED = animal dose mg/kg ×(Animal km /Human km )
 Km factor for each species is constant, the Km ratio is
used
 HED(mg/kg) = animal dose mg/kg × Km ratio

12. PHARMACOKINETICALLY GUIDED DOSE EXTRAPOLATION
 Here NOAEL and its area under the curve are determined
in several animal species, and the animal species that
gives the lowest NOAEL is used as the index species for
scaling.
 The area under the curve gives a measure of systemic
exposure to the drug with a time dimension
 Next, the clearance of the drug in the index species is
scaled allometrically to obtain the estimated clearance in
humans, and the starting dose is given by the product of
the area under the curve in the index species and the
estimated clearance in humans

13. Example of the pharmacokinetically guided approach
 The area under the curve obtained at the NOAEL for a particular
drug is 23.5 mg·h-1·mL-1 in rats. The predicted clearance in
humans is 20.0 L·h-1. The starting dose is given by the equation:
 Starting dose = AUC in index species × estimated clearance in
human
= 23.5 × h⋅mL−1 × 20 = 470 mg
 A suitable safety factor should also be applied. If a factor of 10 is
used, the starting dose is 47 mg.

14. SIMILAR DRUG’ APPROACH
If the drug to be tested is in the same class as another drug
that is already being used in humans, and if the
pharmacokinetics and pharmacodynamics of both drugs are
the same or very similar, the starting dose in humans can be
estimated by the ‘similar drug’ approach

15.  Drug X, a compound never tested in humans, belongs to the
same class as drug A, which is licensed for use in humans.
Preclinical toxicological studies revealed that the most sensitive
species was rats with a NOAEL of 2 mg·kg-1·day-1. The optimal
starting dose of drug A is 30 mg·kg-1·day-1 and the NOAEL of
drug A in rats was 14 mg·kg-1·day-1. The similar drug approach
uses the equation
 Dose of drug X/NOAEL of Drug X= Dose of Drug A /NOAEL of drug
A
 Dose of Drug X = (Dose of Drug A *NOAEL of drug X)/ NOAEL of
Drug A
=10 * 2 /14
=1.42 mg kg-1
= 85 mg in a 60 kg human
 A safety factor should also be applied. If a factor of 10 is used,
the starting dose will be 8.5 mg

16. New methods for predicting pharmacokinetic
parameters
 Method for predicting human volume of distribution
● Average fraction unbound in tissue method
● Proportionality
● Allometry without protein binding
● Allometry corrected for protein binding
16

17. AVERAGE FRACTION UNBOUND IN
TISSUE METHOD
 Fut = 𝑉r Fu
[VDss – Vp (Fu Ve)] – [(1-Fu)(Re/i)Vp]
 Avg Fut of different species is taken equal to human Fut
 Experimentally determine human Fu
Vd(human prediction)= Vp+ [Fu(human) x Ve] + {[1-Fu(human)]x (Re/i)x Vp}
+Vr.Fu(human)/ Fut(avg)
17

18. Proportionality method
Vd (human prediction)= Fu(human). Vd (dog)
Fu(dog)
18

19. Allometry without protein binding
 Physiological parameter used in scaling was total body
weight
 In this method the plots were plotted of total Vd in
preclinical species Vs animal body weight on a log scale for
each other.
Log 10 VD= a.log10 body weight(kg)+ b
 Eqn is obtained by linear regression of data points to
determine the values a and b for each compound. These
were used along with a std value for human body wt(70 kg)
to predict human Vd
19

20. 20
Allometry corrected for protein binding
Values are then plotted as in the previous method to determine relationship
between free Vd Vs total body weight
Human Vd was then calculated from the following eqn
Vd(free) = Vd(total)
fu
Vd(human) = Vd(free).fu(human)

21. 21
 Method for predicting human clearance
● Invitro t1/2 method
● Enzyme kinetic method

22. Invitro t1/2 method
We can calculate the intrinsic clearance data with the help of integrated Michaelis-
Menten equation
Vm . dt = Km + [s] . d[s]
[s]
when [s] = 0.5[s] t=0 the following equation applies
Vm . t1/2 = 0.693 + 0.5[s]t=0
Km Km
The substrate concentration used is well below the Kmax → an assumption
0.5[s]
Km
22
<< 0.693

23. Thus the equation degenerates to
Vm . t1/2 = 0.693
Km
Vm = 0.693 =CL’ int
Km t1/2
The invitro t1/2 is incorporated into the following equation
CL’int = 0.693 *Liver weight
invitro t1/2 * Liver in incubation *fu (inc)
23

24. The liver in incubation value was calculated from the amount of protein in the
incubation and a seal-up factor from protein to gram of body weight.
This equation indicates that the value for binding to protein in the incubation
be included, however in this treatment it was assumed to be zero ie; fu(inc) = 1.
Thus the intrinsic clearance value calculated were based on total concentration
CL’int = 0.693 . 1 × g of liver wt × ml incubation
t1/2(min) kg of body wt mg of microsomal
protein
× 45mg of microsomal protein
g of liver weight
24

25.  Method for predicting human t1/2
● Animal correlation
● Combinations of human volume and clearance predictions
25

26. Animal correlations
 To construct correlation, measured t1/2 value in animal were plotted against
human t1/2 and functions derived from linear regression
 Predictions of t1/2 are then obtained by inserting animal t1/2 values into
regression eqn
26

27. Combination of human volume and
clearance predictions
 Each method for predicting Vd was combined with each method for predicting
Cl to generate predictions of human t1/2 using following eqn
27
Predicted human t1/2 = 0.693 × Predicted human Vd
Predicted human Cl

28. Enzyme kinetic method
 Enzyme kinetic parameters Km and Vmax are measured in liver microsomal
incubations and this was applied to the below eqn
28
Clint= Vmax
Km

29. LIMITS TO EXTRAPOLATING ANIMAL DATA IN
PREDICTING THE HUMAN RESPONSE
 Pharmacokinetic difference
 Idiosyncratic adverse effects in humans
 Underlying pathologic conditions
 Species difference
 ADR
29

30. SCREENING OF IMMUNOMODULATORS

31. IMMUNOMODULATORS
Immunomodulators are the agents that modulate
immune system by suppressing or by stimulating
immune response
• Immunomodulation as part of immunotherapy, in
which immune responses are induced, amplified,
attenuated, or prevented according to therapeutic
goals
●Immunomodulators divided into two parts :-
Immunosuppressant
Immunostimulant

32. Immunostimulant drugs Immunosuppressant drugs
Stimulate response in case of
immunodeficiency
Inhibitory response in case of organ
transplantation
Levamisole Specific T-cell inhibitors- cyclosporin,
tacrolimus
Thalidomide Cytotoxic drugs- Azathiopurines
methotrexate,
Bacillus Calmette Guerin Glucocorticoids –Prednisolone
Interferons Antibodies –Muromonab CD3,
antithymocyte globulin
Interleukin-2

33. SCREENING OF IMMUNOSUPRESSANT DRUGS

34. IN-VITRO METHODS
 Inhibition of histamine from mast cells
 Inhibition of T cell proliferation
 Chemiluminescence in macrophages
 Inhibition of dihydro-orotate dehydrogenase

35. IN-VIVO METHODS
 Acute systemic anaphylaxis in rats
 Anti anaphylactic activity (Schultz-Dale reaction)
 Passive cutaneous anaphylaxis
 Arthus type immediate hypersensitivity
 Delayed type hypersensitivity
 Reversed passive arthus reaction
 Collagen type-Ⅱ induced arthritis in rats
 Adjuvant arthritis in rats

36. IN VITRO METHODS
1.INHIBITION OF HISTAMINE RELEASE FROM MAST CELLS
Purpose and rationale :Histamine release induce from mast cell by calcium
ionophore 48/80. Histamine concentration can be determined with o-
phthalaldehyde reaction
Procedure :
1. Preparation Of mast cell suspension
Wistar rat decapitated and exsanguinated
50ml HBSS inj. Into peritoneal cavity
Massage body abdominal wall is open
Fluid containing peritoneal cell is collected
Centrifuge at 2000 rpm
Cells resuspended in HBSS brought final concentration of 105 mast
cells/100𝜇l

37. Test compound administration and induction of histamine
release
 1ml test drug + mast cell suspension
 Incubate 37°c for 15 min
 Make 3ml vol with HBSS
 Equal vol of calcium ionophore with allergen
 Suspension incubate 37°c 30min
 Centrifuge at 2500 Rpm
Control solutions
 Spontaneous histamine release – only mast cells and solution
determine baseline
 Histamine release – mast cell + solution +calcium ionophore 10-6g/ml
 Test compound control – solutions +test compound

38. EXTRACTION OF HISTAMINE
1ml top layer transfer to
Tube contain 300mg Nacl+1.25 butanol
Sample alkalized by adding 1ml 3N NaoH
Centrifuge for 5min
1ml top layer (butanol) is pipetted out
In 5ml tube containing 2ml n-heptane+0.4ml of 0.12N HCl
Mixing by inverting tube
Separation of aq. and org. phase
0.5ml aqueous phase transfer to tube

39. Induction of o-phthaladehyde complexing reaction :
Sample + 100𝜇𝑙 1N NaOH+100𝜇𝑙 0.2%ophthaladehyde solution After 2min, add 50
𝜇𝑙 3N HCL
Determination Of histamine release :
Determine on fluorescence detector (350nm & 450nm)
Evaluation
Percent histamine release (hist. rel.) can be expressed by the following formula:
sample hist. rel. − spontaneous hist. rel.*100
100% hist. rel. − spontaneous hist. rel.

40. INHIBITION OF T CELL PROLIFERATION
PURPOSE AND RATIONALE
 Activation and/or proliferation of clonal populations of T
cells are critical for the initiation of an antigen specific
immune response.
 Thus, inhibition of T cell activation provides a potent
means for suppressing specific immune response.
 A number of immunosuppressive agents exhibit the ability
to suppress T cell activation.

41. PROCEDURE
Blood leukocytes from normal donors are separated on Ficoll-
Hypaque.
↓
Purification of Peripheral Blood Leukocytes and T Cells Peripheral
Leukocyte suspensions are washed in HBSS and are resuspended in
RPMI 1664 medium containing 10% heatin activated fetal bovine
serum, pooled human serum and 100 U/ml
penicillin/streptomycin.
↓
Highly enriched T cells are obtained by passing leukocytes
through a nylon wool column to remove macrophages and B cells.
↓
These highly enriched T cells are approximately 95% CD3+ cells,
the remaining cells being B lymphocytes

42. MIXED LYMPHOCYTE REACTION
Peripheral blood leukocytes are incubated at 2×105/well with equal
numbers of gamma-irradiated (3000 rads) allogenic peripheral blood
leukocytes and various conc. of test compounds.
↓
Assays are performed in triplicate in 96-well, U-bottom plates.
↓
After 6 days of coculture, the cells are pulsed for 6 h with 1 μC of
[3H]thymidine per well.
↓
[3H]Thymidine incorporation is then measured by scintillation counting.

43. Data are presented as:
% inhibition = CPMexpt − CPMbckgrd *100
CPMctrl − CPMbckgrd
 CPMexpt is mean counts per min of experimental cultures,
 CPMbckgrd is mean counts per min of background well,
unstimulated cultures,
 CPMctrl is mean counts per min of uninhibited,
stimulated cultures.

44. Lymphocyte Stimulation and Proliferation
Peripheral blood leukocytes and isolated T cells are cultured
with anti-CD3 plus PMA, anti-CD28 plus PMA, or 100U/ml
rhuIL-2 in RPMI 1644 containing 10% fetal bovine serum.
↓
Peripheral blood leukocytes or T cells are cultured at 2 × 105
cell per well in a total volume of 200 μl/well.
↓
Assays are performed in quadruplicate in 96-well, U bottom
plates.
↓
[3H]Thymidine is added to each well after 48 h of coculture
and after a 20 h pulse of [3H]thymidine, the cells are
harvested and the amount of [3H]thymidine uptake is
quantitated on a scintillation counter

45. EVALUATION
Dose response curves of inhibition of one-way
mixed lymphocyte reaction and of IL-2 in the
supernatant after stimulation with antiCD3 or anti-
CD28 are established.

46. CHEMILUMINESCENCE IN MACROPHAGES
PURPOSE AND RATIONALE
 The stimulation of macrophages by antigen, complement,
phorbol esters, etc. Leads to elaboration of super oxide
ion and other oxygen metabolites.
 These radicals form the basis for an efficient microbicidal
system in vivo. Inhibition of these process can be regarded
as a measure of immunomodulating effects of compounds.
 The oxygen metabolites can produce light emitting
reactions, which is measurable if it is amplified with
suitable agents such as the cyclic hydrazide luminol

47. PROCEDURE
NMRI mice or Sprague dawley rats are used
A. Positive control
1. Sensitized mice, receiving vehicle
2. Mice, developing an autoimmune disease, receiving
vehicle
3. Rats, developing adjuvant arthritis, receiving vehicle
B. Negative control
1. Mice not sensitized, receiving vehicle
2. Mice, not developing an autoimmune disease, receiving
vehicle
3. Rats without adjuvant arthritis.

48. Ex in-vivo experiment:
 Groups of 6 animals are treated for 6 days orally or
subcutaneously with test compound or the standard
(prednisolone acetate or leflunomide).
 Decapitated and exsanguinated.
 Macrophages are obtained by flushing the peritoneal
cavity with 10 ml saline, containing 250 IU heparin.
 The cells are pooled, washed several times and
suspended.
 For measurement in the luminometer the following
mixture is prepared:

49.  200 μl macrophages (2 × 106)
 100 μl luminol solution (100 μg/ml)
 100 μl phorbolmyristenacetate solution.
Each sample is mixed thoroughly without the
phorbolmyristenacetate solution, put into the luminometer
and counted at 2 min intervals for 10 s. The addition of the
phorbol ester induces the reaction.
In vitro experiment
• 100 μl of macrophage suspension+ 100 μl of the solution of
the test compound incubated for 15 min at 37 °C.
• 100 μl of the 3.5 μM phorbol ester solution
• 100 μl of luminol solution
• luminescence measured in the luminometer

50. EVALUATION:
 The time of maximal counts for the positive control is
recorded.
 For all groups the ratio of counts per 10 s is determined
at that time, compared to the positive control counts per
10 s and the percent change is calculated.
 For statistical evaluation the experimental group is
compared with the positive control group using Student’s
t-test.

51. IN VIVO METHODS
1. Acute Systemic Anaphylaxis in rats
Purpose and rationale :
Detection of shock symptoms and it can be inhibited by corticosteroid and
i.v disodium cromoglycates
Procedure
Female Sprague dawley rat 120g
Immunized by i.m inj. 10mg/kg high purified ovalbumin with 1ml
Bordetella pertussis suspension
IgE Ab are induce and attach to surface of mast cells, After 11
days
Animals challenged by i.v. inj. Of 25mg/kg ovalbumin

52. Formation of Ag-Ab complex in blood/organs
Then immediate anaphylaxis mediators release
Corticosteroid e.g. dexamethasone 1-10mg/kg s.c Or
30mg/kg disodium cromoglycate (before 18h challenged )
Evaluation
Shock symptoms are scored and mortality counted

53. 2. Anti-anaphylactic activity (Schultz-dale reaction)
Purpose and rationale :- Guinea pig are sensitized against egg albumin. Challenge after
3weeks cause isolated organs release mediators e.g. histamine
Procedure :
Guinea pig of either sex sensitized with alum precipitated egg albumin.
Alum egg albumin is prepared by dissolving egg albumin (1mg/ml)in 6% aluminum hydroxide gel,
suspended in saline.
Mixture is stirred and kept at room temperature.
Each animals receives at same time injection of 0.125ml of this mixture in each foot pad and
0.5ml s.c
After 4weeks animals are killed and ileum is dissected out.
Cleaned pieces, about 2-3cm long, are mounted in organ bath containing Tyrode solution at
37℃.
Strips allowed to equilibrate for 15min.

54. Contractility of ileum strip is tested by adding 10-4 g/ml BaCl2 solution.
To one organ bath standard and to other test compound added.
After 3min ovalbumin in final concentration of 2× 10 -6g/ml is added
Contraction recorded with strain gauges by polygraph.
Evaluation :
 The results are expressed as presence or absence of blocking activity
(% inhibition)
 If anti-anaphylactic activity is observed, ED 50 value using different
doses are calculated.

55. 3. PASSIVE CUTANEOUS ANAPHYLAXIS
PRINCIPLE
 Formation of antigen antibody complex induce the
release of mediator from mast cells.
 This results increase in permeability of the vessel walls
and leakage of plasma.

56. PROCEDURE
 Antiserum are injected intradermally in to shaved dorsal
skin of rats.
 After 24hr each animal is challenged with the intravenous
administration of 0.1ml of 2.5% Evans blue dye
containing 25mg/ml of egg albumin.
 Test compound is also administer along with the antigen.
 After 30 min animals are scarified.
 Amount of Evans blue dye that leaked at the site of
reaction is extracted and determined colorimetrically at
620 mili micron wavelength.

57. EVALUATION
Amount of Evans blue that extracted from
passive cuetaneous anaphylactic reaction of
control group is compared with test group.

58. SCREENING OF IMMUNOSTIMULANTS

59. IN VITRO MODELS
 Mitogen induced lymphocyte proliferation
 PFC (plaque forming colony) test in vivo
IN VIVO METHODS
 Experimental auto immune thyroiditis
 Acute graft versus host disease(GVHD) in rats

60. IN VITRO METHODS
MITOGEN INDUCED LYMPHOCYTE PROLIFERATION
PURPOSE AND RATIONALE
 Cultured lymphocytes can be stimulated to DNA synthesis
by various mitogens.
 Measurement of DNA synthesis can be accomplished by
tritiated thymidine which is incorporated into the newly
synthesized DNA.
 Immunmodulating properties can be detected either by
pre-treatment of the animals in vivo or by adding the test
drug to the cultured lymphocytes.

61. PROCEDURE
Ex vivo: Mice or rats are used.
•Animals receive the test compound once a day for 5 days.
•They are sacrificed, spleens are removed and a single cell
suspension of 5 × 106 cells/ml is prepared.
•Mitogens are titrated and 0.1 ml of the cell suspension is
added.
•Plates are incubated at 37 °C for 48–60 h and for another 8
h after addition of 3H-thymidine per well.

62. Cells are harvested on glass fiber filters and after drying the
degree of radioactivity.
IN VITRO
 Animals are sacrificed and their spleens removed.
 A single cell suspension of 107 cells/ ml is prepared and
0.05 ml placed in each microtiter well.
 Then the test compound is added in 0.05 ml.
 At last 0.1 ml of the double concentrated mitogen is
added. Plates are incubated for 48–60 h and for another 8
h after addition of 3H thymidine per well.
 Degree of radioactivity is determined.

63. EVALUATION:
 Stimulation index = proliferation ratio according
to positive control, either with or without mean
spleen weight.
 Statistical evaluation is carried out using the
Student’s t-test.

64. PLAQUE FORMING COLONY TEST IN VITRO
PURPOSE AND RATIONALE
 Identification of antibody producing cells is based on the ability
of the secreted IgM antibody to fix complement and thereby
lyse the indicator erythrocytes.
 Spleen cells or peripheral blood lymphocytes, previously
incubated with antigen, are mixed with sheep red blood cells
(SRBC).
 After addition of compliment and incubation, plaques (clear
areas) caused by the lysis of SRBC appear in the otherwise
cloudy layer. Antibody forming cells can be detected by the
appearance of plaques.
 The number of plaques obtained is proportional to the number
of antibody producing lymphocytes in the cell population.

65. PROCEDURE
 NMRI mice or Lewis rats
Materials
• absorbed guinea pig complement
• SRBC stored in Alsever’s solution
Positive control
Spleen cells incubated with antigen and medium
Negative control
Spleen cells incubated with medium alone.
The animals are decapitated and the spleens are removed
from the peritoneal cavity. A single cell suspension of 15 × 106
cells/ml is prepared.

66.  0.5 ml splenocyte suspension is added to 0.5 ml of a
suspension of SRBC, previously washed in medium and
diluted to 8 × 106 cells/ml.
 1 ml of the solution of the test compound is added and
the linbro wells are incubated at 37 °C in a CO2 incubator
for 5 days. Per group 3 limbro wells are set up.
 On day 5, the 3 wells of each group are pooled, washed in
medium and the number of cells is determined. For each
cell pellet, 875 µl of washed SRBC and 125 µl absorbed
guinea pig compliment are added. The suspension is mixed
thoroughly and filled in chambers constructed of
microslides.
 The chambers are placed in the incubator at 37 °C for 90–
120 min. The plaque forming colonies are counted
immediately after incubation.

67. EVALUATION
The activity of test compounds can be determined using
the following formula:
1. PFC/3 wells
X=plaques × 100
µ l
2. % change in the number of plaques:
x= plaques ×100
plaques pos. control
d% = x-100
3. % change in no of cells:
X = no of cells *100
No of cells pos. control
d% = x-100

68. IN VIVO METHODS
EXPERIMENTAL AUTOIMMUNE THYROIDITIS
PURPOSE AND RATIONALE
 Immunization of rats or mice with porcine thyroglobulin results in
thyroiditis
PROCEDURE
 Female mice (6–8 weeks old) are primed with 50 µg PTg
 given s.c. into four or five sites of injection and are boosted 14 days
later with the same dose of PTg
 The test compounds are administered from day 0 (at priming) until
day 21.

69.  Mice are bled on day 21 and on day 28 after priming.
 The sera are tested for the levels of anti-PTg antibodies
using an enzyme-linked immunosorbent assay (ELISA). On
day 28, the animals are sacrificed and the thyroid glands
prepared.
 Five-micrometer thick sections are stained with Masson-
Goldner’s trichrome solution.

70. EVALUATION
The histological severity of experimental autoimmune
thyroiditis is graded as a function of mononuclear cell
thyroid infiltration indices:
1. Interstitial accumulation of inflammatory cells distributed
between two ore more follicles.
2. One or two foci of inflammatory cells reaching at least
the size of one follicle
3. 10 to 40% of the thyroid replaced by inflammatory cells.
4. More than 40% of the thyroid replaced by inflammatory
cells.
Mean values of treated animals are compared with controls.

71. REFERENCE
 Hans Gerhard Vogel. Drug discovery and evaluation:
Pharmacological assays, vol 2, 3rd edition. Pg no.792-799
 http// www.authorstream.com.
 KD Tripathi, Essentials of Medical pharmacology, Jaypee
publications, Sixth edition, pg no- 173-184.