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Chapter-6 Ex vivo / In vivo studies
Department of Pharmaceutical Sciences, M. M. University, Mullana 98
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After successful formulation of MDTs by modified effervescent method, the selected
formulation of Cefixime (C4) was subjected to ex vivo and in vivo studies. Objective of this
study includes
a. Ex-Vivo Permeability Study: Study the drug permeability of Cefixime from
Formulation C4 in comparison to pure drug and evaluate the permeability
enhancement of Cefixime from the finalized formulation.
b. In-Vivo Study: Study the In vivo parameter of Cefixime in Formulation C4 in
comparison to pure drug after oral administration in animal and to perform
i) Pharmacodynamic studies: To evaluate the Pharmacodynamic effectiveness
of Cefixime in formulation C4 in comparison to pure drug
ii) Assessment of Pharmacokinetic Parameters: To determine the
pharmacokinetic parameters of Cefixime after oral administration of
formulation C4 in rabbit model.
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a. Chemicals: Sodium Chloride, Sodium Citrate, Nutrient Agar (Qualikems, New
Delhi, India.), Acetonitrile HPLC grade, Methanol HPLC grade, Ammonium Acetate
HPLC grade (Ranbaxy Fine Chemicals Ltd, India) were purchased from the standard
concern. . All other chemicals used were of analytical grade.
b. Microbial Stain: Proteus Vulgaris (ATCC No. 13315), procured from Department of
Microbiology, M.M. Institute of Medical Science and Research, M.M. University,
Mullana, Haryana INDIA
c. Animals: 12 Albino male rabbits (approximately 1kg weight) obtained from
Departmental Animal House, M.M. College of Pharmacy, M.M. University, Mullana,
Haryana, INDIA and kept under standard laboratory conditions in 12 h light/dark
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cycle at 25±2 °C. Animals were marked with picric acid solution (for easy
identification) and provided with pellet diet (Lipton, India) and water ad libitum.
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Digital Balance (Shimadzu, Japan), Vortex shaker (Electro Lab, India), Water bath
incubator shaker (Tanco Pvt. Ltd., India), Vaccum Oven (Q5247, Navyug, India), UV
Spectrophotometer (UV 1800, Shimadzu, Japan), HPLC (LC 10AT SHIMADZU) were used.
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6.4.1 Protocol Approval
Protocol no. MMCP/IEC/10/18 approved by Institutional Animal Ethical Committee
(1355/ac/10/CPCSEA), M.M. College of Pharmacy, M.M. University, Mullana.
6.4.2 Methodology
Ex vivo permeability studies of Cefixime in formulation C4 were carried out using non-
everted gut sac technique. The small intestine of freshly sacrificed male albino rabbit was
removed by cutting across the upper end of the duodenum and the lower end of the ileum and
manually stripping the mesentery. The small intestine was washed out carefully with cold
normal oxygenated saline solution (0.9%w/v NaCl) using a syringe equipped with blunt end.
The clean intestinal tract was prepared into 6 ± 0.2 cm long sacs. Each sac was filled with
1ml of pure drug and formulation C4 (equivalent To 10 mg of Cefixime, suspended in 0.1N
HCl) via a blunt needle and the two sides of the intestine were tied tightly with thread. Each
non-everted intestinal sac was placed in a glass conical flask containing 50 ml physiological
solution (Tyrode solution). The entire system was maintained at 370C±0.50C in a shaking
water bath operating at 50 rpm and aerated with oxygen (10–15 bubble/min) using laboratory
aerator. From outside of the sac 1ml samples were withdrawn for 12 h and replaced with
fresh physiological solution. The samples were analyzed using UV–visible
spectrophotometer (UV 1800, Shimadzu, Japan). Amount released per unit area of sac and
6. Ex vivo / In vivo studies
Department of Pharmaceutical Sciences, M. M. University, Mullana 100
percent drug permeation in the receptor compartment were calculated (Ruan et al., 2006 S ).
Studies were performed in six replicates.
66..44..33 Results and Discussion
Table 6.1 Parameters for Permeability studies by non everted gut sac method
Method Non - Everted Gut Sac method
Medium 50ml physiological Solution (Tyrode Solution)
Sample Taken 1 ml
Dilution Factor 10 times
Conc. Conversion factor (Abs-0.018)/0.049 mcg/ml
Theoretical Drug Tablet Formulation C4 equivalent to 10mg cefixime
Table 6.2 Cumulative % drug Permeated from formulation C4 in comparison to pure drug
Time
(hrs.)
Cumulative % Drug Permeated
Pure Drug Cefixime Formulation (C4)
0.25 7.04±0.9 10.51±1.2
0.5 9.73±1.2 10.72±1.1
1 14.89±2.1 20.92±1.5
1.5 18.05±1.9 35.52±2.1
2 21.07±2.4 37.44±3.2
4 27.86±3.2 53.18±3.6
6 33.92±3.3 69.52±4.3
8 43.22±3.1 80.76±3.5
10 57.99±4.3 99.87±3.8
S.D for six trails, P<0.005
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Department of Pharmaceutical Sciences, M. M. University, Mullana 101
Fig 6.1 Comparison of Cumulative % drug Permeated from formulation and pure drug
Selected formulation of Cefixime MDT (C4) was evaluated for Ex-vivo permeability
studies as per the procedure described in Methodology. Result (Table 6.2, figure 6.1) shows
that only <10% Cefixime was permeated through the intestinal membrane from formulation
as well as pure drug in 30 minutes and permeability increased after 1hours. This may be due
to the initiation of dissolution and absorption of Cefixime through intestinal membrane. The
permeability of Cefixime was linear from pure drug and only 57.99±4.3% drug was
permeated even after 10hours. In formulation C4, more than 53.18±3.6% of the drug was
absorbed within 4 hours (T50% = 3.5hrs and T90% = 9hrs) and found 99.87±3.8% permeation
within 10 hours of study. Thus, in formulation C4, the permeability of Cefixime was
enhanced in comparison to pure drug (T1/2 < 3.5hrs in formulation, T1/2 < 9hrs in pure drug).
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Department of Pharmaceutical Sciences, M. M. University, Mullana 102
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6.5.1 Protocol Approval
Protocol no. MMCP/IEC/10/18 approved by Institutional Animal Ethical
Committee (1355/ac/10/CPCSEA), M.M. College of Pharmacy, M.M. University,
Mullana.
6.5.2 Methodology
Selection of Animal: - Six Healthy rabbits (1Kg approx.) were selected and subjected to physical examination.
Standard: - Pure Drug (suspension of 2mg equivalent Cefixime).
Test: - Cefixime Formulation C4 (2mg equivalent suspension)
Delivery method: - 1ml suspension was administered orally in each animal with feeding needle.
Sampling: - 0.5ml blood samples were withdrawn from marginal ear vein of animal at
regular time interval and immediately diluted with anticoagulant solution (3.2% w/v
Sodium Citrate). Samples were centrifuged and plasma was stored at -200C until assayed
for antimicrobial activity.
6.5.3 Evaluation: Antimicrobial study
Method: - Disc Plate Agar Diffusion method
Microbial stain: - Proteus Vulgaris (ATCC No. 13315)
Media: - Nutrient Agar Media
6.5.3.1 Reference Curve
Sterilized nutrient agar media (about 15ml) was poured in each sterilized petridish
and allowed to solidify. The microorganisms were inoculated aseptically by spread plate
method. The drug loaded disc (different concentrations of Cefixime loaded in 5mm
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diameter disc) were made and placed in nutrient agar plate. The petridish were incubated
for 24 hours at 380C. The resultant growth inhibition zone was measured, and tabulated.
These results were drawn as a curve of the concentration verses zone diameter and range
of linearity was determined.
6.5.3.2 Sample assay
The same method was adopted to assess the antimicrobial activity of the antibiotic
in the animal plasma. Plasma samples were loaded in disc (5mm diameter) and placed on
freshly microbial spreaded nutrient agar plate, and incubated for 24 hours at 380C. The
zone of inhibition was recorded. The concentration of unknown samples was read from
the reference curve.
66..55..44 Result and Discussion
6.5.4.1 Reference Curve
The resultant growth inhibition zone in millimeter was measured by calibrated
scale and reference curve (table 6.3, Fig 6.2) was plotted against zone of inhibition (mm)
versus concentration of Cefixime (mcg/ml).
Table 6.3 Zone of Inhibition (mm) by Cefixime with concentration (mcg/ml)
Conc.
(mcg/ml) Zone of
Inhibition* (mm)
Conc.
(mcg/ml) Zone of
Inhibition* (mm)
2 8.00 12 23.00
4 10.00 14 27.00
6 14.00 16 31.00
8 17.00 18 34.00
10 20.00 20 38.00
* Average of three readings
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Fig 6.2 Reference curve of Cefixime (Zone of Inhibition [mm] vs. concentration [mcg/ml])
From the observed reference curve (fig 6.2), linearity was observed over
concentration range of 2-20mcg/ml.
6.5.4.2 Sample assay
The plasma concentration of the orally administered Cefixime pure drug and
in formulation C4 (2mg equivalent) in animal were determined by zone of inhibition
(mm) as shown in figure 6.3A to 6.3F and shows that the Antimicrobial activity of
formulation C4 (ZOI= 14±1.0 in 2.5hrs.) was found more (table 6.3, fig 6.4) in
comparison to pure drug (ZOI= 11±1.0 in 2.5hrs.).
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Fig 6.3 Observed Zone of Inhibition (mm) of Cefixime pure drug and formulation C4
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Table 6.4 Observed zone of Inhibition (mm) with time (hrs) from animal study
Time (hrs.) Zone of Inhibition (mm)
Cefixime Formulation (C4)
0 5±0.0 5±0.0
0.75 8±0.5 9±0.5
1.5 10±1.0 12±1.0
2.5 11±1.0 14±1.0
4 9±1.0 12±1.0
6 7±0.5 10±1.0
8 6±0.5 8±0.5
10 5±0.0 6±0.0
mean S.D for six reading
Fig 6.4 Zone of Inhibition (mm) versus time plot of Cefixime pure drug and formulation C4
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Department of Pharmaceutical Sciences, M. M. University, Mullana 107
Table 6.5 Plasma concentration (Cp) with time (hrs) of Cefixime and Formulation (C4)
Time (hrs.) Mean Plasma Concentration (Cp)
Cefixime Formulation (C4)
0 0.00±0.00 0.00±0.00
0.75 7.55±1.26 10.06±1.26
1.5 12.58±2.52 17.61±2.52
2.5 15.09±2.52 22.64±2.52
4 10.06±2.52 17.61±2.52
6 5.03±1.26 12.58±2.52
8 2.52±1.26 7.55±1.26
10 0.00±0.00 2.52±0.00
mean S.D for six reading
Fig 6.5 Comparative plot of plasma concentration (Cp) with time (hrs) of Cefixime and
Formulation (C4) from antimicrobial study
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Department of Pharmaceutical Sciences, M. M. University, Mullana 108
Table 6.6 Pharmacokinetic parameters in animal after oral administration of Cefixime and
Formulation (C4)
Pharmacokinetic parameters Cefixime Formulation (C4)
Cmax (mcg/ml) 15.09±2.1 22.64±1.8
AUC0-∞(mcg.h/ml) 78.7±9.2 140.0±10.2
Biological Half Life T1/2 (h) 3.58±0.21 4.17±0.32
Tmax(h) 2.5 2.5
Kel (h-1) 0.1938±0.0221 0.1664±0.0143
The peak plasma concentration (15.09±2.1 mcg/ml and 22.64±1.8mcg/ml) were
found to be achieved in 2.5 hrs in animal from Cefixime in comparison to formulation (C4).
The absorption of Cefixime from formulation (C4) was more compared to the Cefixime pure
drug. Further follow up of the plasma concentration shows that in at least 2.5 hours Cefixime
attained peak plasma concentration in pure drug as well as in formulation.
The elimination half life of Cefixime in the animal was found to be 3.58±0.21 hrs.
and 4.17±0.32 respectively and clearance of Cefixime was found almost same in both cases
(0.1938±0.0221 h-1 and 0.1664±0.0143 h-1).
From Pharmacodynamic study, the antimicrobial activity of Cefixime was found to
be enhanced in formulation (C4) in comparison to the pure drug (>77.89% with Cmax
22.64±1.8mcg/ml in 2.5hrs.)
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6.6.1 Protocol Approval
Protocol no. MMCP/IEC/10/18 approved by Institutional Animal Ethical
Committee (1355/ac/10/CPCSEA), M.M. College of Pharmacy, M.M. University,
Mullana.
6.6.2 Methodology
Selection of Animal: - Six Healthy albino male rabbits (1kg approx.) were selected and
subjected to physical examination.
Standard: - Pure Drug (suspension of 2mg equivalent Cefixime).
Test: - Cefixime Formulation C4 (2mg equivalent suspension in distilled water)
Delivery method: - 1ml suspension was administered orally in each animal with feeding needle.
Sampling: - 0.5ml blood samples were withdrawn from marginal ear vein of animal at
regular time interval and immediately diluted with anticoagulant solution (3.2% w/v
Sodium Citrate). Samples were centrifuged and plasma was stored at -200C until assayed
for antimicrobial activity.
6.6.3 Evaluation: HPLC Analysis
HPLC specifications
Instrument: - LC 10AT SHIMADZU with Wakosil II C18 250 x 4.6 mm column
Mobile phase: - Acetonitrile, methanol, 0.5% ammonium acetate buffer in ratio 44:16:40 v/v/v (pH 5.54)
Injection Vol.: - 100 µl
Flow rate : - 0.8 ml/min
Detector : - UV detector at 220nm
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6.6.3.1 Linear Curve
HPLC method was developed for estimation of Cefixime using HPLC
(SHIMADZU LC 10AT) fitted with Rheodyne injector. The column (Wakosil II C18)
was eluted with the mobile phase (acetonitrile, methanol, 0.5% ammonium acetate
buffer in the ratio of 44:16:40 v/v/v) and retention time of cefixime was found.
Linearity of the HPLC method was determined by mathematical treatment of test
results obtained by analysis of samples with Cefixime concentrations across the
claimed range. Area was plotted graphically as a function of Cefixime concentration.
6.6.3.2 Sample assay
The plasma samples were treated with 12.5% tricloro-acetic acid (TCA) for
protein precipitation. The samples were eluted with mobile phase from Wakosil II C18
column and monitored in UV detector. Quantification was achieved by measurement
of the peak area and peak plasma concentration was determined from linear plot.
66..66..44 Result and Discussion
6.6.4.1 Linear Curve
Retention time of Cefixime was found to be 3.17 minutes.
Table 6.7 Peak area versus concentration of Cefixime in HPLC analysis
Sr. No. Concentration (mcg/ml) Peak Area*
1 5 67345
2 10 134054
3 15 200780
4 20 278194
5 30 426976
6 40 545821
*average of 3 reading
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Fig 6.6 Linearity plot of Cefixime (peak area versus concentration [mcg/ml])
From the observed reference curve (fig 6.6), linearity was observed over
concentration range of 5-50mcg/ml.
6.6.4.2 Sample assay
Table 6.8 Level of plasma concentration (Cp) of Cefixime (mcg/ml) from rabbit after oral administration of pure drug and formulation (C4)
Time (min) Mean Plasma Concentration (Cp)
Pure Drug Formulation C4
0 0 0 0.75 5.45±0.8 9.98±0.6 1.5 10.67±1.6 15.23±1.8 2.5 14.64±1.9 19.21±2.1 4 11.67±1.4 16.87±1.5 6 6.34±1.2 10.13±1.1 8 2.67±1.1 5.23±0.8 10 1.3±0.7 2.45±0.5
mean S.D for six reading
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Department of Pharmaceutical Sciences, M. M. University, Mullana 112
Fig 6.7 Plasma concentration (Cp) of Cefixime versus time plot from animal plasma after oral administration of pure drug and formulation (C4)
Table 6.9 Pharmacokinetic parameter of Formulation C4 in comparison to pure drug after oral administration in rabbits
Pharmacokinetic parameters Pure Drug Formulation C4
Cmax (mcg/ml) 14.64±1.9 19.21±2.1
AUC0-∞(mcg.h/ml) 77.9±8.7 121.3±8.4
Biological Half Life T1/2 (h) 3.41±0.45 3.91±0.62
Tmax(h) 2.5 2.5
Kel (h-1) 0.2033±0.0341 0.1772±0.0114
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The peak plasma concentration (Cp) from pure drug was found 14.64±1.9mcg/ml in
2.5 hours (table 6.9) whereas in formulation (C4), Cp was enhanced to 19.21±2.1mcg/ml
indicating more absorption of Cefixime from formulation (C4) was as compared to the
Cefixime pure drug.
From pharmacokinetic study (Fig 6.7, table 6.9), the elimination half life of Cefixime
in the animal was found to be 3.41±0.45 hours and 3.91±0.62 hours from pure drug and
formulation (C4) respectably and clearance of Cefixime was found almost same in both cases
(0.2033±0.0341 h-1 and 0.1772±0.0114 h-1). The AUC0-∞ of formulation was found
121.3±8.4 mcg h/ml in comparison to Cefixime pure drug (77.9±8.7 mcg h/ml), thus
bioavailability of the drug was found to enhanced by more than 50% in formulation (C4) in
comparison to the pure drug (Cmax = 19.21±2.1mcg/ml in 2.5hrs.).