Dissociation constant “pka” It shows the charge on the molecules. Partition coefficient shows the substance which is well partitioned in liquid and water both the parameters are required for understanding the behaviour of the drug, many substances would varied from its physical and chemical properties. So it’s important to determine ionic form of a present molecule is necessary.
Partition coefficient and PKa both in combination can be help full to determine distrubutation of drug compound in a system. Absorption execration and penetration. Affects both the parameters.
Dissociation constant
Ionisation of both acid and base
Pka is not dependent on the concentration for acid
Pka = -log 10 (ka) where ka = (H+) (A-) / (HA)
Concentration ionisation constant, measured by spectroscopy. It is temperature dependent constant
Log p and partition coefficient
It can be shown by
Partition coefficient P = (organic) / (aqueous)
Log P = log 10 (partition coefficient)
Where log D = distributation coefficient
{{54 Earll, Marks. 1999-2006 ;}}
There may be different choose of partition solvent
Octanol the most commonly used solvent
Through which partition or distrubutation ratio can be determined. It’s also determines the distrubutation or partition law. which shows inconsiderateness to concentration and temperature .Concentration studies shows that distribution ratio was clearly concentration dependent in case of” benzoic acid” between water and benzene
The octal water coefficient studies show the resemblance to the body system .so it is mostly used solvent for determination of partition coefficient. Its shows different quantitative structural activity relationship. It shows correlation of biological activity and chemical structure .its lead to high activity, application, implementation in the theory,
It can be shown by BR = a+Bb+cC+......
Where BR is Biological response, B, C are the molecular properties and log Kow, shows large fraction of QSARS relation BR = a+b log Kow
Biological response interferes with the large numbers of reaction to be undertaken in concerned with interaction of xenobiotic activity (in vivo), and in sense of chemical interaction in biological reaction (in vitro). Concepts of pka and partition coefficient studies is relatively important in drug discovery or formulation in pharmaceuticals .it is also showed in vivo chemical studies as Bioaccumulation and relates with the tissue partition coefficient.
{{55 Sangster. 1997 ;}}
Physiochemical properties serve to be the important factor in the drug penetration, solubility, permeability, ionisation, bioavailability, and also adsorption surface.
{{57 Pallasch, Thomas, J. January 22 1988 ;}}
Pka is an essential factor in determining solubility and lipophilicity of drug molecule for better solubility, drug need to be converted into suitable salt form. Traditional method for calculation of pka was potentiometric titration. It is available commercially, due to its high throughput (up to 30 compounds / day) with mini sample requirement method used was Yasuda Shedlovsky Method.
Spectral gradient analysis is recently used technique. UV absorption spectrum is used . Along the GLPKa Sirius Instrument.
Measurement of physiochemical properties can be done along with the properties
Solubility is an important factor in drug absorption. low drug solubility which is always associated with plasma binding, slow tissue distribution, drug-drug interaction .so overtaking factors to determine solubility of a particular drug .Methods such as Potentiometery Titration and Nephetrometric Assay can be implemented but this methods having high throughput, its solubility range is limited .so solubility in buffer solution is measured by LC/MS based solubility determination protocol
Partition coefficient and distrubitation coefficient are the two factors between two immussiable solvent phase (octonal and water) are mostly used to determine lipophilicity of organic compounds.
In a development of drug log D at PH 7.4 is widely used which shows an indication of lipophility of drug molecules at PH of blood plasma. It is necessary to determine the PH due to changing environment of biological system such as gastro instential tracks for proper permeability. Lipophility is one of the factors which are related to ADME properties. So drugs with low or high lipophilicity, shows drug with low lipophility have improper absorption through passive transport and with high lipophlic molecule may get fascinated inside the membrane. Other complication are also related to high lipophility is important. Indirect methods such as REVERSE PHASE HPLC have been proven to be high throughput method. It is an indirect method for lipophilicity determination but traditional methods such as conventional SHAKE AND FLASK method is still used to its simplicity and cost efficiency.
{{56 Yan, Zhengyin.Caldwell, Gary, W. 2004 ;}}
Fluconazole used in the form of semisolid formulation to be applied tropically. It is used as an antifungal agent. To determine partition coefficient is necessary for studies. Distribution of drugs, partition gives information on surface tension, hydrolysis, solubility, dissociation of substance along with structural formula. Partition coefficient of solute between octanol and water is widely used to predict drug pharmacokinetic properties ,techniques involved as traditional shake flask method, potentiometeric titration , liquid chromatography , counter current distribution ,
Drug shows toxic effect and also reduced systemic absorption due to the drug contacted with adsorption force which may be physical or chemical applied to them. Adsorption is important phenomenon in terms of interaction of drug and excipients. The is effected by many different factors such as ph, time of surface area, nature of adsorbent used. “Langmuir and freundlich” adsorption isotherm equation describes adsorption theoretically.
Fluconazole is a bis –triazole antifungal agents, it highly reactive against Cryptococcus neoforman and Candida species in vitro because of their higher infection of Candida species except C albuian they are highly susceptible to oral formulation so needs an alternative antifungal therapy. So partition coefficient of fluconazole is studied in different organic and phosphate buffer to identify the technique which is suitable for tropical use. physiochemical properties such as adsorption , partition coefficient are important to study for development of dosage form , so that behaviour of drug can be understood through which suitability and compatibility of drug can be predicated.{{59 Hajare,Ashok,A.Mali,Mahesh,N.Sarvagod,Sushil.Kurane,Sachin.Patwardhan,Shweta.and.Dange,Arun,S. April-June 2009;}}
Studies show that bioavailability of fluconzole is more concerned with IV and oral administration.
Pharmacokinetics of fluconazole
Forms of administration
1. Administration through intravenous injection by rapid bolus or infusion.
2. Oral administration through tablet, capsules or solution /suspension
Studies proven on voluntaries shows
Tissue penetration of fluconazole
Penetration in the tissue and fluid is important, fluconazole having low lipophility due to low affinity towards plasma proteins. It readily penetrates into body tissue by the means of passive diffusion. Long plasma life gives better distribution phase, which is necessary for fluconazole, available at the site of infection during implementing dosage. It has also properties to penetrate through CSF by the oral means.
Correlation to studied, fluconazole posses good pharmacologic properties due to rapid absorption due to water solubility, high oral bioavailability, and volume of distributation is high due to the relatively high plasma concentration along with high oral bioavailability in feeding and fasting state. In gastroinstenial disease it can be also admistred due to proper absorption, which shows extreme distbutation of fluconazole in body .Due to its low affinity for plasma proteins. {{58 Brammer, K.W.Farrow, P.Rand.Faulkner, J.K. March-April 1990 ;}}
Buffer Solutions
Method: Part one
First prepare four different solutions A, B, C and D in following concentration
Solution A, 30ml of sodium acetate (CH3COONA), with 0.1m was added with 30ml of acetic acid (CH3COOH) with 0.1m.
Solution B was also prepared by adding 20ml of sodium acetate (CH3COONA) with 50ml of acetic acid (CH3COOH) with 0.1m of both.
Solution C, prepared by adding 10ml of sodium acetate (CH3COONA) with 50ml of 0.1m acetic acid (CH3COOH) .
Lastly Solution D was prepared with 50ml of sodium acetate (CH3COONA) with 100ml of 0.1m, CH3COOH.
Next step was to measure the ph by PH meter of solution A, B and C along with its temperature, values are noted.
Part second:
It involves the titration of 50ml of solution D with HCL along the addition of 2ml of HCL each time and measurement of PH to be obtained until the total volume up to 90ml. Reading of PH was obtained by titertaion of solution D with 0.1m HCL.
TABLE
52ml
4.28
54ml
4.17
. . 56ml
4.08
58 ml
3.98
60 ml
3.83
62 ml
3.65
64 ml
3.35
66 ml
2.78
68 ml
2.33
70 ml
2.17
72 ml
1.93
74 ml
1.84
76 ml
1.78
78 ml
1.72
80 ml
1.65
82 ml
1.63
84 ml
1.65
86 ml
1.59
88 ml
1.55
90 ml
1.54
Part third
It involves the titration of 50ml of solution D with 0.1m of NAOH. Adding 2ml of 0.1m NAOH each time along with notification of PH value until the total 90ml volume, 20 different reading where obtained for Alkali.
TABLE
52ml
4.61
54 ml
4.45
56 ml
4.56
58 ml
4.62
60 ml
4.67
62 ml
4.75
64 ml
4.82
66 ml
4.84
68 ml
5.06
70 ml
5.01
72 ml
5.11
74 ml
5.16
76 ml
5.26
78 ml
5.39
80 ml
5.72
82 ml
5.78
84 ml
6.22
86 ml
9.88
88 ml
11.14
90 ml
11.47
Note: each time tip of PH meter is to be cleaned with distilled water in order not to vary the readings.
Readings of both acid and alkali were obtained to plot a graph and calculate the value of pKA and Ka along with the buffer capacity of both. Experiment was performed in the group of three.
Method
To determine partition coefficient of fluconazole .Two different solution where prepared, solution A and Solution B
Part first
Preparation of solution A
Phosphate buffer of PH 7.4, in which 0.1w/v fluconazole was dissolved for preparing stocking solution A.
Solution A was used to prepare different range of calibration of each 1ml containing 5,10,15,20,25,30,35,and 40 ug/ml of fluconazole in phosphate buffer of PH 7.4, seven readings where obtained each of standard lammad max 260nm, with the help of phosphate buffer PH7.4 , it was used as a blank reading to calibrate the absorbance for each time .Reading noted are shown as follows.
Concentration of fluconazole Absorbance of lammad max 260nm
5 0.007
10 0.021
15 0.06
20 0.043
25 0.075
30 0.083
35 0.103
40 0.092
Part second
Preparation solution B was prepared with 0.01% w/v of fluconazole dissolved in 1-octanol. Second part involves the preparation of partitioning solution in four different funnels. First separating funnel should be checked for the leakage, to fit the tap at its bottom, so that after loading the solution it should not be leaked. Four different samples were prepared as follows
First solution involves 5ml solution B with 20ml of 1- octane and 25ml of phosphate buffer.
Second solution, 10ml solution B with 15ml of 1- Octanol and 25ml of phosphate buffer.
Third solution , 15ml of solution B with 10ml of 1- octanol and 25ml of phosphate buffer.
Fourth solution, 20ml of solution B with 5ml of 1-octanol and 25ml of phosphate buffer.
Four solutions are needed to be shaken gently as soon as they are prepared. Shaking for about 30-60min is needed. After shaking check weather two clear layers are appeared in the funnel. Time taken was about 55min of shaking for getting the two different layers. After getting the layers separate attach funnel to the holder stand. Amongst the two different layers, lower layer was water and upper layer was octanol. This layer where separated in two different 50ml tubes as octanol and water. Total four pairs, each of octanol and water. Last part was to recoding the absorbance on UV visible spectrophotometer of lammad 260nm of water and octanol phase. This can be shown as follows
Reading
Organic Buffer
0.041 0.172
0.096 0.150
0.106 0.044
0.133 0.166
Lab Practical Results:
Ph of three solutions where noted as follows
Solution A ph 4.82.
Solution B ph 4.41.
Solution C ph 4.13.
Calculate pka=ph+log [base/acid] pka = -log ka , so Ka = antilog pka
Concentration of base = volume /total volume* molarity.
Solution A
Pka = 4.82
Ka = 1.5*105
Solution B
Pka = 4.01
Ka =1.0*105
Solution C
Pka = 3.42
Ka =2.6* 105
Plot graphs of pH against volume of 0.1M HCl and pH against volume of 0.1M NaOH on the same sheet of graph paper and calculate the buffer capacity with respect to both acid and alkali.
Graph
C:\Users\hp\Desktop\pras.png
Buffer capacity:
Buffer capacity of acid = 15.05ml
Buffer capacity of base =27.02ml
The concentration of fluconazole in the water and octanol phase, Cwat, Corg, respectively, for each composition of the partitioned mixture from the standard curve.
Graph
C:\Users\hp\Desktop\prashant cc.png
Calibration curve, slope is obtained as follows
Slope of the line is 0.0027
Now concentration of fluconazole in organic and buffer
For the solution 1
Concentration of fluconazole in buffer = 15.18
Concentration of fluconazole in octanolol =63.70
For the solution 2
Concentration of fluconazole in buffer = 35.55
Concentration of fluconazole in octanolol =55.55
For the solution 3
Concentration of fluconazole in buffer = 39.25
Concentration of fluconazole in octanolol =16.29
For the solution4
Concentration of fluconazole in buffer = 49.25
Concentration of fluconazole in octanolol =61.48
Calculate the partition coefficient Kow of fluconazole at each of the partitioned mixture,
Kow = Corg/Cwat
Kow =0.172/0.041 = 4.19
Kow =0.150/0.096 =1.56
Kow =0.044/0.106 =0.41
Kow =0.166/0.133 =1.24
Express the average value of the partition coefficient Kow from four experiments, and compare it with values from literatures.
Average value 1.85
, standered range shows the lipophilicity value in the range between -0.89 to 2.21.so average value falls in the range of standard value.
{{60 Alimuddin,Muhammad.Grant,Daniel,Bulloch,Daryl.Lee,Noelle.Peacock,Martin.and.Dahl,Russell. May 6 , 2008;}}
Calculate the distance from the active ser oxygen to the C- terminal carbonyl carbon of the peptide?
Ans: Distance between His 57 and Ser195: 8.30 A
Asp 102 and His 57 :6.47A
C:\Users\hp\Desktop\prashant 1.png
Calculate the distance from nitrogen to the peptide carbonyl oxyg5en, what do these imply?
Ans: 2.82A, shows hydrophobic properties
Peptide specification
C:\Users\hp\Desktop\manaoj 4.png
Identify the location of loops and amino acids type. Compare these to the equivalent residue in trypsin .pdb?
Ans: Amino acids: PRO, SER, GLY, ALA, VAL.
Location: AA- Amino Acid
A A ATOM X, Z, Y ATOM CORDINATE ATOM B FACTOR
PRO 225 C 26.376 9.625 45.862 9.51
SER 221 O 23.446 4.945 48.704 9.61
SER 214 CA 28.692 7.173 36.496 8.10
GLY 216 CA 28.971 5.629 43.557 12.57
ALA 185 CA 22.264 10.587 49.513 7.42
VAL 188 CA 17.455 6.480 46.705 7.05
C:\Users\hp\Desktop\prashant05.png
Calculated the surface in the context of residue 16-245.from chymotrypsin?
Ans : The surface context residue from 16 to 245 from chymotrypsin can be drawin as follows
Area : 8021
Volume:28018
C:\Users\hp\Desktop\prashant mol.png
(Due to some errors of software it shows two images)
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