Pharmacokinetic Evaluation of Amoxicillin/Clavulanicacid And

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PHARMACOKINETIC EVALUATION OF AMOXICILLIN/CLAVULANICACID AND

ANTACID INTERACTION

Anab Fatima B.Pharm, M.Phil.(Pharmaceutics)

Thesis submitted for the award of degree of

Ph.D

In Pharmaceutics

DEPARTMENT OF PHARMACEUTICS

FACULTY OF PHARMACY

UNIVERSITY OF KARACHI.

In the name of ALLAH

The most Beneficient and tha most Merciful……………….

ALLAHUMA SALLEY ALA SYEDENA MAULANA MUHAMMEDUN WA ALA AALEHYY WA ASHABHI BARIK WASALLIM WA SALLO

ALEHEY

SURAT AL-FATIHAH

1. IN THE NAME OF ALLAH,THE MOST GRACIOUS,THE MOST

MERCIFUL.

2. ALL THE PRAISES AND THANKS BE TO ALLAH,THE LORD OF

THE A LAMIN(MANKIND,JINN AND ALL THAT EXIST).

3. THE MOST GRACIOUS,THE MOST MERCIFUL.

4. THE ONLY OWNER(AND THE ONLY RULING JUDGE)OF THE

DAY OF RECOMPENSE(THE DAY OF RESURRECTION).

5. YOU(ALONE) WE WORSHIP,AND YOU(ALONE) WE ASK FOR

HELP(FOR EACH AND EVERY THING).

6. GUIDE US TO STRAIGHT WAY.

7. THE WAY OF THOSE WHOM YOU HAVE BESTOWED YOUR

GRACE,NOT(THE WAY) OF THOSE WHO EARNED YOUR

ANGER NOR OF THOSE WHO WENT ASTRAY.(AAMEEN)

DEDICATED TO:

My beloved Parentral Grand Father(late),Grand Mother(late),Parents and all those persons whose prayers bring me to

this stage.

ACKNOWLEDGEMENT “If ALLAH assist you, then there is none that can overcome you” (Holy Quran 3:160) All praise to Almighty Allah who gives me strength, confidence and courage to complete the thesis. I am heartiest thankful to my research supervisor Prof.Dr.S.Baqir.S.Naqvi for his valuable guidance, sincere advice, moral support and encouragement throughout the research project. He was really much helpful during research work for managing expensive chemicals and accessories for rapid completion of the project.

• I am really so much thankful to Dr.Rabia Ismail Yousuf and Mrs.Rehana Saeed Assistant Professor Department of Pharmaceutics, University of Karachi for their kind and humble cooperation throughout my research project.

• I am also thankful to Dr. Mohammed Harris Shoaib, Mrs. Sabahat jabeen,Mr.Iyad Naeem,Dr.Shaista Assistant Professors and Ms.Fauzia Israr lecturer Department of Pharmaceutics,Faculty of Pharmacy,University of Karachi for their sincere suggestions and help whenever required during this research project. • Worthful acknowledgement to Prof.Dr.Dilnawaz Sheikh and Prof.Dr.Rafi Sheikh for their resilient support and reinforcement during the research work. • Special thanks to Prof.Dr. Javeid Iqbal Dean ,Faculty of Pharmacy,Hamdard University for his co-operation and valuable suggestions during my research project. • I am also thankful to each and every teacher including Prof.Dr.Fauzia, Prof.Dr.Nighat and Dr.Shahnaz for providing me knowledge in any way. • I appreciated all non-teaching staff of department of Pharmaceutics,University of Karachi for their co-operation during this work. • Thanks to my friend Miss. Shaheen Parveen who sincerely encouraged me during my research work. • I am also obliged to library incharge Mr.Azhar for providing me books during this research work. • I am thankful to Mr.Amjad and their Lab staff of Liaqat National Hospital for providing me plasma for my research. • I am so much thankful to Mr. Jibran and Mr.Ilyas from Imam Clinic for helping me to provide plasma from Husaini blood bank and for conducting my clinical study at Imam Clinic. • I am also indebted to whole administration of Imam Clinic including Dr.Ali Imam, Dr.Noman Palekar, Dr.Hisala and other HR and nursing staff for their humble Cooperation during my clinical study. • I am also thankful to Dr.Sajjad, Mr.Ghulam Abbas, Mr.Asim of Dr.Tahir Shamsi lab and Mr.Ayaz from Hamdard University for providing their support and help in sampling of blood during my study. • I am really thankful to Dr.Tanveer Abbas, Assistant Professsor department of Microbiology,University of Karachi for telling me useful links for literature survey. • Special thanks for my parents prayers that gave me strength during my work without which this work would not be completed. • Special thanks for my all family members especially my brothers Sheikh Abdul Khaliq,Sheikh Abdul Rehman and Sheikh Mohammed Jiyad for their kind cooperation during my research project. • I sincerely acknowledge and appreciate those volunteers namely Farrukh Najam, Mehdi Raza, Syed Zaad Ali, Mohammed Rafiq, Mohammed Asif Basheer, Syed Mohammed Mohsin Rizvi, Mohammed Uzair Shah, Waqas Ali, Adil Waqas and H.M.Bilal Anjum who participated in this research project. • Words for those who helped me in any way for the completion of this research work.

May ALLAH Bless Them All.(Aameeen)

TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………………….

KHULASA………………………………………………………………………………………

1.INTRODUCTION……………………………………………………………………………..1 1.1 Drug Interaction……………………………………………………………………………...2

1. 2.Drug-Drug Interactions(DDIs)……………………………………………………………...3

1.3. Pharmacokinetic Determination of Drug-Drug Interactions…………..9

1.4. Pharmaceutical analysis of drugs in dosage forms……………………..10

1.5. ANALYSIS OF ANTIBIOTIC USING HPLC…………………….…12

1.6. HISTORY/STORY OF PENICILLIN…………………………………14 1.6.4. PHARMACOKINETIC OF PENICILLIN…………………………………………..17

1.6.5. AMOXICILLIN/CLAVULANIC ACID(AUGMENTIN)……………………………18

2. OBJECTIVE OF THE STUDY……………………………………………………26-27

3. LITERATURE REVIEW…………………………………………………………..28-62

4. EXPERIMENTAL……………………………………………………………………...63

4.1.PHYSICO-CHEMICAL TESTS OF DIFFERENT CO-AMOXICLAV BRANDS…….64

4.1.1. WEIGHT VARIATION TEST………………………………………………………..64

4.1.2 THICKNESS VARIATION TEST……………………………………………………65

4.1.3. HARDNESS TEST…………………………………………………………………….65

4.1.4. DISINTEGRATION TEST……………………………………………………………65

4.1.5. DISSOLUTION TEST…………………………………………………………………65

4.1.6. CONTENT ASSAY FOR CO-AMOXICLAV TABLETS BY HPLC………………67

4.2. PHARMACOKINETIC EVALUATION OF CO-AMOXICLAV TABLET……..68

4.3. Validation Parameters………………………………………………………………….74

4.4. System suitability parameters………………………………………………………….74

4.4.1. Specificity ……………………………………………………………………………….74

4.4.2. Linearity…………………………………………………………………………………74

4.4.3. Intra-Day and Inter-Day Accuracy and Precision……………………………………74

4.4.4. Lower Limit of Quantification (LLOQ) and Limit of Detection (LOD)…………….75

4.4.5. Recovery…………………………………………………………………………………75

4.4.6. Stability………………………………………………………………………………….75

4.4.7. Freeze and Thaw stability……………………………………………………………...75

4.4.8. Long Term Stability…………………………………………………………………….76

4.5. Pharmacokinetic Analysis……………………………………………………………...76

4.5.1. Compartmental…………………………………………………………………………76

4.5.2. Non-Compartmental……………………………………………………………………76

4.7. SERUM BACTERICIDAL TEST (SBT)…………………………………………….77

5.RESULTS…………………………………………………………………………………80- 139

% RELATIVE BIO-AVAILABILITY………………………………………………………161

7.DISCUSSION……………………………………………………………………………162- 177

8.REFERENCES …………………………………………………………………………230-245

9.APPENDIX…………………………………………………………………………………..246

NATIONAL BIOETHICS COMMITTEE(NBC) PAK ISTAN APPROVAL…………..247

PUBLICATION FROM RESEARCH WORK……………………………………………..248

LIST OF PUBLICATIONS……………………………………………………………...... 249

LIST OF TABLES

TABLE 1……………………………………………………………………………………6 & 7

THE MOST DANGEROUS DRUG INTERACTION.……………………………………………………………………………6 & 7

TABLE 2………………………………………………………………………………………...83

DETAILS OF DIFFERENT AMOXICILLIN/CLAVULANIC ACID PRODUCTS AVAILABLE IN PAKISTAN………………………………………………………………….83

TABLE 3………………………………………………………………………………………...84

WEIGHT VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN………………………………………………………………….84

TABLE 4…………………………………………………………………………...……………86

THICKNESS VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN………………………………………………………………….86

TABLE 5………………………………………………………………………………………...87

HARDNESS VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN…………………………………………………………………87

TABLE 6………………………………………………………………………………………...90

DISSOLUTION OF DIFFERENT BRANDS OF AMOXICILLIN/CLAVULANIC ACID TABLET(250/125 mg) AVAILABLE IN PAKISTAN……………………………………….90

TABLE 7………………………………………………………………………………………91

HPLC ASSAY OF DIFFERENT BRANDS OF AMOXICILLIN/CLAVULANIC ACID TABLET(250/125 mg) AVAILABLE IN PAKISTAN…………………………………….....91

TABLE 8(a)……………………………………………………………………………………..92

CALIBRATION CURVE OF AMOXICILLIN STD in Human Plasma(Linearity,Accuracy and Precision)…………………………………………………92

TABLE 8(b)…………………………………………………………………………………….93

Back Calculated Concentration of Amoxicillin Standard in Human Plasma……………..93 TABLE 9(a)……………………………………………………………………………………..96

CALIBRATION CURVE OF CLAVULANIC ACID STD in Human Plasma (Linearity, Accuracy and Precision)………………………………………………………………………..96

TABLE 9(b)…………………………………………………………………………………….97

Back Calculated Concentration of Clavulanic Acid Standard in Human Plasma………97

TABLE 10(a)…………………………………………………………………………………100

INTRA-DAY PRECISION AND ACCURACY OF AMOXICILLIN IN HUMAN PLASMA(n=3)………………………………………………………………………………...100

TABLE 10(b)…………………………………………………………………………………..100

INTRA-DAY PRECISION AND ACCURACY OF CLAVULANIC ACID IN HUMAN PLASMA(n=3)……………………………………………………………………...…………100

TABLE 11(a)……………………………………………………………………………...101-102

INTER-DAY PRECISION AND ACCURACY OF AMOXICILLIN IN HUMAN PLASMA(n=5)……………………………………………………………………………101-102

TABLE 11(b)……………………………………………………………………………...103-104

INTER-DAY PRECISION AND ACCURACY OF CLAVULANIC ACID IN HUMAN PLASMA(n=5)…………………………………...……………………………………….103-104

TABLE 12(a)…………………………………………………………………………………..105

LOWER LIMIT OF QUANTIFICATION (LLOQ) FOR AMOXICILLIN……………..105

TABLE 12(b)…………………………………………………………………………………..106

Back Calculated Concentration of Amoxicillin……………………………………………..106

TABLE 13(a)…………………………………………………………………………………..107

LOWER LIMIT OF QUANTIFICATION (LLOQ) FOR CLAVULANIC ACID………107

TABLE 13(b)…………………………………………………………………………………..108

Back Calculated Concentration of Clavulanic Acid ……………………………………….108

TABLE 14(a)…………………………………………………………………………………..109

ABSOLUTE ANALYTICAL RECOVERY OF AMOXICILLIN…………………….....109 TABLE 14(b)…………………………………………………………………………………..110

ABSOLUTE ANALYTICAL RECOVERY OF CLAVULANIC ACID………………..110

TABLE 15(a)…………………………………………………………………………………..111

RELATIVE ANALYTICAL RECOVERY OF AMOXICILLIN………………………111

TABLE 15(b)…………………………………………………………………………………..112

RELATIVE ANALYTICAL RECOVERY OF CLAVULANIC ACID………………..112

TABLE 16(a)…………………………………………………………………………………..113

FREEZE AND THAW (F/T) STABILITY OF AMOXICILLIN…………………………113

TABLE 16(b)…………………………………………………………………………………..114

SUMMARY OF AMOXICILLIN STABILITY IN HUMAN PLASMA………………….114

TABLE 17(a)…………………………………………………………………………………..115

FREEZE AND THAW (F/T) STABILITY OF CLAVULANIC ACID…………………...115

TABLE 17(b)…………………………………………………………………………………..116

SUMMARY OF CLAVULANIC ACID STABILITY IN HUMAN PLASMA…………..116

TABLE 18(a)……………………………………………………………………………...117-118

LONG TERM STABILITY OF AMOXICILLIN……………………………………117-118

TABLE 18(b)……………………………………………………………………….……..119-120

LONG TERM STABILITY OF CLAVULANIC ACID…………………………….119-120

TABLE 19……………………………………………………………………………………..121

DETAILS OF THE VOLUNTEERS PARTICIPATED IN PHARMACOKINETIC STUDIES………………………………………………………………………………………121

TABLE 20……………………………………………………………………………………...122

PLASMA CONCENTRATION TIME PROFILE OF CO-AMOXICLAV (250/125) TABLETS IN 10 HEALTHY VOLUNTEERS……………………………………………...122 TABLE 21(a)…………………………………………………………………………………..123

COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF AMOXICILLIN………………………………………………………123

TABLE 21(b)………………………………………………………………………………...... 124

NON-COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF AMOXICILLIN………………………………………………………124

TABLE 22(a)…………………………………………………………………………………..125

COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF CLAVULANIC ACID……………………………………………….125

TABLE 22(b)…………………………………………………………………………………..126

NON-COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC

PARAMETERS OF CLAVULANIC ACID……………………………………………….126

TABLE 23……………………………………………………………………………………...127

PLASMA CONCENTRATION TIME PROFILE OF CO-AMOXICLAV (250/125) TABLETS + ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS………………………127

TABLE 24(a)…………………………………………………………………………………..128

COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF AMOXICILLIN +ANTACID………………………………………..128

TABLE 24(b)…………………………………………………………………………………..129

NON-COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF AMOXICILLIN +ANTACID………………………………………..129

TABLE 25(a)…………………………………………………………………………………..130

COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF CLAVULANIC ACID+ANTACID…………………………………130

TABLE 25(b)…………………………………………………………………………………..131

NON-COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF CLAVULANIC ACID +ANTACID………………………………….131 TABLE 35….…………………………………………………………………………………159

SERUM BACTERICIDAL TEST DETERMINATION OF SIC OF CO-AMOXICLAV ON STAPHYLOCOCCUS AUREUS…………………………………………………………….159

TABLE 36……………………………………………………………………………………...160

SERUM BACTERICIDAL TEST DETERMINATION OF SIC OF CO-AMOXICLAV ON

ESCHERICHIA COLI………………………………………………………………………..160

TABLE 37……………………………………………………………………………………...161

% RELATIVE BIO-AVAILABILITY………………………………………………………161

LIST OF FIGURES

FIGURE 1……………………………………………………………………………………….15

STRUCTURE OF PENICILLIN……………………………………………………………...15

FIGURE 2……………………………………………………………………………………….19

STRUCTURE OF AMOXICILLIN…………………………………………………………..19

FIGURE 3……………………………………………………………………………………….19

STRUCTURE OF CLAVULANATE POTASSIUM………………………………………..19

FIGURE 4……………………………………………………………………………………….85

WEIGHT VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN…………………………………………………………………85

FIGURE 5………………………………………………………………………………………87

THICKNESS VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN…………………………………………………………………87

FIGURE 6………………………………………………………………………………………89

HARDNESS VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN……………………………………………………………….,.89

FIGURE 7…………………………………………………………………………………..94-95

VALIDATION CHARTS OF AMOXICILLIN LINEARITY, ACCURACY AND PRECISION…………………………………………………………………………………94-95

FIGURE 8……………………………………………………………………………………98-99

VALIDATION CHARTS OF CLAVULANIC ACID LINEARITY, ACCURACY AND PRECISION…………………………………………………………………………………98-99

FIGURE 9……………………………………………………………………………….132-134 PLASMA CONCENTRATION TIME PROFILE COMPARISON OF AMOXICILLIN(250mg) ALONE AND AMOXICILLIN(250mg) TABLET+ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS…………………….132-134

FIGURE 10(a)………………………………………………………………………………...135

MEAN PLASMA CONCENTRATION TIME PROFILE OFAMOXICILLIN (250mg) TABLET ALONE IN 10 HEALTHY VOLUNTEERS……………………………………135

FIGURE 10(b)…………………………………………………………………………………135

MEAN PLASMA CONCENTRATION TIME PROFILE OFAMOXICILLIN TABLET ALONE(250mg) +ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS………………...135

FIGURE 11………………………………………………………………………………136-138

PLASMA CONCENTRATION TIME PROFILE COMPARISON OF CLAVULANIC ACID ALONE AND CLAVULANIC ACID(125mg) TABLETS + ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS…………………………………………………………….136-138

FIGURE 12(a)…………………………………………………………………………………139

MEAN PLASMA CONCENTRATION TIME PROFILE OF CLAVULANIC ACID(125mg) TABLET ALONE IN 10 HEALTHY VOLUNTEERS………………..…..139

FIGURE 12(b)…………………………………………………………………………………139

MEAN PLASMA CONCENTRATION TIME PROFILE OF CLAVULANIC ACID TABLET ALONE(125mg) +ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS……139

ABBREVIATIONS AND SYMBOLS

WHO: World Health Organization FDA: Food and Drug Administration HPLC: High performance liquid chromatography EMEA: European Medicine Agency. BCS: Biopharmaceutical classification system. %AUC extra: Percentage of AUC extra with respect to AUC total. α:Overall distribution rate constant. ABE: Average bioequivalence. ANOVA: Analysis of Variance

AUC0-∞: Area under curve from time 0 to infinity.

AUC extra: Extrapolated AUC

AUC last :AUC from t=0 to last(last sampling time)

AUC total :AUC last + AUC extra.

AUMC extra :Extrapolated area under momental curve.

AUMC last :AUMC from t=0 to last.

AUMC total :AUMC last + AUMC extra. Cl: Total clearance

Cl R :Renal clearance C max: Maximum plasma concentration. Cº:Concentration at t=0. HVD: Half value duration. Ka: Absorption rate constant. Kel: Elimination rate constant MRT: Mean residence time T½:Half life T½Ka:Absorption half life T½Kel:Elimination half life T max: Time to reach maximum plasma concentration V c:Apparent volume of central compartment V d: Volume of distribution V ss: Apparent volume of plasma compartment V z: Apparent volume of distribution during the terminal phase. MBC: Minimum bactericidal concentration MIC: Minimum inhibitory concentration SBC: Serum bactericidal concentration. SBT: Serum bactericidal test. SIC: Serum inhibitory concentration. ADME: Absorption,distribution,metabolism and elimination ADRs: Adverse drug reactions

ABSTRACT

Every day various Pharmaceutical products being marketed by various Pharmaceutical companies and various problems arises after the product gets approved and marketed.As coprescription of various drugs is a common practice of health care practitioners in our country resulting in significant drug-drug interactions in local population and are very much challenging because one drug may alter bioavailability of any of the other drug at its receptor site. Mostly they resulted due to any significant change in ADME(absorption,distribution,metabolism and elimination).These interactions lead to serious adverse effects and sometimes lead to death.Some other factors like self-medication,lack of professional pharmacist at pharmacy, racial and ethnic differences contribute to development of ADRs(Adverse drug reactions).It also increase professional liability of Pharmacist that by knowledge of pharmacokinetic tools they point out various drug-drug interactions and provide appropriate guide line for better pharmacotherapy.

Drug-drug interaction study can be analyzed by various pre-clinical and clinical experimental ways.The present study mainly focused on compartmental and non-compartmental method of analysis.In this method pharmacokinetic drug-drug interaction analyze followed by statistical comparision of some parameters.It is relatively a simple and rapid method.If most of pharmacokinetic parameters analyzed showed a significant statistical difference demonstrate the manifestation of drug-drug interaction.

During the present study pharmacokinetic interaction between oral combination of amino penicillin and a beta-lactamase inhibitor i.e. Co-amoxiclav(250 mg amoxicillin + 125 mg clavulanic acid) with an antacid(10 ml) containing aluminium hydroxide,magnesium hydroxide and simethicone were evaluated.As it is most common practice of using this combination in local population not only prescribed but also as self medication.The data obtained was used to compare pharmacokinetic parameters of Co-amoxiclave alone and with simultaneous administration of antacid.Initially Physico-chemical test were performed for augmentin to check the quality of product.

The study was conducted according to FDA guide line .The study design was approved by the National Bioethics Committee , Ministry of Health, Government of Pakistan.Islamabad after critical ethical review and a written consent duly signed by volunteers has been taken.The study was a single dose,two treatment,two sequence cross over design on twelve healthy volunteers.An equal number of volunteers were assigned to each sequence.Blood samples were collected at 0.5,1,1.5,2,3,4,6, and 8 hrs. plasma was separated and concentrations were determined by validated HPLC techniques. Then different pharmacokinetic parameters were calculated by applying both compartmental and non-compartmental methods of analysis. The results of Co-amoxiclav alone were taken as standard pharmacokinetic results reported in Pakistani population while the results of Co-amoxiclav + antacid ( aluminium hydroxide, magnesium hydroxide and simethicone combination) were used to estimate extent of interaction.The (Mean±S.D) values of Cmax of amoxicillin alone and with antacid found to be 8.88±0.09 and 7.84 ± 0.06 µg/ml at Tmax of 2 ±0.00 hrs and 3 ± 0.00 hrs and for that of Clavulanic acid was 2.66 ±0.03 and 2.44 ± 0.02 µg/ml at Tmax of 1.5 hr and 2 hr respectively.For amoxicillin the values of AUC0-t(AUClast) and AUC

0-∞ were 33.33±0.70 and 37.89±0.70 alone and 36.25±0.43 and 41.64±1.15mg/ml.hr with antacid and for Clavulanic acid values were 7.05±0.11 and 7.70±0.16 alone and 7.54±0.35 and 8.27±0.81 mg/ml.hr with antacid.The values of absorption and elimination half lives were also calculated i.e. T½ Kel and T½ Ka and it were determined to be 1.32±0.09hr and 1.23±0.06hr for amoxicillin alone and 1.58±0.06hr and 1.62±0.03hr with antacid and for Clavulanic acid values were 1.25±0.17hr and 0.97±0.02hr alone and 1.58±0.08hr and 1.41±0.12hr with antacid sequentially.

The analysis of compartmental parameters also provided the values of apparent Clearance(CL) and Vd/F was decreased with antacid.Similarly for Clavulanic acid apparent Clearance and Vd/F was reduced with antacid.

Similarly values of rate constant of Co-amoxiclav at different phases were also compared with and without antacid.The values of absorption rate constant and elimination rate constant for amoxicillin alone were 0.55±0.026 hr¯¹and 0.51±0.026hr¯¹ and were 0.42±0.01hr-¹ and 0.41±0.008 hr-¹with antacid while the values for same parameters for Clavulanic acid were 0.70±0.016hr-¹ and 0.57±0.016hr-¹ alone and 0.43±0.06hr-¹ and 0.45±0.03hr-¹ with antacid.

Non-compartmental parameters mean residence time(MRT) and area under first moment time curve(AUMC) were also estimated.The values of MRT and AUMC for amoxicillin alone found to be 1.93±0.08 h and 137.16±4.00 mg/L.(h)² and it increase to 4.55±0.18 h and 206.32±5.09 mg/L.(h)² with antacid.For clavulanic acid values of MRT and AUMC were 3.61±0.10h and 25.02±0.65 mg/L.(h)² alone and 2.45±0.20 hr and 43.02±2.03 mg/L.(h)² with antacid respectively.As Co-amoxiclav follows one compartmental model therefore the values of K12 and K21 and distribution rate constant(α) did not calculated.Statistical analysis such as one way ANOVA for all pharmacokinetic data and unpaired t-test for Cmax was performed by SPSS 16.0 version.The analysis showed significant difference when co-amoxiclav used along with antacid.The combination was not proved to be suitable in various pharmacokinetic parameters and showed interactions.

Therefore in highly populated countries like Pakistan such type of studies must be conducted to know about the pharmacokinetic parameters within local populations and also to find out severe type of interactions relating to co-prescriptions of various drugs and the ways to cope up with the problems relating them.Such type of studies on local population also provide tool for effective and safe therapy.

KHULASA

INTRODUCTION

1. INTRODUCTION

Once a pharmaceutical product has been marketed by different pharmaceutical companies they claim a sense of balance between chemistry,pharmacology and pharmacokinetic of the product and because of various ethical issues pharmacokinetic and metabolic studies were mostly carried out only in animals and in vitro systems.When the drug product is approved and marketed ,only then we can estimate its safety profile.When these formulations are prescribed by the health care professionals we are unable to break the cycle of co-prescription of different drugs which ultimately resulted in various type of clinically significant interactions(Sorin E etal).Although it’s not possible for all health care practitioners to recognize all these interactions but it’s important to develope awareness of possible mechanisms and principles involved.

Several studies showed a confound interrelation-ship between various factors and drug response by human body(fig 1)

DRUG RESPONSE

Pharmacokinetics------pharmacodynamics------pathophysiology ↓

Drug delivery—input(dosing rate)------Drug concentration↔ Drug effects↔disease parameters

In plasma(biophase)

↓output(clearance)

Drug elimination

Fig1:Schematic diagram of Interrelation ship between drug response and various factors.

1.1 .Drug Interaction.

According to Leon Shargel “Drug interaction refers to an adverse drug response produced by the administration of a drug or co-exposure of the drug with another substance,which modifies the patient’s response to the drug.”A precipitant drug is the drug,chemical or food element causing the interaction and an object drug is the drug affected by the interaction.

Drug interactions includes:

1. Drug-drug interaction. 2. Drug-herbal interaction. 3. Food-drug interaction. 4. Pharmacogenetic interaction. 5. Chemical-drug interaction like interaction of drug with alcohol or tobacco. 6. Drug-laboratory test interaction such as chemical interaction.

1.2. Drug-Drug Interactions(DDIs):

Drug-drug interactions can occur by two ways i.e. either one drug will alter the concentration of another drug which is called as pharmacokinetic interaction or one may change the biological response of another called as pharmacodynamics interactions.

Pharmacokinetic drug interactions are very challenging to calculate and complex too because interacting drugs have unrelated actions.Most oftenly these interactions are due to alteration in ADME(absorption,distribution,metabolism and excretion) which ultimately bring change into amount and duration of drug’s availability i.e. bioavailability at the receptor site.

There are various mechanisms by which drugs interact kinetically with each other.These include:alteration in gastrointestinal absorption or in pH(either of G.I or urinary),complexation effect of food and alteration of G.I motility, distribution and metabolism(www.ibpassociation).

1.2.1. Absorption

The bioavailability(rate and extent of systemic drug absorption) of many drugs alter by another drug resulting in increased or decreased drug bioavailability at the absorption site.e.g tetracycline and quinolones antibiotics form complexes with divalent cations(calcium, magnesium or aluminium present in antacid) resulting in decreased bioavailability of antibiotics.On the other hand bioavailability of digoxin improves when taken after erythromycin which reduce floral inactivation of digoxin.This drug interaction not only occur in G.I but also can occur at other absorption sites like skin where epinephrine (a vasoconstrictor) reduce the percutaneous absorption of transdermal lidocaine or fentanyl. Sometimes these drug interactions which affect bioavailability in the G.I.T occur as a result of competition for carrier mediated drug absorption or alteration of intestinal blood flow.

1.2.2. Distribution

The ability of a drug to plasma protein binding and displacement interactions or tissue or cellular interactions will affect the distribution of the drug e.g. valproic acid reduces hepatic clearance of phenytoin and prevent its liver metabolism by dislocating phenytoin from plasma albumin protein binding sites. Similarly clearance of digoxin decrease by quinidine as a result of displacement of digoxin from both α₁-glycoprotien and albumin tissue binding sites resulting in high plasma concentration of digoxin.

1.2.3 Drug elimination and Clearance

Drug metabolism or hepatic clearance of some drugs can be affected by enzyme induction, enzyme inhibition, substrate competition for the same enzyme and alteration in hepatic blood flow by another drug. For example Phenytoin decrease tacrolimus levels by increasing metabolism and Cimetidine decrease atorvastatin clearance and increase drug levels. Sometimes interacting drugs share same drug metabolizing enzymes e.g. fluconazole inhibits the hepatic metabolism of Warfarin greatly increased the risk of bleeding. Any change in GFR, tubular reabsorption, active drug secretion, renal blood flow or nephrotoxicity may affect renal clearance of many drugs.For example Probenecid blocks the active tubular secretion of penicillin and some cephalosporin antibiotics.In the same way alkalinization of urine by antacid enhance the reabsorption of amphetamine with the reduction in clearance while alkalinization of urine pH increase ionization of salicylates ,reduce reabsorption and increase clearance.

It was estimated by Lazorou etal that incidences of adverse drug reactions(ADRs) increase and sometimes deaths are caused by these clinically significant drug interactions.There are so many reasons found for ADRs as many drugs are used as combination to treat patients, also great number of prescriptions are filled out for one person and the rate of ADRs increase exponentially if patient is on four or more medications(Jacubeit T etal).Various efforts are needed to reduce poly pharmacy but in some patients the number of drugs cannot be reduced therefore an understanding of possible drug-drug interaction is important for health care practitioners. It is also habit of some individuals to see more than one physician and it will be difficult for one prescriber to be aware of all drugs prescribed to the patients by multiple prescribers, for example if a physician prescribe an antihistamine to one patient and another prescribe anti-anxiety to the same patient then there may chances of excessive depressive effects.

The parallel use of prescription drug with a non-prescription drug (like aspirin, antacid, decongestants etc) by various patients by themselves reported various drug interactions. During questioning the patient neglect to mention the use of non-prescription drug as they are taking them from such a long period in a routine manner that they do not consider them to be drug.

The incidences of pharmacokinetic drug interaction also increased by the tendency of some individuals to abuse or knowingly misuse drugs.

In Pakistan OTC term is used for all kinds of medicines and they are in easy access from a literate person to a lay- man without prescription. More over most of pharmacies in Pakistan running by illiterate or lay-man personel. Even in case of antibiotics there is greater misuse and people are taking them on their own without consultation with the physician or Pharmacist. The role of Pharmacist is very important in the whole world but in Pakistan do not understand the importance of Pharmacist whose main responsibility is to understand the use of drug, their interactions and side-effects. They can play an important role in the detection and prevention of drug-related problems by maintaining patient medication record. It is important to encourage patients to purchase all medications whether prescription or non-precription from pharmacy owned by a professional Pharmacist, the role of federal and provincial Government cannot be refused in this matter. Along with antibiotic people also are taking other drugs along with them although they don’t need them. They will ultimately result is severe metabolic drug-drug interactions which are matter of great concern both for physicians and general public as a whole.

In February drug information and poison control center of GC university Faisalabad published a list of most dangerous drug interactions:

The Most Dangerous Drug Interactions: Drugs Side effects Comments

Alcoholic • Medications such as: 99.9% of all medications are not well tolerated Beverages hydrocodone, Vicodin, while drinking alcohol, but there are some drugs that you absolutely should avoid while Lorcet, or any other drinking. It is important to remember that medications created with alcohol acts as a central nervous system depressant, which normally slows heart rate, codine as a base. pulse, breathing, and blood pressure. If you are going to be taking a medication that will act as • Sublingual, buccal, or a depressant, it is not wise to compound the dermal nitroglycerines. effect with an alcoholic beverage.

• Various different asthma medications

• Antifungal antibiotics

• Certain antidepressants

Dairy Products: • Avoid with antibiotics There are certain components in milk that bind such as erythromycin, to the active ingredients in your antibiotics that will render the medications completely useless. tetracycline, Play it safe, and avoid eating any dairy one to vancomycin, and cipro. two hours before and after taking your meds.

• If it ends in “cycline” you should probably avoid taking with dairy.

Tyramine • Do not mix traditional Certain foods like chocolate, aged cheese, red containing MAOI’s (monoamine wines, Chinese food, avocados, and certain fish foods: contain a chemical called Tyramine. This oxidase inhibitor) with chemical compound can cause your body to foods containing release large amounts of adrenaline into your blood stream that will result in an increased tryramine…ever!

Ask your doctor if you are on a

traditional or non-traditional heart rate and blood pressure. This can cause a MAOI, it can make a huge stroke or a heart attack. Play it safe, and ask your doctor before you take any MAOI’s, or difference better yet, request that your doctor prescribe a non-traditional MAOI (SSRI’s like Prozac). This newer class of drug can offer the same benefit as the traditional MAOI’s, but without the diet restriction, that can be very difficult to maintain.

Leafy Greens • Lower your intake or Mixing a blood thinner and various greens can and avoid vegetables like; cause dangerous blood clots to form in your bloodstream, which can lead to heart attack and “flower/floret” spinach, broccoli, lettuce stroke. veggies: mixtures, radicchio, and cauliflower when taking blood thinning drugs such as Warfarin, Coumadin or Dicumarol.

• If eliminating these foods from your diet is impossible, talk to your doctor about lowering or raising your dose as appropriate.

Oral • Mixing your oral A little known fact is that taking an antibiotic contraceptives: contraceptives with while on an oral contraceptive can weaken the effects or sometimes, completely negate the ANY antibiotic. effects.

Table 1: The most dangerous drug interactions

Most of the enzymes involve in the metabolism of drugs are polymorphic i.e. they have more than one variants of genes although CYT isozymes have similar characteristics but due to polymorphism there will be variation in metabolism of drugs from population to population in efficacy, toxicity or side effects of different interacting drugs. In Pakistan the pharmacokinetic data of different drugs being used by physicians based on data obtained from other populations and it is not promising that it will produce same effects in local population as that of where drug is being manufactured.It may possible that it will produce severe interaction when given with other drugs as there may difference in pharmacokinetic parameters from population to population .Therefore it is important to develop and established guide lines to perform pharmacokinetic interaction studies in local population.

Another very important thing is “spacing between drugs”. One of the major cause for pharmacokinetic drug-drug interaction is that peoples are taking two or more drugs at the same time e.g. an antacid and H2-blocker should not be given at the same time as they may interact with each other. A proper information regarding timing of drugs should be given by Pharmacist to the patient to reduce such incidents.

It can be said that with the advent of newly approved drugs our therapeutic armamentarium has been inflated but it also increase the potential for drug-drug interactions.These drug-drug interactions causes a change in pharmacokinetic of drugs which can cause secondary amendment in pharmaco-dynamic of drugs. These drug-drug interactions depends on those factors which are related to drugs (like dosage of antibiotic, base line concentration of interacting drugs and its therapeutic index),patient (inter-individual variance) and administration(scheduling of other drug, duration of therapy with antibiotic).An inter-individual variability in the extent of metabolism resulting in a major alteration in the rate of elimination and plasma concentration time profile ultimately develop severe drug-drug interaction in some individuals(Mastour etal).All these factors dramatically increase the professional responsibility of Pharmacist for proper selection of drugs specifically where various competitors are present and to determine pharmacokinetic parameters and bioequivalence studies of drugs before their introduction into market. The role of the pharmacist is very important to choose right drug with right dose according to genetic factors and clinical response. By the Knowledge of pharmacokinetic tools Pharmacist study mechanism of

drug-drug interaction and implement them for better management of pharmacotherapy including anticipation of clinically significant drug interactions.

1.3. Pharmacokinetic Determination of Drug-Drug Interactions:

Drug-drug interaction study can be analyze by various ways including compartmental and noncompartmental method of analysis. The non-compartmental pharmacokinetic analysis is the most commonly used method. In this method pharmacokinetic drug-drug interaction analyze followed by statistical comparision of some parameters.It is relatively a simple and rapid method. The parameters which are calculated in this method include AUC(observed and/or total), Cmax, Tmax and t ½.The difference between the calculated parameters before and after interaction evaluated statistically with the help of ANOVA or t-test for paired values. If most of pharmacokinetic parameters analyzed showed a significant statistical difference demonstrate the manifestation of drug-drug interaction.

By applying bio-equivalence study test over pharmacokinetic parameters of drug administered alone(reference) versus along with another drug (test) we can find prevailing significant statistical difference in these parameters. But these finding need further evaluation of clinical parameters like therapeutic efficacy, signa of side effects etc for their clinical significance.

In compartmental analysis we can obtain additional information beside that provided by noncompartmental analysis but the data obtained in this method cannot be fitted in a suitable model so it is not a preferable way of pharmacokinetic determination of drug-drug interaction. In the compartmental pharmacokinetic method of analysis we can estimate that interaction between two drugs may be due to two factors i.e. either increase of bioavailability or a decrease in apparent clearance of the first drug by the second one so in this we can relate different physiologic phases of drug metabolism with bioavailability and clearance(Sorin E etal)

There are various factors which affect the bioavailability of a drug like drug product formulation, physiological factors affecting bioavailability and Physico-chemical factors (Myers 1988).

1.4. Pharmaceutical analysis of drugs in dosage forms:

A very high level of analytical and chemical support requires at every stage of drug manufacturing in order to ensure safety and quality of drug. There are various recommended procedures provided by pharmacopeia for the analysis of pharmaceutical substances, excepients and dosage forms and their interactions with other drugs prior to their use in patients. During various steps of drug development and manufacturing and to determine pharmacokinetic parameters the most important used analytical technique is the separation technique: High Performance Liquid Chromatography: HPLC(Shulamit Levin 2010).It became most important analytical method for identification and quantification of drugs for discovery, development and manufacturing in the pharmaceutical and bio-medical analysis. The assay of pharmaceutical formulations also requires studies of dissolution properties of solid dosage forms. Therefore in order to study the mechanism of drug interaction pharmacokinetic is a useful tool and with the help of this, important facets of human pharmacology can be explicated.

1.4.1 Dissolution:

Dissolution may be defined as “Process by which a solid substance enters in the solvent to yield a solution, stated simply, dissolution is the process by which a solid substance dissolve. Actually it is controlled by the affinity between the solid substance and the solvent”(Banakar 1992).

Whenever a tablet is ingested after passing through esophagus it reaches to stomach and initially tablet dissolve in the stomach where an aqueous environment exist. By controlling the rate of dissolution the duration of drug effect can be controlled. There are several dosage forms available having same active ingredient but with different rate of dissolution. Drugs with longer duration of action not readily dissolved and may not only improve compliance but also maintain concentration within therapeutic concentration range over a long period of time.

The rate of dissolution is described by Noyes-Whitney equation as:

Where:

 dW/dt = rate of diffusion  A =surface area of solid  C =concentration of solid in the bulk dissolution medium  Cs = concentration of solid in the diffusion layer surrounding the solid.  D = diffusion coefficient  L = diffusion by layer thickness

It is cleared from above equation that by changing surface area(by altering the particle size e.g. micronization) of the solid the rate of dissolution may be modified. A reduction in the dose may be required in order to achieve same therapeutic effect for many drugs by reducing the particle size however it should be noted that it must not affect the solubility of the drug. By selecting a suitable polymorph of a compound rate of dissolution may also be modified because different solubility and dissolution rate characteristics exhibited by different polymorph. The amorphous form dissolve quicker as compare to crystalline form which require more energy to leave lattice during dissolution. Anhydrous and hydrous form of drug exhibit different dissolution rate and anhydrous dissolve faster then hydrous but sometimes anhydrous form shows lower solubility.

Solubility can also be controlled by chemical modification i.e. esterification e.g. in gastric fluid searate andestolate esters of drugs are poorly soluble but these are hydrolyze by esterases in the GIT wall and blood to release drug from parent drug.

A barrier to modify the rate of dissolution is coating on tablet or a pellet e.g. enteric coating prevent the release of drug before reaching intestine and allow to dissolve in the basic environment of the intestine(Kaplan Pharmacology 2010).

1.4.2. Bio-analytical studies of drugs and their interactions:

In pharmacokinetic studies the assay of drugs in biological fluids samples in order to determine ADME(absorption, distribution,metabolism and elimination) is very essential. The biological samples are physiological fluids such as plasma, serum,tissue extract and urine from experimental animals or human subjects. In these biological analysis the HPLC methods developed, validated and transferred and these methods involves quantification of drugs in biological samples, its assay, impurities, stability, dosage form content uniformity, dissolution and there interaction studies between drugs(Shulamit Levin 2010).During method validation the important parameters tested

defined by ICH,USP and FDA and other health organizations are selectivity, precision, accuracy, linearity, limit of detection, limit of quantitation and robustness(CDER,ICH,USP-NF).Generally the serum and plasma contain an equivalent concentration of drug as that contained in human blood which may vary from high µg/ml to low as ng/ml. The knowledge of pharmacokinetic parameters like maximum plasma concentration, plasma half life and volume of distribution used to determine dosing schedule to achieve desired therapeutic levels within therapeutic range and then the difference between calculated parameters before and after drug-drug interaction will evaluated statistically and compared by using ANOVA and t-test for paired values. These analytical methods(HPLC,GC with MS) can be used to assay drug in biological fluids at very low sensitivity limit in the ppb(parts per billion) range. In the last decades reversed phase(RP) HPLC methods using ultraviolet, flouresence or electrochemical detection provide a way of eagerly determining ppb levels of drugs(Smith 1988).

1.5. ANALYSIS OF ANTIBIOTIC USING HPLC:

HPLC is a chromatographic technique used for same purpose of identification, quantification and purification of individual components of the mixtures (Wikepedia).These assay methods have remarkable role in determining the quality control of various bulk drugs, pharmaceutical formulations and their interactions. They are also useful in determination of drugs and their metabolites in biological samples when coupled with mass spectrometery. Due to high sensitivity and selectivity when coupled with mass spectrometery become the leading method in bio-assay, pharmacokinetic and metabolic studies.(Wieslawa Misiuk,2010).Many antibiotics(like betalactamase) have serious stability problems and their analysis required a very rapid and highly specific methods whether in formulated or in unformulated forms. In characterization of their analysis HPLC techniques provide valuable tools in every aspects of developing quality products and determining their interactions(Shalini,2002).

Mainly all HPLC techniques follows the same basic priciples,i.e.

1.Extraction of drug from plasma with a particular solvent, some times double extraction is required where drug is highly water soluble.

2.Separation of drug into solid phase by the help of HPLC. 3.Detection of effluent by Spectrometery.

4.Quantitation of amount of antibiotic by peak height analysis or peak area analysis.

In routine operation HPLC utilizes different types of stationary phases(i.e. sorbents) contained in columns, a pump and a detector providing characteristic retention time and the amount of each analyte passing through the detector. Small size columns(250 mm or shorter and 4.6 mm i.d. or smaller packed with smaller particles) can be utilize in HPLC and also higher mobile phase pressure can be use (up to 2000psi) as compare to other liquid chromatography. The Higher pressure provided by pump(rather then gravity) to move the mobile phase and sample through the densely packed column and the increased density by the use of smaller sorbent particles provide better separation of samples in the columns. The composition of mobile phase may vary and mostly it depends on the degree of interaction between analytes and stationary phase(e.g. hydrophobic interaction in reversed phase HPLC).The nature of column and the sample components will determine the choice of mobile phase components i.e. additives (like salt or acids) and gradient conditions. There are various important parameters which influence the detection sensitivity and separation selectivity in gradient elution like internal diameter of column, particle size and pore size of stationary phase and pump pressure(Wikepedia).

In routine practice for the estimation of false peaks(bubbles) and for removal of microparticulate particles mobile phase is degassed by sonication and filtered. The organic stationary phase mainly composed of C8 and C18 aliphatic chains and chemically used to microparticulate, porous silica of 5-10 micrometer mean particle diameter, densely and uniformly cased in stainless steel sleeve. For an efficient column as a rule of thumb a single theoretical plate must be 2-5 times mean particle diameter of support phase with overall efficiency of 300 to 5000 theoretical plates. Normally internal diameter of sleeve is 2-5 times with the column length vary from 3cm to several meters. Mobile phase flow at the rate of 1-3ml/min requiring pressure upto 2000psi to maintain the flow and to attain chromatographic time and pH range below 7-7.5 although non-siliceous packing material are also available which work well in the basic range. A preferred amount of 5-100 µl of sample are injected and retention time is less than 30 min. Current HPLC systems routinely work in nanogram range with between day coefficient of variation usually less than 5 -7 %.(Synder and Kirkland 1979).The biological fluids contain a variety of proteins, carbohydrate and lipids and they not only produce deleterious effects on injector, column packing materials and pump but can also interfere with the separation and quantities of analyte molecula in the study therefore in the

assay of biological fluids including antibiotic it is necessary to remove proteins as much as possible by a suitable de-proteinizer like trichloroacetic acid or acetonitrile etc(Lorian 1991).

1.6. HISTORY/STORY OF PENICILLIN:

In the year 1929 a pioneer man Alexander Fleming first time observed the antibacterial effect of Penicillin miraculously. He found that when an agar plate was streaked with the bacterium Staphylococcus aureus then a fungal colony grown around as a contaminant and he also found bacterial colonies transparent around fungus as their cells were lysing. He was so much devoted person and he spent a lot of time to find way to treat wound infection, at that time he instantaneously recognized the importance of fungal metabolite which could be used to control growth of bacteria. Later the fungal contaminant was identified as Penicillium notatum that’s why given the name as Penicillin. Fleming identified its antibacterial activity against many Gram- positive bacteria in the laboratory and he also prepared a locally applied, crude preparation from this culture filtrate of Penicillin to treat eye infection. Unfortunately he was unable to purify this compound because of its instability until the period of second world war(1939-1945).After that two british Scientists, Florey and Chain contribute this effort working in USA managed to produce the antibiotic on industrial scale for general public use.All these three scientists got noble prize for their work and it become considered as wonder drug which saved millions of lives during that time and although the emergence of resistance limits its effectiveness but still upto yet it is consider as front-line antibiotic to treat various bacterial infections(www.pharmainfo.com).

1.6.1. CHEMISTRY/STRUCTURE OF PENICILLIN:

Penicillin is not a single compound but actually it is a group of closely related compound ,all of these compounds have same basic ring like structure(beta-lactam) derived from two amino acids (valine and cysteine) by a tripeptide intermediate.The third amino acid of tripeptide replaced by an acyl group(R) and different type of Penicillin confers specific properties by the nature of this acyl group(www.pharmainfo.com).All Penicillins are acidic in nature and are acid labile and in neutral and basic solutions their beta-lactam ring opens and result in inactivation or degradation

of Penicillin but over the years this family of antibiotic has been grown and at least 20 kinds of Penicillins are available today. Newer Penicillins are resistant to stomach acid such as Penicillin V or have broader spectrum such as ampicillin and amoxicillin(YuryBayarski).

Figure 2: Structure of Penicillin

1.6.2. MODE OF ACTION OF PENICILLIN:

The bactericidal effects of all Penicillins is exhibited by inhibition of bacterial cell wall synthesis. They prevent the cross linking of peptides on the mucosaccharide chain.The enzyme transpeptidase is inhibited irreversibly by Penicillin in a way that it react with serine residue in the transpeptidase and as a result the growth of cell wall is inhibited (yocumetal 1980).A hypothesis was given by Tipper D etal that Penicillin is a structural analog of acyl-D-analyl-Dalanine terminus of the penta-peptide side chain of nascent peptidoglycan and the active site of cell wall transpeptidase was irreversibly acylate by Penicillin due to its highy reactive betalactam structure.The Penicillin mode of action considered as much interesting as it prevent the cross linking small peptide chains in peptidoglycan that is the main polymer of bacteria. It does not affect already existed cells but newly produced cells grow abnormally and their wall rigidity no longer maintained and made them vulnerable to osmotic lysis (www.pharmainfo.com).The Penicillin derivatives may act as bactericidal(kill the bacteria) or they may act as bacteriostatic (stop the growth of bacteria).If there is an adequate concentration of Penicillin in the body then

they act as bactericidal against sensitive micro-organisms, the presence of inadequate concentration of Penicillin resulted in bacteriostatic activity which may or may not control the infection (YuryBayarski).The bacteriostatic response of Penicillin was observed in certain bacterial species like Streptococcus mutants and in mutants of Streptococcus pneumonia(Tipper DJ).One of the structurally novel beta-lactam antibiotic i.e. CP35587 which is a modified Penicillin developed by Joseph Presslitz and it showed that transpeptidase is not a killing site for Penicillin in E-coli and K.pneumoniae and this is not a mode of action in case of CP35587 a bactericidal Penicillin. Hence the mode of action of beta-lactam antibiotics vary among species.

1.6.3. CLASSIFICATION OF PENICILLIN:

On the basis of ability to kill various types of bacteria and from narrow to broad range of effectiveness the Penicillins are classified as:

1.6.3.1. Natural Penicillin(Penicillin G.Penicillin V:

In the Penicillin family natural Penicillin were the first agent to be presented for clinical use. Their chemical structure based on the original Penicillin G structure. They have good coverage against Gram-positive strains of Streptococci, Staphylococci and some Gram-negative bacteria like meningococcus. A modified form of Penicillin is Penicillin V with good acid stability and immediate solubility and absorption specifically used against Staphylococcci, Streptococci and Pneumococci. They can be hydrolyze by beta-lactamase enzyme.

1.6.3.2. Antistaphylococcal/Penicillinase resistant Penicillin:

Because of increasing resistance in Staphylococci against natural Penicillin anti-staphylococcal Penicillin or Penicillinase resistant Penicillin were developed. The group comprised of methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillinetc.A narrow spectrum of activity produced by Penicillinase resistant Penicillin as compared to natural Penicillin. They are developed to produce their antimicrobial activity only against Penicillinase producing strains of Gram-positive cocci, particularly Staphylococcul species.The use of methicillin has been banned in USA because of reports of occurrence of interstitial nephritis.

1.6.3.3. Aminopenicillin:

The first Penicillins discovered to be active against Gram-negative bacteria such as E-coli and H- influenza were aminopenicillin i.e. ampicillin, amoxicillin and bacampicillin. They are acid stable and may be taken with food. Amoxicillin showed much higher and sustained blood antibiotic levels than other Penicillin and better absorbed from gastrointestinal tract. Amoxicillin and ampicillin were indicated to treat mild infections like otitis media, sinusitis, bronchitis,UTI and bacterial diarrhea. Amoxicillin is a good drug of choice for otitis media.

1.6.3.4. Extended Spetrum /Anti-pseudomonal Penicillin:

This group include both alpha-carboxycillin (carbenicillin and ticarcillin) and acyl amino Penicillins (piperacillin,azlocillin and mezlocillin).The spectrum of activity of these antibiotic is similar to amino-penicillin but with some enhanced activity against various Gram-negative organisms of family Enterobacteriacae. They may used alone or in combination with aminoglycoside to treat several mild to moderate infections. They are also like aminopenicillin are susceptible to degradation by beta-lactamase enzyme(Yury Bayarski).

1.6.3.5. Beta-Lactam/Beta-Lactamase Inhibitor Combination:

Many Penicillins like ampicillin, amoxicillin,ticarcillin and piperacillin available in combination with one of several beta-lactamase inhibitors: Clavulanicacid,salbactam or tazobactam. This combination extended the activity of these Penicillins against beta-lactamase producing strains of S.aureus and some of Gram-negative bacteria(Katzung).Amoxicillin/Clavulanic acid is only an oral combination with bata-lactamase inhibitor and it has good antimicrobial coverage against beta-lactamase producing bacteria(Yury Bayarski) .

1.6.4. PHARMACOKINETIC OF PENICILLIN:

Different Penicillins exhibit different behavior of absorption after oral administration and it mainly depends on their acid stability and protein binding. Both methicillin and nafcillin were not suitable for oral administration just because of acid labile nature of methicillin and erratic nature of nafcillin. Serum concentration in the range of 4-8 µg/ml after 500mg oral dose produced by dicloxacillin, ampicillin and amoxicillin considered as acid stable and relatively well absorbed. Presence of food may impaired oral absorption of most oral Penicillin (Except amoxicillin) could be overcome by 1-2 hours before or after a meal.

A rapid absorption of most Penicillin observed after parentral administration.Intramuscular injection of large dose produced irritation and local pain therefore intravenous route is preferred. After 30 min of intravenous injection of 1g of Penicillin serum concentration of 20-50µg/ml observed. Very little amount of free drug is present in serum which can be determined by protein binding. Nafcillin produced lower level of free drug in serum as it is highly protein bound Penicillin as compared to less protein bound Penicillin (e.g Penicillin G,ampicillin).In body fluids and tissues Penicillin were widely distributed , polar in nature and the concentration in extracellular fluid has been greater than within cells.

A prolonged blood and tissue concentration resulted by delayed absorption of formulated benzathine and procaine penicillin. Serum levels in excess of 0.02 µg/ml for 10 days in excess of 0.003 µg/ml for three weeks were maintained after single I.M injection of 1.2 million units of benzathine penicillin.Similarly peak concentration of 1-2 µg/ml and clinically useful concentration were maintained for 12-24 hours after a single I.M dose of 600,000 units of procaine penicillin.

In most tissues the concentration of Penicillin similar to those present in serum. In the ratio of 315% of those found in serum excreted in the sputum and milk. Penicillin has poor penetration into eye, prostate and the CNS but penetration improved in case of inflamed meninges and could achieved a concentration of 1-5 µg/ml at 18-24 million units of daily parentral dose which was sufficient enough to kill susceptible strains of pneumococci and meningococci.

Major amount of Penicillin excreted through kidney(10% is by GFR and 90% by tubular secretion) and a small amount were excreted by other route. Penicillin G have normal half life of 30 min which could be exceeded upto 10 hours in renal failure. Ampicillin and extended spectrum Penicillin secreted slowly and have half life of one hour. If renal function was compromised with creatinine clearance of 10ml/min or less then the dose of Penicillin must be adjusted accordingly upto one-fourth to one third of the normal dose. Nafcillin mainly cleared by biliary excretion and oxacillin, dicloxacillin and cloxacillin eliminated by both kidney and biliary excretion therefore no dosage adjustment required in renal failure. In newborn the clearance of Penicillin less efficient than adults, so dose adjusted by weight alone resulted in higher systemic concentration for longer periods (Katzung).

1.6.5. AMOXICILLIN/CLAVULANIC ACID(AUGMENTIN):

This antibiotic is an oral combination consisted of semisynthetic aminopenicillin amoxicillin and a beta-lactamase inhibitor i.e. Clavulanate potassium (potassium salt of clavulanic acid).Amoxicillin is an analog of ampicillin which is derived from basic penicillin nucleus, 6aminopenicillanic acid. Clavualnic acid is derived by fermentation of Streptomyces clavuligerus and possessed the ability to inactivate a wide variety of beta-lactamase enzymes.

1.6.5.1. Chemistry:

Molecular formula of amoxicillin is C₁₆H₁₉N₃O₅S.3H₂O and the molecular formula is 419.46.Amoxicillin has chemical formula as (2S,5R,6R)-6-[(R )-(-)-2-Amino-2- (phydroxyphenyl)acetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- carboxylic acid trihydrate which can be structurally represented as:

Figure 3: Structure of Amoxicillin

The molecular formula of clavulanate potassium is C₈H₈KNO₅ with the molecular weight of 237.25.Chemically it is potassium(Z)-(2R,5R)-3-(2-hydroxyethylidine)-7-oxo-4-1- azabicyclo[3.2.0]heptane-2-carboxylate, represented as:

Figure4:Structure of Clavulanate Potassium

1.6.5.2. Clinical Pharmacology:

Amoxicillin/Clavulanic acid is a broad spectrum antibiotic and its bactericidal effect covered a broad spectrum of Gram-positive and Gram-negative aerobic and anaerobic pathogens including:

Gram-positive:

• Streptococcus group A,B,C,G(Strep.pneumoniae,viridans,milleri,faecalis,pyogenes,anthracis,bovis)

• Staphylococcus aureus • Epidermidis(penicillin-susceptible and penicillin-resistant but methicillin-resistant strains of MRSA,MRSE) • Enterococcus faecalis • Corneybacterium spp. • Listeria monocytogenes • Nocardia asteroids Gram-negative:

• Aeronomas spp. • Bordetella pertussis • Brucellaspp • Campylobacter jejuni • Citrobacter spp.(moderately susceptible) • Eschericia coli • Haemophillus influenza • Helicobacter pylori • Klebsiellaspp • Moraxella catarrhalis • Morganella spp.(moderately susceptible) • Neisseria gonorrhoeae • Meningitides • Pasteurelamultocida • Proteus mirabilis • Vulgaris • Salmonella spp.

• Shigellaspp • Vibrio cholera • Yersinia enterocolitica(moderately susceptible) Anaerobic:

• ActinomycesIsraeli • Bacteroides spp.(including B. fragilis) • Clostridium spp.(Except C. difficile) • Peptostreptococcus spp. • Eikenellacorrodens • Fusobacterium spp. • Propionibacterium spp. • Peptococcus spp.

Resistant or partially resistant organisms:

• Pseudomonas aeruginosa • Enterobacter spp. • Serratia spp.

• Morganii • Mycoplasmae spp. • Chlamidiae spp. • Rickettsiae spp.

1.6.5.3. Pharmacokinetic:

The pharmacokinetic parameters of both components i.e. amoxicillin and clavulanic acid were similar and they did not affect pharmacokinetic parameters of each other.

1.6.5.3.a. Absorption: After oral administration amoxicillin/clavulanic acid rapidly absorbed and not affected by the presence of food. About 45 min after ingestion reached peak plasma concentration. 1.6.5.3.b. Distribution: Amoxicillin showed bioavailability of approximately.90% while that of clavulanic acid showed approximately 60% with plasma protein binding as 17-20% for amoxicillin and 22-30% for clavulanic acid respectively. High concentration of drug found in

plasma, sputum, bronchial secretion, lung tissue, the middle ear,prostate,peritonealabscess,gallbladder,uterus,ovary,adiposetissue,muscles,bones,skin,biol o gical fluids (including in the synovial, peritoneal, pleural, bile pleurent discharge). 1.6.5.3.c. Metabolism, Excretion:Like all penicillin, amoxicillin excreted through kidney by tubular secretion and glomerular filteration. Clavulanic acid metabolized in the liver and excreted by GFR and somewhat in the form of metabolites. Correction of dosing regimen(reduced dosage and /or increased interval between doses) required in patients with severe renal or hepatic insufficiency because of reduced clearance. 1.6.5.4. Dosage and Administration:

The normal adult oral dosag of amoxicillin/clavulanic acid is 500/125mg tablet every 12 hours or 250/125mg tablet every 8 hours. This dose should be 875/125 mg every 12 hours or 500/125 mg every 8 hours in case of severe or respiratory tract infections. Patients with impaired renal functions having GFR of < 30 ml/min should not receive 875/125 mg tablet. Depending on severity of infection and patients having GFR 10-30 ml/min received a dose of 500/125mg or 250/125 mg every 12 hours. If GFR rate fall below 10 ml/min then patient received 500/125 mg or 250/125 mg every 24 hours depend on severity of infection. Patients on hemodialysis received 500 /125 mg or 250/125 mg every 24 hours.An additional dose must be given during and at the end of dialysis. Patients with impaired hepatic functions must be dosed with caution and a regular monitoring of hepatic function must be carried out.

In pediatric patients weighed 40 kg or more dose must be adjusted according to adult recommendations.

Both amoxicillin and clavulanate tablet taken without regard to meal but absorption of clavulanic acid enhanced when both are administered at the start of the meal. Amoxicillin/clavulanic acid taken at the start of the meal to minimize the potential of gastrointestinal intolerance.

1.6.5.5. Indications: • Lower respiratory tract infections(caused by beta-lactamase producing strains of H.influenza, M.catarrhalis). • Otitis media(by beta-lactamase producing strains of H. influenza, M.catarrhalis) • Sinusitis(caused by beta-lactamase producing strains of H. influenza, M.catarrhalis) • Skin and skin structure infections(caused by beta-lactamase producing strains of S.aureus,

E.coli and Klebsiella spp).

• Urinary tract infections(caused by beta-lactamase producing strains of E-coli, Klebsiellaspp and Enterobacter spp). 1.6.5.6. Adverse Reactions:

Genarally amoxicillin/clavulanate potassium tablet is well tolerated and most of side effects observed in clinical trials are mild to transient and because of drug related side effects less than 3% of patients discontinued therapy. The most frequently reported adverse effects are :

• Diarrhea/loose stool (9%) • Nausea (3%) • Skin rashes and urticarial (3%) • Vomiting (1%) Other less frequent reported side effects are abdominal discomfort, flatulence and headache(www.drug.com).

1.6.6. Antacid/Anti-acid:

Those substances that neutralizes stomach acidity called as Antacid/anti-acid.Mainly they are available in various combination of three basic compounds, magnesium, calcium, aluminum which neutralize stomach acid(Health guide 2012).

1.6.6.1. Mode Of Action:

A neutralization reaction performed by antacid, they increased pH and reduce acidity in the stomach. When the concentration of gastric hydrochloric acid goes higher, it reached the nerves in the gastrointestinal mucosa and as a result they signal pain to the CNS (Wikepedia).

1.6.6.2. Indications:

Antacid have rapid onset and short duration of action so they can only be indicated for rapid relief of gastric discomfort for a short span of time(Pharmainfo.net).

• Relieve of heart burn • Gastro-esophageal reflux disease/acid indigestion

• Upset stomach • Sour stomach • Flatulence(by additional component simethicone) • GERD(by addition of alginic acid in antacid formulation) 1.6.6.3. Classification:

Antacids are divided into two classes:

1. Chemical Antacid: Those antacids which act by chemical neutralization of g astric acid(like sodium bicarbonate). 2. Non-absorbable antacid: Those act by adsorption of gastric acid(like calcium and magnesium salts). 1.6.6.4. Adverse Effects:

Antacids may have following adverse effects. Different antacids can produce different type of

adverse effects like:

1.6.6.4.a. Alkalosis: It usually caused by carbonate antacids and as a result it not only altered the

excretion of other drugs but also produced kidney stones. Gastric distention, headache and

decreased muscle flexibility occurred by carbon dioxide produced due to chemical reaction

between carbonate and hydrochloric acid.

1.6.6.4.b. Hypo phosphatemia and osteomalacia: Insoluble aluminum-phosphate-complex formed

due to presence of aluminum hydroxide in antacids and developed the risk of hypophosphatemia

and osteomalacia. Renal insufficiency greatly increased the risk because of poor gastrointestinal

absorption of aluminum.

1.6.6.4.c. Constipation:The drug containing aluminum cause constipation and are therefore

contraindicated in pregnancy.

1.6.6.4.d. Hypermagnesemia: In renal failure patients accumulation of magnesium may lead to

hyper-magnesemia along with the risk of cardiovascular and neurological complications.

1.6.6.4.e. Diarrhea: Magnesium hydroxide have laxative properties so it may cause diarrhea in

certain patients.

1.6.6.4.f. Arterial hypertension/ Heart failure: The increase intake of sodium containing antacids

may be harmful in case of arterial hypertension, heart failure and renal diseases (Wikepedia).

1.6.6.5. Drug Interactions:

Various medications interact with antacids and their absorption is inhibited by antacids. Antacids(absorbable) altered the pH of stomach or urine sufficient enough so that alter the absorption or excretion of drugs. Sometimes complex formation with other medications may also altered bio-availability of drugs. The drugs whose absorption is reduced by antacids are tetracyclin,antifungal,ciprofloxacin,propranolol,captopril,ranitidine,famotidine,fexofenadine,quin olones and iron supplements.

Some drugs can be potentiated by antacids like valproicacid, sulfonylurea(anti-diabetic), quinidine, levodopa and digoxin (Pharmainfo.net).

OBJECTIVE OF THE STUDY

2. OBJECTIVE OF THE STUDY

In the present study different Co-amoxiclav brands were selected and purchased from local market.These brands were manufactured in Pakistan.The main objective of the study include:

1. To estimate the quality of different brands of amoxicillin /clavulanic acid 375mg available in market. 2. Validation of a linear, precise and specific HPLC method for simultaneous analysis of amoxicillin/clavulanic acid tablet in Pakistani human plasma. 3. To determine pharmacokinetic parameters of co-amoxiclav after single administration with or without antacid in healthy human volunteers. 4. To compare the pharmacokinetic data of the current study in local population with the previous reported data. 5. To apply various statistical module to estimate the pharmacokinetic parameters of coamoxiclav 375 mg tablet. 6. To determine serum bactericidal activity of Co-amoxiclav.

LITERATURE SURVEY

3. LITERATURE REVIEW

The liquid chromatography combined with electrospray ionization tandem mass spectrometry in both positive and negative ion modes was used by Ashrai-Khorassani etal(2005)to separate and detect amoxicillin,clavulanic acid and ticarcillin in three matrices:water,cation adjusted Muller Hinton Broth (CAMHB) and 5% Lysed horse blood(LHB) in CAMHB. For sample clean up direct precipitation of protein with acetonitrile (CHзCN ) was used and an octadecyl silica (C 18) bonded phase column used for chromatographic separation.Using a gradient elution programme the mobile phase consisted of 1% aqueous formic acid and 1% formic acid in CHзCN with ampicillin as an internal standard.An excellent linearity showed by mass spectrometer in the calibration of all drugs and amoxicillin and clavulanic acid were easily quantified in a range of 10-15 ng/ml.Ashrai- Khorassani etal successfully used the method to quantify these drugs in a series of multi-well panels.

In order to determined amoxicillin and clavulanic acid quantitatively in pharmaceutical dosage forms, the sample components were separated by a reversed phase C18 column, with mobile phase contained a mixture of methanol-phosphate buffer(pH 3.2-3.4)(5:95,v/v) a simple and sensitive HPLC method with electrochemical (EC) detection was described by Aghazadeh A etal(2001).By applying a potential of +1.25 V antibiotic were detected amperometrically and over a concentration range of 15.625-500 ng high linearity for amoxicillin( r=0.9999) and clavulanic acid (r= 0.9979) were demonstrated.The limit of detection for amoxicillin was 0.8 ng ml(-1) and 15ng ml(-1) for clavulanic acid.In oral suspension and tablets the method was convenient and reproducible to analyze two components for other pharmaceutical dosage forms.

The method for simultaneous determination of amoxicillin and potassium clavulanate in tablet and suspension preparation was presented by Abounassif etal(1991).The method was isocratic

specified reversed phase column and a buffered mobile phase(MeoH + KH2po4-buffer pH 6+H2O,15:1:84) at the rate of 1.0ml/min with UV detection at 235nm.Replicate injection of the sample and standard preparations were used to test the suitability of the chromatographic system developed and within 2% relative standard deviations(RSDs) were observed.By utilizing proposed method recovery experiments conducted showed results of 101.5% +/- 1.72(n=6) and 101.22% +/- 1.93(n=6) for amoxicillin in tablets and powder for oral administration,respectively and for clavulanic acid 100.33 +/- 1.90(n=6) and 99.61 +/- 1.32(n=6) in the tablet and suspension powder respectively.The proposed method also compared with USP method and proved to be satisfactory.In terms of precision and accuracy no significant difference between two methods,indicated by F and t-test observed.

Pharmacokinetics and clinical efficacy of amoxicillin and clavulanic acid was studied by AlRroomi etal(1984) in the cure of 32 children with urinary tract infection.A favourable clinical and bacteriological response was observed in 21(80%) of 26 children with proved urinary tract infection.Amoxicillin resistant organisms were treated successfully in fifteen of these children.In the study serum and urine concentration of amoxicillin and clavulanic acid were measured after first oral dose. Brogden RN etal reviewed that clavulanic acid is a beta-lactamase inhibitor produced from Streptomyces clavuligerus and in combination with certain beta-lactam antibiotics extended their spectrum against bacteria which owed their resistance to the production of beta-lactamases.It extended the antibacterial activity of amoxicillin against beta-lactamase producing strains as well as amoxicillin resistant species such as Bacteroides fragilis.Clavulanic did not decrease and rarely extended the susceptibility of amoxicillin-sensitive bacteria.After oral administration clavulanic acid adequately absorbed and the basic pharmacokinetic parameters were similar to those of amoxicillin. For the determination of amoxicillin and clavulanic acid a sensitive and specific UPLC/MS method was developed by Cirić B etal.Adult healthy volunteers received as per os one tablet of amoxicillin(875 mg) in combination of clavulanic acid (125mg) before taking samples which were pretreated withby direct deproteinization with perchloric acid.For both amoxicillin and clavulanic acid the limit of quantification was 0.01µg/ml and the method was reproducible day by day(RSD <70%).For concentration of 1,5 and 20 µg/ml the analytical recoveries for amoxicillin ranged from 98.82% to 100.9% and the recoveries for clavulanic acid were 99.89% to 100.1% for concentration

of 1,2 and 5 µg/ml.After a single oral administration of amoxicillin/clavulanic acid combination the assay was successfully applied to a pilot pharmacokinetic study in healthy volunteers.Within the range of expected values upon the literature data for HPLC-UV and LC-MS methods the plasma concentrations of both amoxicillin and clavulanic acid were determined.Therefore it was concluded by Ciric B etal(2010) that the method had few advantages compared to LC/MS-MS and was faster used run time of 5 min with lower limit of quantification(LOQ) and could be applied for pharmacokinetic studies of both amoxicillin and clavulanic acid.

The bone penetration of a beta-lactam by population pharmacokinetics and pharmacodynamic profiling via Monte carlo simulations was firstly evaluated and presented by Cornelia Landersdorfer etal(2009).A single intravenous infusion of 2000 mg/200mg amoxicillinclavulanic acid injected to 20 uninfected patients undergoing total hip replacement before surgery.Specimens of bone(pulverized under liquid nitrogen with a cryogenic mill,including an internal standard) and blood samples were collected.The liquid chromatography-tandem mass spectroscopy used to analyze the drug concentration in serum and total bone.They used NONMEM and S-ADAPT for population pharmacokinetic analysis and a target time of nonprotien bound drug concentration above the MIC for ≥50% of the dosing interval for near maximal bactericidal activity in serum.For amoxicillin the median of the ratio of AUC for bone/AUC for serum was 20%(10th to 90th percentile for between–subject variability[variability],16 to 25%) in cortical bone and 18%(variability,11 to 29%) in cancellous bone and for clavulanic acid 15%(variability 11 to 21%) in cortical bone and 10%(variability 5.1 to 21%) in cancellous bone was observed.The equilibration half lives between serum and bone were 12min for amoxicillin and 14 min for clavulanic acid.Amoxicillin achieved robust(≥90%)probabilities of target attainment(PTAs) for MICs of ≤12mg/litre in serum and 2-3 mg/litre in bone and population PTAs above 95% against methicillin-susceptible Staphylococcus aureus in bone and serum for a 30 min infusion of 2000mg/200mg amoxicillin-clavulanic acid every 4hrs.The bone AUC of amoxicillin-clavulanic acid was 5-10 times lower than in serum and it achieved a rapid equilibrium and favorable population PTAs against pathogens frequently encountered in bone infection. A prospective pharmacokinetic study of oral co-amoxiclave was conducted by Chierakul etal(2006) in patients with melioidosis to determine the optimal dosage and dosing interval in this infection.Two 1g tablets of Augmentin(1750 mg of amoxicillin and 250 mg of clavulanic acid) was administered to 14 adult patients suffered from melioidosis and then serial plasma

concentration were measured.Multiple doses of co-amoxiclav were administered at different dosage and dose intervals and Monte Carlo simulation was used to predict the concentration of each drug.Along with chequerboard MIC data from 46 clinical isolates and four reference strains of Burkholderia pseudomallei the proportion of the dose interval above MIC(T> MIC) was calculated from 10000 simulated subject plasma concentration profiles.The result showed median(range) maximum plasma concentration of amoxicillin and clavulanic acid as 11.5(3.340.2)mg/L and 5.1(0.8-12.1)mg/L and with median range elimination half lives were 94(73-215) and 89(57-140) min,respectively.The simulation data showed T>MIC of< or =50% in 0.7%, 2.8%, 8.6% and 33.2% of patients when co-amoxiclav 1750/250 was given at 4, 6, 8 or 12 hourly dosing intervals respectively havingcorresponding proportion for T >MIC of > or=90% for 95.8%, 78.6%, 50.2% and 10.8% respectively.The result concluded that in melioidosis coamoxiclav(750/250) should not given at dosing interval greater than 6 hours.

Carsenti-Etesse H(1998) studied the pharmacokinetic parameters and killing rates in serum of volunteers received amoxicillin,cefadroxil or cefixime alone or in combination with niflumic acid or paracetamol which often combined with antibiotics to avoid inflammation and pain in acute ear,nose and throat diseases.Between these two classes of drugs pharmacokinetic parameters were described in experimental models exceptionally in humans and interactions were investigated with three antibiotics(amoxicillin 500mg x 2,cefixime 200mg, cefadroxil 500 mg x 2 and one placebo capsule).In a randomized cross over double blind trial interactions were investigated on pharmacodynamic parameters and on rate of killing in the serum of six healthy volunteers received the antibiotic associated or not with the product.After oral administration of antibiotics alone or in combination with the drugs blood samples were collected at 0.25,0.50,1,1.5,2,4,6 and 12 hr with a wash out period of at least 1 week between eleven sequences.Bio-assay and HPLC methods used to measured antibiotics and all serum samples obtained at peak level, 4 and 6 h were tested for killing rate.By the trapezoidal rule method area under time kill curve was calculated and relative bioactivity in percent was defined as(AUC control-AUC test)/AUC control x 100.The antibiotics or associated drugs did not show pharmacokinetic interaction in the AUC and T1/2 of the plasma concentration regardless of the dose as determined by HPLC or microbiological assay.The antibiotic killing rate was found to be time dependent for these beta-lactam antibiotics. The pharmacokinetic behavior of amoxicillin/clavulanic acid(4:1) combination was studied in 15 turkeys and 15 chickens by Carlos etal(1995) after intravenous and intramuscular administration

of single doses(25 mg/kg body weight).The purpose of the study was to verify any difference in the disposition kinetics of amoxicillin and clavulanic acid between turkeys and chickens.Both compartmental and non-compartmental methods were used to analysed the plasma concentration- time data.A two compartment open model best described the disposition curves for both drugs after intravenous administration in turkeys and chickens.Both species showed similar apparent volume of distribution for amoxicillin and clavulanic acid but the body clearances were significantly slower in turkeys than in chicken.After intravenous administrationthe elimination half life of amoxicillin was similar in turkeys(1.12±0.09 h) and chickens(1.03±0.11 h) but that of clavulanic acid differed significantly(P<0.05) between turkeys(1.12±0.03 h) and chickens(0.98±0.05 h).Both drugs had a significantly longer half life(P<0.05) in turkeys and chickens after intramuscular administration than after intravenous treatment.After intramuscular injection the bioavailability was high and similar with both drugs but higher values were obtained for chickens than turkeys. The intravenous and oral pharmacokinetics of amoxicillin and clavulanic acid combination(20 mg/kg of sodium amoxicillin and 5 mg/kg of potassium clavulanate was determined by Carceles etal(1995) in six goats.The pharmacokinetics of both drugs could be described by an open twocompartment model after intravenous administration.The volume of distribution of amoxicillin was greater(0.19±0.01 l/kg) than clavulanic acid(0.15 ± 0.01 l/kg) as compare to distribution and elimination constants which were higher for clavulanic acid indicating that clavulanic acid eliminated more quickly than amoxicillin.The pharmacokinetic behavior of both drugs were best described by an open one-compartment model with first order absorption after oral administration.The oral administration showed longer as twice elimination half-lives (2.15± 0.20 h and 1.94 ± 0.16 h for amoxicillin and clavulanic acid respectively) than intravenous administration (1.20 ± 0.16 h and 0.86 ±0.09, respectively) and the manifest an apparent flip-flop situation.The bioavailability was 27% for amoxicillin and 50% for clavulanic acid.

In a population pharmacokinetic-pharmacodynamic model (PK-PD) in 8 healthy volunteers a PK- PD drug-drug interaction between acenocoumarol and amoxicillin/clavulanic acid antibiotic was assessed by Delavenne X etal.During study each subject received single dose of 8mg of acenocoumarol on day 1 then from day 3-9 one gram of amoxicillin plus 250 mg of clavulanic acid was given.Each subject received a single dose of 8mg of acenocoumarol concomitantly with antibiotic on day 8.During 48 hr following each acenocoumarol administration 11 blood samples

were collected and at each sampling time the plasma concentration of acenocoumarol and prothrombin time were measured. Delavenne X etal(2009) firstly identified the structural PK model by pooling data from the trial with individual data from other acenocoumarol PK trial,also used indirect response model to fit PD data.With the help of nonmem software models were built using a non-linear mixed effect modeling approach and on PK and PD parameters covariates were tested including antibiotic treatment.A two compartment,first order input model with log normal inter-individual variability was used to fit acenocoumarol PK data.There were significant improvement in the fit of PK data by weight and antibiotic treatment with a 15% decrease in acenocoumarol clearance with concomitant antibiotics(P < 0.05).A successful application of a n indirect response model to the PK-PD data of acenocoumarol was found without any marked affect of any covariate including antibiotic treatment.At PK level drug-drug interaction was demonstrated without any PD corollary.

A pharmacokinetic study with oral amoxicillin/clavulanic acid was conducted in a randomized, open,single dose by Dinis PB,etal(2000) in 23 adult patient with chronic rhinosinusitis selected for surgery.All patients were randomly allocated to receive a tablet of 875/125 mg amoxicillin/clavulanate 2-4 hrs before surgery began.For the determination of both amoxicillin and clavulanic acid concentration levels tissue samples were collected at specific sinonasal sites during operation.The resulting data showed adequate tissue levels of amoxicillin throughout sinuses which were high enough to cover common susceptible pathogens but clavulanate was detected in only half of the sinonasal tissue samples.It was concluded by Dinis PB etal that the kinetics of oral clavulanic acid apparently failed to provide a widespread anti-beta lactamase activity capable of enhancing the activity of amoxicillin in all parts of sinuses.In the treatment of acute rhinosinusitis amoxicillin/clavulanic acid maintained a central role because amoxicillin was still the most effective oral beta-lactam against Streptococcus pneumonia, a particularly virulent and increasingly resistant upper respiratory tract pathogen.In an unpredictable fashion the data showed expected concomitant anti beta-lactamase activity. Two randomized double blind crossover studies and one randomized crossover study were performed by Deppermann etal(1989) to report possible drug-drug interaction between antacid(aluminum magnesium hydroxide, 10ml per dose for 10 doses), antimuscarinic drugs(pirenzipine, 50mg per dose for 4 doses),and H2-blockers(ranitidine, 150mg per dose for 3 doses) and amoxicillin(1000 mg), cephalexin(1000 mg),doxycycline(200mg) and

amoxicillinclavulanic acid(625 mg).serum and urine concentration were determined by means of bioassay,and open one or two compartment models (statistics were calculated by the Wilcoxon test) in ten healthy volunteers.The bioavailability of amoxicillin,cephalexin and amoxicillinclavulanic acid did not influence by antacid,pirenzipine and ranitidine with very negligible difference observed in the pharmacokinetic parameters, but G.I absorption of doxycycline(area under the concentration-time curve.38.6 +/- 22.7 mg.h/litre,fasting;6.0 +/- 3.2 mg.h/litre,with antacid) significantly reduced (P less than 0.01) by antacid. The results showed subtherapeutic levels of doxycycline. The efficacy and tolerability of clarithromycin extended release(ER) to amoxicillin/clavulanic acid was compared by Ernie Riffer(2004) in patients diagnosed with acute bacterial sinusitis. At least 12 years old 437 ambulatory patients having signs/symptoms and radiographic finding of acute sinusitis were randomized in a controlled,multicenter,investigator-blinded study and allowed to receive amoxicillin/clavulanate 875/125mg twice daily or clarithromycin ER 1000mg once daily for 14 days.After the completion of treatment clinical and bacteriological response rates were determined at a test-of-cure visit conducted up to 10 days.During follow up visit the radiological response was assessed and the clinical cure rate was 98% (184/188)in clinically valuable patients with clarithromycin ER group and 97%(179/185) with amoxicillin/clavulanate group(95% CI for the difference in rates[-2.4%,4.7%]) and it was sustained(96% for each treatment group).The amoxicillin/clavulanate group(95% CI for difference in rates[12.0%.2.9%]) and clarithromycin ER group showed the pathogen eradication rates as 98%(61/62) and 94%(61/65) respectively with similar radiological success rate of 94%(172/183) in both groups(95% CI for difference in rates [- 4.9%,4.9%]). After initiation of study drug symptomatic improvement was observed as earlier as 2-5 days with a statistically significantly higher resolution rate of sinus pressure(p=0.027) and improvement/resolution rate of nasal congestion(p=0.035) during treatment with clarithromycin ER.For each treatment group the resolution/improvement rate at the test of cure visit was ≥94% for the primary acute sinusitis and with clarithromycin ER(p=0.010) there was a statistically significant higher resolution/improvement rate of purulent nasal discharge.The quality of life got a positive and rapid impact by both study drug and a high level of satisfaction and probability of using study antibiotic again reported by patients,low health care resource use,fewer sinusitis related physician and outpatient visit required by patients in the clarithromycin ER group(p=0.055).Therefore in terms of incidence of drug related adverse events the treatment groups were comparable.

The pharmacokinetics of amoxicillin/clavulanic acid (4:1) combination was studied by Escudero etal(1998) after intravenous and intramuscular administration of single doses(25mg/kg) body weight to 50 pigeons.Compartmental pharmacokinetics and non-compartmental methods were applied for the analysis of plasma concentration time data.A two compartment open model best described the disposition curve for both drugs after intravenous administration.Amoxicillin and clavulanic acid showed apparent volume of distribution of 1.77 litres/kg and 1.30 litres/kg respectively with no significant difference in the body clearance of both.After intravenous and intramuscular administration amoxicillin illustrated the elimination half lives of 1.22(0.09) hour and 1.52(0.09) hour and those of clavulanic acid were 1.15(0.08) and 1.49(0.08) hour respectively.The half life(P<0.05) of both drugs were significantly longer after intramuscular administration as compared to intravenous treatment.Both drugs showed similar and high bioavailability after intramuscular injection(75.98% for amoxicillin and 74.61% for clavulanic acid).Peak concentrations were proportional to the dose of both products administered(5.81 mg/litre of amoxicillin and 1.89 mg/litre of clavulanic acid) and mean peak plasma concentration of clavulanic acid(0.29 hour) was reached earlier than amoxicillin(0.38 hour).Therefore Escudero etal proposed that after single administration of intramuscular dosage regimen of 105 mg/kg of the combination (84mg/kg) of amoxicillin and 21 mg/kg of clavulanic acid every 12 hours will achieve minimum concentration > or = 0.5-1 mg/litre(MIC of most susceptible pathogens). The pharmacokinetics of amoxicillin/clavulanic acid(4:1) combination was studied by Elisa Escudero etal(1998) after intravenous and intramuscular administration of single doses(25mg/kg body weight) to 50 pigeons.Compartmental and non-compartmental pharmacokinetic methods were used to analysed the plasma concentration-time data.Two compartment open model best described the disposition curves for both drugs and the apparent volume of distribution of amoxicillin and clavulanic acid were 1.77 litres/kg and 1.30 litres/kg respectively.There was no significant difference in the body clearance of amoxicillin and clavulanic acid and amoxicillin after intravenous and intramuscular administration showed elimination half lives of 1.22(0.09) hour and 1.52(0.09) hour respectively and 1.15(0.08) hour and 1.49(0.08) hour for clavulanic acid.Both drugs had a considerably longer half life(P<0.05) after intramuscular administration than that after intravenous administration.After intramuscular injection the bioavailability was high and similar for both drugs(7598 % for amoxicillin and 74.61% for clavulanic acid).An earlier mean peak plasma concentration of clavulanic acid (0.29 hour) was reached than amoxicillin(0.38 hour) and

peak concentration were proportional to the dose of both products administered(5.81 mg/litre of amoxicillin and 1.89 mg/litre of clavulanic acid).It was therefore proposed from a single administration that an intramuscular dosage regimen of 105 mg/kg of the combination (84 mg/kg of amoxicillin and 1.89 mg/litre of clavulanic acid) every 12 hours will achieve minimum concentration≥0.5 mg/litre.

The pharmacokinetic behavior of amoxicillin/clavulanic acid combination(25mg/kg) and alone amoxicillin(20mg/kg),clavulanic acid(5mg/kg) was studied by Escudero etal(1996) after intravenous administration of single dose to 10 goats.The objective of the study was to conclude the difference in the plasma kinetics of these drugs when administered alone or in combination.Both compartmental and non-compartmental pharmacokinetic methods were used to analysed the plasma concentration time data and a biexponential equation(two compartment open model)best described the disposition curves for both drugs alone and in combination.Amoxicillin showed elimination half lives as 1.05±0.09h alone and 1.13±0.19 h in combination, and that of clavulanic acid as 0.87±0.07 h and 0.85±0.09 h respectively with similar apparent volume of distribution in the two treatment.The body clearances of amoxicillin were 0.12±0.01 l/h.kg alone and 0.11±0.01 l/h.kg in combination and of clavulanic acid were 0.12±0.02 l/h.kg alone and 0.12±0.01 l/h.kg in combination with amoxicillin.No significant difference was observed in the half lives and body clearance of amoxicillin and clavulanic acid when administered alone and in combination, hence it was concluded that the iv administration of amoxicillin and clavulanic acid as a combined product did not alter the disposition kinetics of either drug. The pharmacokinetics of amoxicillin and clavulanic acid was studied in 15 sick children by Elias Jones etal(1990) after a 30 min iv infusion of 50 mg/kg amoxicillin and 5 mg/kg potassium clavulanate.Levels of both compounds were assayed in plasma and at the end of infusion mean peak concentrations were 121.0 mg/l for amoxicillin 12.0 mg/l for clavulanate, upto a mean of 15.8 and 1.92 mg/l respectively after 2h.The data showed mean(β phase) T ½ 0.88 h for amoxicillin and 0.79 h for clavulanate.In some individuals the elimination half life of clavulanate was much shorter because of higher plasma clearance.Therefore the data recommended that for treatment of some infections due to β-lactamase producing organisms in severely ill children require more frequent iv administration of amoxicillin and potassium clavulanate than in less severely affected children.

With UV detection using internal standard a simple,rapid and sensitive isocratic reversed phase HPLC method was developed and validated by Foroutan SM etal(2007) for simultaneous determination of amoxicillin and clavulanic acid in human plasma.With a minimum quantification limit of 15 and 30 ng ml(-1) respectively the assay enabled the measurement of amoxicillin and clavulanic acid for therapeutic drug monitoring.It was a simple,one step extraction procedure and analytical recovery was complete with in the method.In reversed phase conditions a Chromolith Performance(RP-18e,100 mm x 4.6 mm)column with an isocratic mobile phase consisted of 0.02M disodium hydrogen phosphate buffer-methanol(96:4, v/v) with pH 3.0 and UV at 228nm was used for separation.Less than 9.0% of coefficient of variation for inter-day and intra-day assay were found.

A new pharmacokinetically enhanced formulation of amoxicillin/clavulanate(2000/125 mg) developed by File TM etal(2005) designed to combat infections caused by S.pneumonia including penicillin-resistant(PRSP, penicillin minimum inhibitory concentrations(MICs) > or = 2mg/l) isolates and those with elevated amoxicillin/clavulanic acid MICs,while maintaining coverage of beta-lactamase producing pathogens.In adult patients with CAP a pooled efficacy analysis of four randomized (1:1) and one non-comparative clinical trials of amoxicillin/clavulanate 2000/125 mg, given twice daily,was conducted along with conventional amoxicillin/clavulanate formulations as comparative agents.The efficacy(eradication of the initial pathogen or clinical cure in patients for whom no repeat culture was performed) in patients with S.pneumonia infection was 92.3%(274/297) for amoxicillin/clavulanate,2000/125 mg and 85.2%(46/54) for comparator(P=0.11) at follow-up(days 16-39).A successful treatment was observed in 24 of 25 PRSP-infected patients receiving 2000/125 mg and both amoxicillin/clavulanate 2000/125 mg and comparator were well tolerated.Few patients withdrew from the study. The pharmacokinetics and urinary excretion of clavulanic acid was studied in eight healthy adult volunteers by Ferslew etal(1984) after oral administration of 500mg amoxicillin and 125 mg potassium clavulanate.The concentrations of clavulanic acid in the serum and urine were assayed by using HPLC and the resulting pharmacokinetic parameters were t1/2β=1.019±0.090 hour,t1/2α=0.276±0.031 hour,lag time=0.321±0.018 hour,t-max=1.042±0.80 hour,Cmax=2.098±0.441µg/ml and AUC=4.897±0.979µg Xhr/ml.After administration the

clavulanic acid(as percentage of dose administered) showed cumulative urinary excretion as:14.05±2.87 within 2 hours,25.77±3.98 within 4 hours and 27.85±4.27 within 6 hours.

The antibacterial activity and kinetics of amoxicillin and clavulanic acid combinations were tested by Fuglesang JE etal(1983) against 11 E.coli and five Klebsiella aerogenes strains.The strains were highly resistant due to beta-lactamase production apart from one E.coli.In all strains synergy was demonstrated by agar dilution and detected against beta-lactamase producing strains under simulated in vivo conditions with constantly decreasing concentrations simulating in vivo pharmacokinetics.The antibacterial activity determined by MICs and bacterial kill kinetics in vivo simulation model were correlated acceptabily.When antibiotic dosage was increased a higher bacterial kill rate was observed beyond the MICs where an antibacterial effect was seen,not demonstrable by traditional agar dilution tests.A greater relative amount of amoxicillin compared to clavulanic acid in combination allowed a reduction in the total amount of antimicrobial agents with the same degree of antibacterial activity.

The use of HPLC to provide a rapid assay of the components of Augmentin in body fluids described by Foulstone and Reading(1982).They assayed clavulanic acid by reacting the sample with imidazole which produces a derivative absorbing at 311nm.In the human serum and urine this derivative chromatographs on reverse phase HPLC columns clear of interfering components.A very low concentration as 0.1 µg/ml of clavulanic acid was detected by this method.In this assay system no interference was observed from amoxicillin penicilloic acid, or the acid and alkali degradation products of clavulanic acid. Amoxicillin did not produce any derivative and readily assayed by HPLC in body fluids.In order to simplify the methodology same chromatographic conditions were employed for the assay of amoxicillin and the clavulanic acid derivative however,amoxicillin was determined of the antibiotic/ml.For the detection of any underlying peaks which may co-chromatograph an alkali blanking procedure for amoxicillin and clavulanic acid was also described and ultrafiltration procedure to remove protein from serum samples before HPLC was applied successfully for the assay as it prolong column life and reduce interference in the amoxicillin assay.The resulting data from HPLC was compared with those obtained from microbiological assays. The effects of antacid(containing magnesium) on the pharmacokinetics of linezolid were evaluated by Gabriela etal(2006).In a crossover,randomized study single dose of 600mg linezolid was

administered orally alone or 10 min after administration of antacid Maalox 70mVal containing 600mg magnesium hydroxide and 900mg aluminium hydroxide in nine healthy males and nine healthy females.The plasma concentrations of linezolid were determined by HPLC and pharmacokinetic parameters were calculated for both treatments,although the pharmacokinetic of linezolid did not influence by coadministeration of antacid.The maximum concentration in plasma(C-max)(linezolid plus antacid versus linezolid alone)were 1.01(0.99 to 1.02) and 0.99(0.96 to 1.02) repectively with the ratios(90% confidence intervals) of the individual values of the area under the concentration-time curve.In any of the pharmacokinetic parameters(the time to C- max,lag time,Volume of distribution[V/F],and clearance[CL/F]). no significant difference was observed between the treatment groups but a significant sex difference was observed for AUC,C- max,V/F and CL/F which could be almost completely explained due to difference in body weight between males and females.In any condition no clinically relevant adverse effects were detected.It was demonstrated by the study that the oral absorption of linezolid was not affected by antacid containing magnesium hydroxide and aluminium hydroxide and they can be safely administered with linezolid.

A rapid,capillary electrophoresis method was evaluated by Genowefa etal(2003) for determination of amoxicillin and clavulanic acid in Augmentin as well as ampicillin and sulbactam in Unasyn preparation for injections.The mobile phase composed of phosphate-borate buffer at pH 8.66 containing 14.4% sodium dodecyl sulphate and the method was validated.In concentration of amoxicillin 0.05-3.03mg/ml and ampicillin 0.05-3.08 mg/ml as well as clavulanic acid 0.02-2.02 mg/ml and sulbactam 0.05-2.08 mg/ml the reproducibility,precision,accuracy and assay linearity were established.When compared with alternative HPLC method,this new method was fast,inexpensive and limits consumption of organic solvents used for drug analysis.No significant difference was found by student’s t-test statistical analysis between the results obtained by the two methods t(calculated) 0.32 and 1.69 for amoxicillin and clavulanic acid and 0.67 and 1.93 for ampicillin and sulbactam were smaller than tabulated. A micellar electrokinetic chromatography method was developed by Genowefa etal(2002) for simultaneous assay of ticarcillin and clavulanic acid in Timentin i.v injection preparation.The separation of both components of timentin was excellent by this method,the method was validated and had confirmed specificity,reproducibility,precision and accuracy.The established limit of detection and quantitation for Timentin were in the concentration 0.04 and 0.08 mg/ml

respectively.Two most commonly used methods i.e. UV and HPLC were compared with the elaborated technique and the obtained results and their statistical analysis proved the same precision of all methods without showing any marked differencebetween CE and HPLC.

A simple and accurate HPLC method with UV detection at 220nm was validated by Guillaume etal(2002) for the simultaneous determination of amoxicillin and clavulanic acid in human plasma.Methanol was used for deprotienization of plasma samples.A mobile phase consisting of acetonitrile –phosphate solution-tetramethyl ammonium chloride solution and a reversed phase C8 column was used to achieve a good chromatographic separation between both compounds.A linear calibration curves were represented over a concentration range of 0.625-20mg/l for amoxicillin and 0.3125-10 mg/l for clavulanic acid with determination coefficient>0.998.The limit of quantitation were 0.625 and 0.3125 mg/l for amoxicillin and clavulanic acid respectively therefore the method was accurate(bias <7%) and reproducible (intra and inter-day R.S.D <15%) and for both components analytical recoveries from human plasma ranged from 91 to 102%.This validated method was rapid (total run time <10min) and requires only a 100µl sample and was successfully conducted in a pilot pharmacokinetic study involved healthy volunteers after a single oral administration of amoxicillin/clavulanic acid combination(500/125 mg).Hence the assay was considered suitable for biomedical application. The previous studies showed that high doses of amoxicillin, equivalent to those produced by 500 and 750 mg oral doses in humans(area under the plasma concentration time curve) were effective against a penicillin-resistant strain of Streptococcus pneumonia.It was further confirmed by Gillian etal(1998) in an experimental respiratory tract infection in immunocompromised rats.Amoxicillin’s antibacterial activity was unexpectedly enhanced in vivo against penicillinresistant and susceptible S.pneumonia strains when subtherapeutic doses of amoxicillin were coadministered with β-lactamase inhibitor potassium clavulanate.The reason was obscure hence it was proposed that observed effect might be due to differential binding of clavulanic acid and amoxicillin to penicillin binding proteins. The concentration of amoxicillin and clavulanic acid was determined by Gould IM etal(1994) in bronchial mucosal biopsy samples obtained at bronchoscopy following five different dosing regimens.Almost from 50 patients undergoing diagnostic bronchoscopy , bronchial biopsy and serum samples were obtained.Before 1-3 hours of bronchoscopy ten patients each received 375 mg,625 mg,750mg and 3.25 g oral and 1.2 g i.v co-amoxiclave.By means of HPLC using a

microbore column,solid phase extraction, and preconcentration to improve sensitivity ten folds over previous methods were used to determined the concentrations of amoxicillin and clavulanic acid.It was found that the concentration of both amoxicillin and clavulanic acid in bronchial mucosa were dose related and were well above the MIC 90 of co-amoxiclave for the common bacterial respiratory pathogens including Haemophilus influenza,Micrococcus catarrhalis and Streptococcus pneumonia for all dosing regimens with mean mucosal levels as 200% and 118% of the corresponding serum levels for co-amoxiclave.Even at the lowest dose of 375 mg orally amoxicillin and clavulanic acid were concentrated in bronchial mucosa and were likely to produce sufficient tissue levels in the lungs to inhibit all the common community acquired respiratory payhogens.

A rapid and specific HPLC method was developed and validated by Han-Gon choi etal(2004) for the simultaneous determination of cefatrizine and clavulanic acid in the plasma of beagle dog.The procedure involved sample pretreatment reaction of clavulanic acid with 1,2,4-triazole resulted in the formation of derivative with UV detection at 314 nm.In a reversed phase C-18 column this derivative was separated without any interference by other components in plasma.Cefatrizine did not form any derivative and detected at 269 nm and sulfanilamide used as an internal standard,The sulfanilamide,derivative and cefatrizine showed retention times of 3.5,4.9 and 6.0 min respectively.The assay showed linearity from 2 to 100 µg/ml for cefatrizine and from 1 to 50 µg/ml for clavulanic acid.For cefatrizine the precision expressed as R.S.D ranged from 4.2 to 18.2% and 5.5 to 15.8% for clavulanic acid ,similarly accuracy ranged from 97.9 to 120%(lower limit of quantitation) for cefatrizine and from 97.7 to 119.2% for clavulanic acid.It was successfully employed method to follow the time course of the concentration of cefatrizine and clavulanic acid in beagle dogs following oral administration of cefatrizine and clavulanic acid. In order to determined amoxicillin and clavulanic acid simultaneously a rapid and accurate HPLC method with UV detection at 220 nm was validated by Hoizey G etal(2002).By direct deproteinization plasma samples were pretreated with methanol,a reversed phase C8 column was used with mobile phase consisted of acetonitrile-phosphate solution-tetramethyl ammonium chloride solution.Within concentration ranges of 0.625-20mg l(-1) for amoxicillin and 0.3125-10 mg l(-1) for clavulanic acid the calibration curves were linear with determination coefficients>0.998.The method was accurate(bias < 7%) and reproducible(intra and inter day R.S.D < 15%) with a quantitation limit of 0.625 and 0.3125 mh l(-1) for amoxicillin and clavulanic

acid respectively and for both components analytical recoveries from human plasma ranged from 91 to 102%.The method was rapid(total run time < 10min) and only 100 microl sample was required,hence it was fully validated to allowed simultaneous determination of amoxicillin and clavulanic acid.In a pilot pharmacokinetic study of healthy volunteers after single oral administration of amoxicillin/clavulanic acid combination(500/125mg) the method was successfully applied and proved to be suitable for biomedical applications.

An oral sustained release formulation for amoxicillin that maximized the duration of active drug concentration in the extracellular fluid was developed by Hoffman A etal(1998),therefore increased the dosing interval with assured antimicrobial activity.The underlying principle was based on the pharmacodynamic properties which were non-concentration dependent and required long exposure of the pathogen to the drug with minimal post antibiotic effect.The new matrix tablet formulation consisted of hydrophilic polymer and designed to release 50% of its content within the first 3 hrs and to complete the drug release process over 8 hrs in vitro due to pharmacokinetic constraints i.e short biological half life and limited absorption window with poor colonic absorption.HoffmanA ,etal evaluated the pharmacokinetic of the new formulation in 12 healthy vulunteers and compared to a conventional gelatin capsule with both formulation containing 500mg amoxicillin.An HPLC method with a flourometric detector used to determined the plasma concentration of active amoxicillin and penicilloic acid.The area under concentration time curve and maximal serum amoxicillin concentration were found to be lower than the immediate release formulations.The resulting data suggested that a larger dose of new formulation should be given(e.g 750 mg or 1g 12 hourly) in order to achieved a twice daily dosing regimen to provide therapeutic concentrations for the whole 12 hr dosing interval.This was recommended on the basis of large interindividual differences of the extent of amoxicillin absorption investigated and to assured that poor absorber also got benefit from full antibiotic efficacy.Therefore increased patient compliance and improved therapeutic outcome leaded by this dosing regimen.

The pharmacokinetics of ticarcillin 5.0g and clavulanic acid 0.2g alone and in combination(30 or 50g ticarcillin +0.2 g clavulanic acid as 3.2 or 5.2 g timentin) were examined by Hoffken etal(1985) after 15 min infusion in ten healthy volunteers.Against two ticarcillin resistant strains i.e Klebsiella oxytoca and Pseudomonas aeruginosa, the serum bactericidal activity of 5.0 g ticarcillin alone and 5.2g Timentin was determined in the first and sixth hour after administration.An open 2-

compartment model used to described the serum kinetics of both ticarcillin and clavulanic acid and both showed similar kinetic behaviour in serum with T1/2β of 74.8±11.5 min for ticarcillin and 76.6±4.6 min for clavulanic acid.The total clearance of clavulanic acid was 158±23 ml/min and it was clearly exceeded that of ticarcillin 112±9 ml/min.In 24 hour urine sample recovery rate for clavulanic acid was 41.3% as compared to 79.4% for ticarcillin.A limited change in the kinetics of ticarcillin and clavulanic acid was observed co-administration of both substances and serum bactericidal action of ticarcillin and clavulanic acid was significantly enhanced and detected for Klebsiella species and not for Pseudomonas species and only in first hour.

A pilot study was performed by Havard etal(1982) involved thirteen patients with a mean age of 67 years suffered from lower respiratory tract infections.They were treated with Augmentin(500mg amoxicillin +250mg clavulanic acid) given orally every 8 hours for 7 days.Ten cases showed good clinical response, one failed and two patients could not assessed.No serious side effect observed Except one patient had to stop treatment(due to diarrhea).The result showed that in patients the peak levels of amoxicillin and of clavulanic acid in blood and sputum were achieved at a later time than occur in healthy volunteers.

A new per oral amoxicillin/clavulanate therapeutic system composed of immediate release tablet and controlled release floating capsule was developed by Janez Kerc etal(2007) by an in vivo bioavailability study.For amoxicillin and clavulanic acid of the new therapeutic system the pharmacokinetic parameters were:AUCt, AUCi,(AUCt/AUCi),Cmax,Tmax,kel,T½ and additionally for amoxicillin T4 and T2 were calculated from plasma levels.The enhanced pharmacokinetic parameters were confirmed by a newly developed therapeutic system containing 1500mg of amoxicillin and 125 mg of clavulanic acid and also prolonged time over MIC of amoxicillin in relation to a regular immediate release amoxicillin/clavulanate formulation was confirmed.

For the determination of amoxicillin in human plasma a simple,economical and reproducible HPLC mass spectrometric(MS) method was developed and validated by Khuroo AH etal(2008).In terms of selectivity,precision/accuracy,recovery,dilution integrity,matrix effect,effect of anti- coagulant and stability the method was validated.By solid phase extraction(HLBOasis cartridge) sample preparation was carried outthen after being processed sample was chromatographed on

Hypersil Gold(4.6 x 50 mm), 3 microm C18 column with the mobile phase consisted of 10mM ammonium formate buffer (pH 5.0) and acetonitrile(10:90, v/v).With turbo-ion spray in positive ion mode amoxicillin was detected by MS-MS detection and showed linear weighed(l/X2) calibration curves over the range of 0.17 to 17.0 µg/ml.The intra-day accuracy and the intra day precision was from 1.3% to 8.8% and 94.1% to 108.5% respectively while the inter day precision and inter day accuracy were from 1.85 to 6.2% and 95.1% to 105.9%.The observed data showed mean recovery of 66.3% for amoxicillin and 71.6% for internal standard(ampicillin).with different anti-coagulant(citrate,monobasic sodium phosphate,dextrose,and adenine-citrate,and adenine and ethylene diamine tetraacetic acid)the stability of amoxicillin was studied at -15ºC and -50ºC and no significant degradation was observed for 60 days. In an in vivo bioavailability study a new per oral amoxicillin/clavulanate therapeutic system composed of immediate release tablet and controlled release floating capsule was developed and evaluated by Kerc J etal(2007).In this new therapeutic system the pharmacokinetic parameters for amoxicillin and clavulanic acid i.e AUCt,AUCi,(AUCt/AUCi),Cmax,Tmax,Kel,T(1/2) and additionally T(4) and T(2) were calculated from the plasma levels.The pharmacokinetic parameters of the newly developed therapeutic system containing 1500mg of amoxicillin and 125mg of clavulanic acid were enhanced and it was confirmed by the study.The study further confirmed the prolonged time over MIC of amoxicillin in relation to a regular immediate release amoxicillin/clavulanate formulation.

A simple,fast and sensitive high performance liquid chromatography(HPLC) mass spectrometric (MS) method was developed by Kyung-Hwan etal(2004) for simultaneous determination of amoxicillin and clavulanic acid in human plasma using terbutaline as internal standard.The analytes were separated on a C8 reversed-phase column with formic acid-water- acetonitrile(2:1000:100) after precipitation of the plasma protein with acetonitrile and detected using electroscopy ionization(ESI) mass spectroscopy in negative selected ion monitoring(SIM) mode.In clinical studies the method was validated and successfully applied to analysis of amoxicillin and clavulanic acid with the limit of quantitation 0.12µg/ml for amoxicillin and 0.062µg/ml for clavulanic acid and it was five times lower than that of the published HPLC-UV method. In order to provide more effective therapy against resistant pathogens than provided by currently available formulations Kaye CM etal(2001) developed a new oral pharmacokinetically enhanced

formulation of broad spectrum antibiotic amoxicillin/clavulanate.In 24 healthy males and 31 females subjects with a mean age 35yrs(range 18-58yrs) and a mean body weight 69kg(range 5186kg),a single oral dose of pharmacokinetically enhanced amoxicillin/clavulanate(2000/125 mg; 16:1 ratio) was administered at the start of the meal.The blood samples were collected at frequent intervals up to 12 hours after dosing and plasma was assayed for amoxicillin and clavulanate concentrations using validated procedures.The time above the MIC(T>MIC) would be approximately > or = 40% over a 12 hr dosing interval, as the new formulation consisted of 1 layer of immediate-release amoxicillin and clavulanate and another of sustained-release amoxicillin in a proportion for that of an amoxicillin MIC of 4microgram/ml.It was appeared to be a slower decline with the pharmacokinetically enhanced formulation than is usually seen with conventional formulations and there was evidence of a second amoxicillin absorption phase after the expected sharp peak in plasma amoxicillin concentration.The mean T>MIC for an amoxicillin MIC of 4 µg/ml was 49.4% of a 12 hr dosing interval, a value that cannot be achieved with existing approved doses and formulations of amoxicillin/clavulanate.The plasma amoxicillin concentrations were very low(approx: 0.05µg/ml) by 12 hrs and suggested no expectation of notable dose-to-dose accumulation on repeat dosing with a BID regimen.The existing formulations of amoxicillin/clavulanate and new formulation showed similar terminal half-lives as 1.27 hrs for amoxicillin and 1.03 hrs for clavulanate with no serious adverse events or death reported during study.It was therefore concluded and suggested that the enhanced pharmacokinetic profile of amoxicillin/clavulanate was seen and the formulation was likely to be highly effective for oral treatment of infections caused by bacteria including beta-lactamase producing organisms and strains with amoxicillin MICs< or = 4µg/ml.

In order to determined amoxicillin in human plasma Krauwinkel WJ etal(1993) developed a HPLC method using solid phase extraction.Amoxicillin after concentrating on a C8 cartridge, a reversed phase column packed with 5 micron Chromspher C18 was used to elute the cartridge on line.The method required mobile phase consisted of methanol-0.08M phosphate buffer(pH 7.6)(20:80),contained 0.01M tetrabutylammonium dihydrogen phosphate and monitored at UV spectrophotometry of 234nm.The consistency of method was proved and considered useful in the bioavailability studies for the development of new amoxicillin formulations.

The first evaluation of the bone penetration of a beta-lactam by population pharmacokinetic and pharmacodynamic profiling via Monte carlo simulation was presented by Landersdorfer CB etal(2009).A single intravenous infusion of 2000/200 mg amoxicillin-clavulanic acid before surgery was given to 20 uninfected patients and blood and bone specimens were collected and under liquid nitrogen with a cryogenic mill including an internal standard bone samples were pulverized.By means of liquid chromatography tandem mass spectrometry the drug concentration in serum and total bone were analyzed.A target time of the non-protein bound drug concentration above the MIC for > or = 50% of the dosing interval for near maximal bactericidal activity in serum and population pharmacokinetic was determined by NONMEM and SADAPT.The data showed the median of the ratio of the area under the curve (AUC) for bone/AUC for serum was 20%(10th to 90th percentile for between subject variability,variability 16-25%) in cortical bone and 18%(variability,11-29%) in cancellous bone for amoxicillin and 15%(variability, 11 to 21%) in cortical bone and 10%(variability 5.1-21%) in cancellous bone for clavulanic acid.Amoxicillin achieved robust(> or = 90%) probabilities of target attainment(PTAs) for MICs of < or = 12mg/litre in serum and 2-3 mg/litre in bone and population PTAs above 95% against methicillin-susceptible Staphylococcus aureous in bone and serum for a 30min infusion of 2000mg/200mg amoxicillin/clavulanic acid every 4 hr.A rapid equilibrium and favorable population PTAs against pathogens commonly encountered in bone infection was achieved by amoxicillin/clavulanic acid and the AUC of amoxicillin/clavulanic acid was 5-10 times lower in bone than serum.

An accurate,precise and sensitive HPLC assay method for the determination of amoxicillin in human plasma samples was developed by Luis Renato Pires etal(2003).Two amoxicillin capsules(500mg) formulations(amoxicilina from Brazil as a test formulation and amoxil from SK & B as reference formulation) compared for there bioavailability in 24 volunteers of both sexes.Concentrations of amoxicillin were analyzed by combined reversed phase liquid chromatography with UV detection of 229nm.Cold methanol was added for extraction of amoxicillin and cefadroxil(internal standard) from plasma.Column used for attainment of separation was Lichrosorb® 10µm,C18 reversed phase column at room temperature.The mobile phase consisted of mixture of 95% phosphate buffer(0.01 mol/L),pH 4.8 and 5% acetonitrile.An open randomized 2-period cross over study design was conducted.A wash out period of one week was given between the doses administered.Samples were obtained from plasma over an 8hour period.The C-max and area under the curve(AUC 0-8h) ratios(test/reference) were calculated for

the assessment of bioequivalence between the two formulations.US Food and drug administration proposed statistical interval of 80-125%.At a flow rate of 1.3ml/min,the internal standard and amoxicillin were eluted at about 4.2 and 5.2 min respectively.Plasma showed absolute recovery of amoxicillin as 90.0% at 3µ/ml, 98.6% at 25µ/ml and 95.3% at 50µg/ml.The relationship between peak height ratio and plasma concentrations(r²≥ 0.999) were excellent in the assay with the limit of quantification of 1g/ml.The individual percentage ratio for geometric mean of Amoxicilina/amoxil was 101.4% for AUC 0-8h and 99.9% for C max having 90% confidence interval of 98.3-104.4% and 95.7-103.9% respectively.As the 90% confidence interval of both lies in the range of 80% -125% proposed by FDA therfore it was concluded by Luis Renato Pires etal that Amoxicilina 500mg capsules was bioequivalent to Amoxil® 500mg capsule. The pharmacodynamics of amoxicillin/clavulanic acid was studied by Lowdin E etal(2002) against different strains of Haemophilus influenza in an in vitro kinetic model.During study bacteria were exposed to amoxicillin/clavulanic acid at an initial inoculums of 106 CFU/ml with an initial concentration of approx.15/3 mg/ml,8/3 mg/ml which was simulated with the peak levels in human achieved after a dose of 875/125mg amoxicillin and 500/125 mg with a half life of 1hr.In addition to that experimentally a 2000/125 mg of pharmacokinetically enhanced formulation of amoxicillin/clavulanic acid was also given bid.After initial dose of 875/125 mg a repeated dose was given at 12 hr and the pharmacokinetically enhanced formulation or at 8 and 16 h after the dose of 500/125mg.An in vitro kinetic model consisted of a spinner flask with a filter membrane fitted in between the upper part and the bottom part in order to prevent bacterial dilution was used to perform experiment.At a constant rate with a pump the medium is removed from the culture flask and samples were taken repeatedly at intervals of 1-2 h up to 24 h during experiment for viable counting.One of the strains of H.influenza was also exposed to a constant concentration corresponding to the peak serum levels obtained after a dose of 500/125 mg.In the in vitro kinetic model the concentrations of amoxicillin was as expected and there was a tendency for a greator bactericidal effect with 500/125mg tid as compared to 875/125 bid at the end of experiment(24h) with difference in CFUs between the two dosing regimens of 2.6 log10 CFU for H.influenza LH 2803 and 1.8 log10 CFU for other clinical strains but these differences did not affect statistical significances(P=0.075 and 0.10 respectively).With the pharmacokinetically enhanced formulation in comparision with the bid regimen both at 8,16 and 24 hrs and 8 and 16 hr with the tid regimen statistically significant higher bactericidal effect was seen in the experiment.It was concluded by the study that neither the standard dosing regimen of amoxicillin(875/125 mg b.i.d. or 500/125

mg) used in which the time that the free(non-protien bound) concentration the MIC(T>MIC) exceeding was less than 50% was sufficient to achieve a complete bactericidal effect during the first 24h of treatment.The pharmacokinetically enhanced formulation gave a longer T>MIC(73- 79%)of amoxicillin even though the concentration of clavulanic acid was only detectable for 45% of the dosing interval and complete killing of all strains was obtained after 24 h.

A new, simple,precise,rapid and accurate RP-HPLC method was developed by Malathi etal(2009) for the simultaneous estimation of cefpodoxime proxetil and clavulanic acid from pharmaceutical dosage forms.In the method mobile phase consisted of acetonitrile:50mM potassium dihydrogen phosphate buffer(pH 3.0,70:30 v/v) at a flow rate of 1.0 ml/min carried out on a Zorbax Eclipse XDB 5 µ C18(150x 4.6 mm) column with UV detection at 228 nm.The internal standard was aspirin and resulting data showed retention time of clavulanic acid,cefpodoxime proxetil and aspirin as 4.43.6.44 and 5.6 min respectively.In terms of accuracy,precision,linearity,limit of detection,limit of quantification and solution stability the developed method was validated.For the estimation of these drugs in combined dosage forms the proposed method can be used.

Moore TD etal(1996) determined the stabilities of amoxicillin(16 µg/ml) and clavulanate(8µg/ml),alone and in combination in BACTEC medium(Middlebrook 7H12B medium) by means of HPLC and bioassay.The half life of amoxicillin(trihydrate and sodium) along with clavulanate in nonradiolabelled 7H12B medium was 6.7 days by HPLC while that of clavulanate half life was 2.0 days in combination with amoxicillin.The half lives of amoxicillin trihydrate and clavulanate in radiolabelled 7H12B medium were comparable(7 and 2 days respectively) by bioassay to those determined by HPLC.The HPLC showed half life of 1.88 days for clavulanate alone while it was 1.87 days by bioassay and this was adjusted by a “topping up” procedure or adding one half the concentration of clavulanate every 2nd day for accurate amoxicillin/clavulanate MIC testing with the BACTEC mycobacterial susceptibility system.

The pharmacokinetic properties of amoxicillin and clavulanic acid were reviewed and discussed by Navarro etal(2005).The non-linear absorption processs for amoxicillin supported by reported data.A reduction in peak plasma concentration (C-max)/dose ratio,prolongation of time to reach C-max and variation for high dose of amoxicillin observed due to gastrointestinal absorption of this antibacterial caused by saturable transport mechanisms,limited solubility and the presence of

an absorption window.It was also suggested by the given data that absolute bioavailability of clavulanic acid reduced due to possible interaction between amoxicillin and clavulanic acid.The intrinsic pharmacodynamics of each drug along with the synergism produced by amoxicillin/clavulanic acid association are also reviewed and analysed by Navarro etal.A review of the principles of pharmacokinetics/pharmacodynamic analysis were applied to amoxicillin regarding new pharmacokinetically enhanced formulation with consideration given to data obtained from in vitro studies and animal models.The application of pharmacokinetic /pharmacodynamic analysis to the scarce pharmacokinetic data were also included with theoretical consideration concerning the efficacy of this formulation.Navarro etal also highlighted the broad pharmacokinetic variability of both amoxicillin and clavulanic acid administered together or at high doses of amoxicillin and considered this aspect to improve predictions based on pharmacokinetic/pharmacodynamic analysis for new formulations.It was also proposed that a more realistic prediction in clinical practice can obtained by methodological recommendations such as Monte Carlo simulation. A replicate design approach to a bioequivalence study of amoxicillin/clavulanic acid combination was suggested by Nasir etal(2004) followed a 250/125 mg oral dose to 23 subjects and to compared the analysis of individual bioequivalence with average bioequivalence.A crossover ,2-treatment 2- sequence 4-period study was conducted.The results of average bioequivalence was shown but results from individual bioequivalence approach had no success in showing bioequivalence.It was therefore concluded by Nasir etal that the individual bioequivalence approach was a strong stastical tool to test for intra-subject variances and also subject-by-formulation interaction variance compared with the average bioequivalence approach.

A review of published literature detailing the clinical use of amoxicillin/clavulanic acid was undertaken by Neu HC etal(1993)(spanning perion 1997 to 1992) to assessed the clinical efficacy of the product and to determined whether any changes had occurred during this time.A total of over 38,500 patients were treated with amoxicillin/clavulanic acid in 415 publications attained selection criteria.The data analyzed confirmed amoxicillin/clavulanic acid efficacy over a wide range of clinical indications and annual and triennial grouping of publications suggested a little variation in the clinical effectiveness of amoxicillin/clavulanic acid.In comparative and uncontrolled trials the clinical efficacy rates(cured or improved) with amoxicillin/clavulanic acid were 88% and 92% respectively.Most common but relatively infrequent adverse event were

gastrointestinal side effects and therefore it was analysed that amoxicillin/clavulanic acid continue to be a useful antibiotic for upper and lower respiratory tract infections,skin structure infections,dental,head and neck infectins and selected urinary tract infections.

The pharmacodynamic effects of a pharmacokinetically enhanced formulation of amoxicillin 2000 mg bid was compared with amoxicillin 875 mg bid,875 mg tid and 500 mg tid by Odenholt I etal(2004) against Streptococcus pneumonia with different susceptibility to amoxicillin in an in vitro kinetic model.In the mode contained amoxicillinl simulating the human serum concentration– time profile of the pharmacokinetically enhanced formulation twice daily(c-max 17mg/L after 1.5h),the strains of S.pneumonia with amoxicillin MICs of 1,2,4 and 8 mg/L at an initial inoculums of approx. 10(5) cfu/ml were exposed.The clinical isolates were also depicted to amoxicillin with concentration-time profile correlating to human dosage of 875mg bid(c-max 15mg/L after 1 hr),875mg tid and 500mg(c-max 8mg/L after 1 hr)tid with simulated half life of 1 hr.During 24 hr repeated samples were taken regularly.In comparision it was found that the pharmacokinetically enhanced formulation was more effective at reducing bacterial counts than any other formulation evaluated and enhanced formulation showed eradication of strains with a MIC of < or = 2mg/L but regrowth occurred with other dosing regimens.The enhanced formulation kept the bacterial count < or = 10(2) cfu/ml for at least 14 out of 24 hr tested in the experiment with the strain with a MIC of 4 mg/L as compared no any other formulation reduced the bacterial count down to < or = 10(2) cfu/mL at any point.Therefore the least effective dosage regimen for all strains was 875 mg twice daily.

The pharmacokinetics of linezolid and co-amoxiclave after single and multiple dose administration was investigated by Olaf Burkhardt etal(2002) in 12 healthy volunteers(six female and six males).Co-amoxiclav was given in tablets of 1000mg(875+125 mg) once a day for 7 days while linezolid given as 600mg tablet twice a day for 7 days with a washout period of 4 weeeks between administration of two antibacterial agents.Samples of blood and urine were collected on days1 and 7 before and at different time point upto 24 h after medication.By mean of validated HPLC methods the concentrations of antibiotics in serum and urine were measured and in resulted data linezolid exhibited a mean Cmax of 14.5±4.6 mg/L after T max of 47.5±20.1 min on day1, with a significant increase to 24.0±6.9 mh/L on day 7(P<0.01).A significant increase was revealed in the AUD tot(total area under data) from 140.5±28.3 mg.h/L on day 1 to 220.2±42.6 mg.h/L on day

7(P<0.01) but no significant difference was observed in terminal elimination half life between days 1 and 7(9.53±2.87 versus 7.97±3.08h) or in total clearance(71.6±17.6 versus 81.5±14.7 ml/min,1.73m²).The results were in agreement with the assumption of a limited accumulation of linezolid under the dosage regimen given.In females the serum linezolid concentrations were higher than in males with significant difference in the volume of distribution V/f between females and males(41.6± 4.2 versus 52.2±3.3 L/70 kg; P< 0.01).In this study the pharmacokinetic parameters of amoxicillin and clavulanic acid were similar to previously published data with no accumulation of co-amoxiclave, During study no adverse event was observed.

To compare the bioavailability of two amoxicillin capsule(500mg) formulations(amoxicilina from Brazil as a test formulation and amoxil from smithkline beecham laboratories,Brazil as reference formulation) an accurate,precise and sensitive HPLC assay was developed by Pires de Abreu LR etal(2003) in 24 volunteers of both sexes.By combined reversed phase liquid chromatography and UV detection of 229nm the concentration of amoxicillin were analyzed and amoxicillin was extracted from plasma by addition of cold methanol along with cefadroxil as internal method.The Lichrosorb 10 micron C18 reversed phase column at room temperature used to obtain separation with 95% phosphate buffer(0.01 mol/L) and 5% acetonitrile mixture as mobile phase at pH 4.8.An open randomized 2-period crossover balanced design study was conducted with a 1-week wash out period between the doses.Over an 8-hr period plasma samples were obtained and by calculating individual peak plasma concentrations(C-max) and AUC(08hr) ratios(test/reference) the bioequivalence between the two formulations was assessed.The statistical interval proposed was 80-125% as established by the US Food and drug administration Agency.At a flow rate of 1.3ml/min the internal standard and amoxicillin eluted about 4.2 and 5.2 min respectively with mean absolute recovery of AMO was 90.0% at 3µg/ml,98.6% at 25µg/ml and 95.3% at 50µg/ml.There was an excellent relationship observed between peak height ratio and plasma concentrations(r(2) > or =0.999) in the assay with 1g/ml LOQ based on 200 litre of plasma.The geometric mean of amoxicilina/Amoxil 500mg capsules individual percentage ratio was 101.4% for AUC(0-8h) and 99.9% for C-max and the 90% C.I were 98.3104.4% and 95.7-103.9% respectively.The conclusion proved method to be suitable for pharmacokinetic,bioavailability and bioequivalence studies.Both amoxicilina 500mg capsules and Amoxil capsules were bioequivalent in terms of rate and extent of absorption because 90% C.I for both C-max and AUC(0-8hr) lied within 80-125% interval proposed by Food and Drug administration.

Pajchel G etal(2002) evaluated a rapid,capillary electrophoresis method for the determination of amoxicillin/clavulanic acid in augmentin as well as ampicillin/sulbactam in unasyn preparation for injection.The composition of mobile phase was phosphate-borate buffer at pH 8.66 contained 14.4% sodium dodecyl sulfate and the method was validated.The result showed established reproducibility,precision,accuracy and assay linearity in concentration of amoxicillin 0.05-3.03 mg/ml and ampicillin 0.05-3.08mg/ml as well as clavulanic acid 0.02-2.02 mg/ml and sulbactam 0.05-2.08 mg/ml.When compared with alternative HPLC method used for drug analysis this new method was fast,inexpensive and limited consumption of organic solvents.There were no significant differences found in statistical analysis by student’s t-test(calculated)0.32 and 1.69 for amoxicillin and clavulanic acid and 0.67 and 1.93 for ampicillin and sulbactam smaller than t(tabulated).

The in vitro synergy of amoxicillin/clavulanic acid combination has not always translated in vivo into clinical superiority compared with amoxicillin alone evaluated by Paulo-Borges etal(2000).The superiority of the combination in the treatment of both acute otitis media and acute sinusitis was disputed by conflicting reports which might be due to inadequate target tissue pharmacokinetics and this possibility was explored by Paulo-borges etal by conducting a randomized,open,single dose,sinus tissue pharmacokinetic study with oral amoxicillin /clavulanic acid.Before 2-4 hrs of surgery 23 adult patients with chronic rhinosinusitis were randomly allocated to receive a tablet of 875/125 mg co-amoxiclave the samples were collected at specific sinonasal sites during the operation for the determination of both amoxicillin and clavulanic acid concentration levels.The levels of amoxicillin was adequate throughout the sinuses to cover common susceptible pathogens but the presence of clavulanic acid was detected only half of the sinonasal tissue sample.The resulting data showed that the kinetics of oral clavulanic acid apparently failed to gave a wide spread antilactamase activity capable of enhancing the activity of amoxicillin in all parts of the sinuses but despite this fact amoxicillin/clavulanic acid played a central role for the treatment of acute rhinosinusitis.This is because against Streptococcus pneumonia , a particular virulent and increasingly resistant upper respiratory tract pathogen amoxicillin was still most effective oral lactam.The data also showed that a concomitant anti-s- lactamase activity can be expected to occur in an unpredictable fashion.

Amoxicillin/clavulanate was initially launched as three times daily dosage for the treatment of a range of community acquired infections then a decade later it was considered necessary to introduce a twice daily dosage in terms of convenience,compliance and to maintained itself competitive with other newly launched antibacterial, it was reviewed by Richard Bax(2007).The amount of amoxicillin was increased relative to clavulanate in the twice daily formulations of amoxicillin/clavulanatein order to provide equivalent bacteriological and clinical efficacy with no change in the safety profile.By means of randomized clinical trials equivalence of two dosing regimens was confirmed in adults(in skin and soft tissue,urinary tract and lower respiratory tract infections,sinusitis and recurrent tonsillitis) and pediatrics(in lower respiratory tract infections,otitis media and recurrent tonsillitis).Due to reduced daily dose of clavulanate an improvement in the safety profile,specifically gastrointestinal effects was noted for all patients,particularly in children

The pharmacokinetic properties of amoxicillin and clavulanic acid was studied by Reyns T etal(2007) after single intravenous and oral dosage of 20mg/kg of amoxicillin and 5mg/kg of clavulanic acid in healthy,fasted pigs.Validated HPLC method used to determine the plasma concentration of the drugs and compartmental and non-compartmental analysis were employed to calculate the pharmacokinetic parameters.The plasma concentration-time curves were best portrayed by three compartmental open model for amoxicillin and two compartmental open model for clavulanic acid after I.V administration.Both drugs rapidly eliminated from plasma as amoxicillin (with t(1/2 gamma)=1.03h and a clearance of 0.58 L/h.kg) and clavulanic acid (with t(1/2 beta) of 0.74h and a clearance of 0.41 L/h.kg).The apparent volume of distribution was 0.34 L/Kg for both drugs.A non-compartmental model was used after oral administration and resulting elimination half-lives of amoxicillin and clavulanic acid were not significantly different i.e.0.73 and 0.67h respectively.The average maximal plasma concentration of amoxicillin and clavulanic acid were 3.14 and 2.42 mg/L and these were reached after 1.19 and 0.88h respectively.The per oral route showed average bioavailability of 22.8% for amoxicillin and

44.7% for clavulanic acid.

The pharmacokinetic characteristics of ticarcillin and clavulanic acid were determined by Reed.MD etal(1995) after first dose(n=22) and again after achieving steady state conditions(n=16)

in a group of infants and children.The subjects ranged in age from 1 month to 9.3 yrs in the study, all but 3 patients were 6 months of age or older.50 mg of ticarcillin and 1.7 mg of clavulanic acid (30:1 ratio)/kg of body weight given intravenously to each patient every 4 hours.For ticarcillin average elimination half life,steady state volume of distribution,and body clearance were 1.1 hours,0.22 L/kg and 2.7 ml/min/kg respectively and for clavulanic acid 0.9hours,0.4L/kg and 6.2 ml/min/kg respectively.In the urine 71% of ticarcillin 50% of the clavulanic acid were excreted unchanged over 4 hour sampling period with a corresponding renal clearances averaged 2.1 and 3.2 ml/min/kg respectively.First dose and steady state evaluations did not show any difference in the pharmacokinetic behaviour of either agent but in comparision the pharmacokinetic behaviour of clavulanic acid was significantly different from that of ticarcillin.Therefore for the treatment of most systemic infections that occur outside CNS the obtained pharmacokinetic data combined with known invitro susceptibilities of important clinical pathogens support a dose of 80mg of ticarcillin and 2.7 mg of clavulanic acid.

The single dose pharmacokinetics of ticarcillin and clavulanic acid (Timentin) were evaluated by Richard F.Jacobs etal(1985) in children and young adults with cystic fibrosis after 0.5 hour i.v infusion of both 3.1 and 3.2 gm formulation(representing 3.0gm ticarcillin combinedwith 100 mg and 200 mg clavulanic acid,respectively) in a cross over design.Ticarcillin component was used in the dose of 75 mg/kg and model-dependent and non-compartmental pharmacokinetic parameters were congruous.One compartment open model adequately characterized the disposition of ticarcillin and clavulanic acid.The data showed the elimination half life,apparent steady state volume of distribution and total body clearance of ticarcillin for 3.1 gm formulation from serem were 1.19 hours,0.231 L/kg and 0.150 L/hr/kg respectively and 1.21 hours,0.211 L/kg and 0.123 L/hr/kg respectively for 3.2 gm formulation.86% and 93% of the dose of 3.1 and 3.2 gm formulation ticarcillin were excreted unchanged in urine during first 6 hour after infusion and then concomitant renal clearance values were 0.120 and 0.112 L/hr/kg for 3.1 and 3.2 gm formulations respectively.During the 6 hour post infusion period for both formulation approx. 50% of a clavulanic acid dose was excreted unchanged but for ticarcillin no marked difference observed between 3.1 and 3.2 gm formulations.A significant difference between the two formulation was observed for clavulanic acid in comparision of the area under the serum concentration vs time curve and dose size(P<0.01).In both formulations a linear inverse relationship were identified between demographic factor(e.g, age,weight,height,body surface area) and both the apparent

volume of distribution and total body clearance of ticarcillin and clavulanic acid.Hence it was evaluated that ticarcillin/clavulanic acid combination in either 3.1 or 3.2 gm formulation was suitable for microbiologic and clinical assessment in patients with cystic fibrosis.

A simple,rapid and sensitive isocratic reversed phase HPLC method with UV detection has been developed and validated by Seyed Mohsen Foroutan etal(2007).They performed simultaneous determination of amoxicillin and clavulanic acid in human plasma using internal standard.The method facilitate therapeutic drug monitoring of amoxicillin and clavulanic acid with the minimum quantification limit of 15 and 30 ng/ml,respectively.The assay extraction procedure was one step with complete analytical recovery.They maintained a reversed-phase condition for separation using a Chromolith Performance (RP-18e, 100mm x 4.6 mm) column.The mobile phase with adjusted pH of 3.0 was isocratic containing 0.02 M disodium hydrogen phosphate buffer-methanol (96:4,v/v wave length 228nm).The coefficient of varitation were less than 9.0% for inter-day and intra-day assay.(1)

The pharmacokinetic properties of amoxicillin when used alone or in combination with clavulanic acid may be different and may also show interaction between these two agents,commented by Sayed Abolfazl Mostafavia etal(2006).The absolute bioavaiability of clavulanic acid might decrease due to interaction between theses two agents.The study conducted was open, randomized and replicated Latin square under fasting condition.Theycompared pharmacokinetics of new formulation of amoxicillin-clavulanic acid with a reference formulation.A single dose was administered in 15 healthy male volunteers each received an equimolar dose of new suspension formulations of amoxicillin-clavulanic acid(312 mg/5ml or 156 mg/5ml) or Augmentin (312 mg/5ml) as reference product.A wash out period of one week has been given between the administration of these antibiotics.Blood sampling has been done before and after drug administration of each formulation at different time interval upto 6 hour and then with the help of validated HPLC method plasma concentration of antibiotics were measured.The C-max of 7.5±1.6 mg/l after T-max of 75±25 min for amoxicillin and C-max of 2.5±0.6 mg/l after T-max of 61±15 min has been exhibited by three formulations.The formulations showed AUC0-inf (total area under curve) 1278±172 g.min/ml for amoxicillin and 354±66 g.min/ml for clavulanic acid.The data did not show significant difference among these formulations and found to be similar to previously published data.Hence the investigator from this study proved that two generic formulations are

bioequivalent with the brand Augmentin with regard to pharmacokinetic parameters and considered equally effective in medicinal practice without any pharmacokinetic interaction between amoxicillin and clavulanic acid.

The relevant pharmacokinetic,pharmacodynamic and clinical information associated with the use of combination of amoxicillin/sulbactam was reviewed by Soutric J etal(2006).It was a modern antimicrobial combination and proved to be useful for the treatment of several infections caused by different micro-organisms mainly with the beta-lactamase producing species.

A randomized,controlled trial was designed by Sethi etal(2005) to show that a short, 5-day course of pharmacokinwtically enhanced amoxicillin-clavulanate at dose of 2,000/125 mg(Augmentin XR) is clinically as effective as longer 7 day course of conventional amoxicillin/clavulanate at 875/125 mg(both given twice daily) in the treatment of acute exacerbation of chronic bronchitis(ACEB).In order to compared with conventional formulation amoxicillin-clavulanate at 2000/125 mg was designed to extend the therapeutic levels of amoxicillin in serum over the 12 hour dosing interval,to eradicate bacterial strains for which amoxicillin MICs were < or = 4µg/ml while retaining efficacy against beta-lactamase producing pathogens.Almost 893 patients were randomized and received study medication (amoxicillin/clavulanate at 2000/125 mg was given to 443 patients and 875/125 mg given to 450 patients) and at least one pathogen identified at screening in overall 141 patients received amoxicillin/clavulanate at 2000/125 mg and 135 received the comparator formulation.In the preprotocol population(PP) amoxicillin/clavualanate at 2000/125mg was as effective clinically at the test of cure(days 14 to 21,primary efficacy endpoint) as amoxicillin/clavulanate at 875/125 mg(clinical success rate of 93.0 and 91.2% respectively;treatment difference, 1.8;95% confidence interval CI, -2.2,5.7.Both formulations showed high bacteriological success in the bacteriology PP population(amoxicillin/clavulanate 2000/125mg 76.7%,amoxicillin/clavulanate at 875/125 mg ,73.0%;treatment difference 3.8;95% CI,-7.5,15.0).A similar incidence of events were observed and both therapies were well tolerated and due to adverse events less than 5% patients withdrew from study.Hence sethi etal proved similar clinical effectiveness of shorter 5day course of amoxicillin/clavulanate at 2000/125 mg with longer 7-day courses of amoxicillin/clavulanate at 875/125 mg with high bacteriological efficacy and no difference in tolerability.

A bioequivalence study was demonstrated by Sourgens etal(2004) between the new tablet formulation(test) taken as intact tablet and after prior dispersal,versus the originator product viz. augmentan ® film coated tablet(875/125) used as reference.An open,single dose, crossover design study was conducted in 48 healthy volunteers.The C-max and AUC 0-∞ were used as primary parameters of evaluation for both amoxicillin and clavulanic acid with 90% C.I of the ratios Solutab® tablet/Augmentan® within the range of 0.8-1.25 in the bioequivalence study.Individual plasma concentration-time curve and compartmental analysis were used for calculation of duration of plasma concentration exceeding the amoxicillin MICs.The bioavailability characteristics of the test tablet taken intact or in dispersed form and the reference tablets were very similar as shown by resulting data and the study also confirmed the appropriateness of using a b.i.d dosage schedule for both formulation for some major pathogens.

The pharmacokinetic analysis of individual steady state plasma amoxicillin concentration was done by Sedivy j etal(2004) both in i.v infusion phase and in oral phase of AMC,administered on the basis of quantitative susceptibility of the detected microbes.On the model of Staphylococcus aureus the in vitro growth/killing dynamic parameters were evaluated.As a surrogate criterion of efficacy therapeutic protocol optimization leading to prediction of the earliest time to reduce the number of viable bacteria to 10-6 was performed.The study performed in 17 patients suffering from infected diabetic foot ulcers and the model microbial dynamic parameters based on individual plasma amoxicillin oscillation.The reduction in number of viable bacteria was reached significantly earlier after continuous i.v AMC infusion than after same daily AMC dose administered intermittently.Highly frequent oral therapy of AMC( not longer than 8 hrs dosing interval) was also sufficiently effective in case of highly susceptible staphylococcal strain.The time required for effective reduction of microbes was extended by decreasing plasma amoxicillin concentration exponentially.It was concluded that the individualized optimization of amoxicillin/clavulanate dosage on the basis of growth/killing microbial dynamic parameters and antibiotic concentration/time fluctuation enhanced the antimicrobial effect and the treatment of infected non-critical ischemic diabetic foot ulcer.

The concentration of cefaclor (CFC) or amoxicillin/clavulanic acid (AMX/CA) was evaluated by Scaglione ,Caronzolo etal(2003) in middle ear fluid.They utilized traditional method for preserving stability and clearing cell contents to those obtained.The oral regime were followed with CFC(20

mg/kg of body weight) or AMX/CA (20 mg/kg)(4:1 ratio) for treatment of sixty seven children infected with effusive otitis media.The resulting concentration in the total fluid was appeared lower than those in cell-free fluid.

Many microbial infections can be effectively controlled by combining clavulanic acid with amoxicillin and overcome the bacterial drug resistance due to β-lactamase production,the use of this combination was greatly insisted by Susan E.Orosz etal(2000).Amoxicillin/clavulanic acid were administered to blue fronted Amazon parrots(Amazona aestiva) in this study in a multiple dosing trial. Drug combination of 125mg/kg was given to birds at 0800,1600 and 2200 hours on day 1-5.The result showed larger AUC in parrots as compared to humans but half lives were similar to those of humans. Therefore it was suggested that at this dosing interval this drug combination achieves levels that may be effective against many bacterial species.

Stein GE etal(1984) reviewed the pharmacokinetics,microbiology,therapeutic use,adverse effects and dosage of amoxicillin/potassium clavulanate.Amoxicillin increased the absorption of clavulanic acid which alone had weak antibacterial activity after oral administration.Food had no influence on absorption of amoxicillin/clavulanic .Both acute uncomplicated and complicated UTI and exacerbation of chronic bronchitis caused by amoxicillin resistant organisms in adults effectively cured by amoxicillin/clavulanic acid.The efficacy of amoxicillin/clavulanic acid was comparable to cefaclor for treating uncomplicated UTI in adults and children,acute bronchitis and bronchopneumonia and acute sinusitis,otitis media and skin and soft tissue infections in children.The most common side effects seen with amoxicillin/ clavulanic acid were diarrhea,nausea,vomiting and skin rash.The first three might be lessened by taking the combination with food.

The pharmacokinetics of intravenous Augmentin was investigated in a two compartment model by Staniforth etal(1984).Firstly in a crossover design study a bolus injection of 1.2g Augmentin administered to 8 healthy volunteers with and without probenecid which showed an increase in serum concentration of amoxicillin in the presence of probenecid with no effect on clavulanic acid.An infusion over 30 min of 2.2g Augmentin given to further 8 volunteers in another study and a peak concentration in excess of 100µg/ml for amoxicillin and 14 µg/ml for clavulanic acid were recorded at the end of infusion.The obtained serum and urinary concentration of amoxicillin and

clavulanic acid in both studies were well above those regarded as essential to achieve a therapeutic effect. The pharmacokinetic parameters of a parentral formulation of amoxicillin-clavulanic acid were determined by Schaad Urs B etal(1983).The ratio in formulation was kept 5:1 for amoxicillin and clavulanic acid respectively.For study 12 pediatric patients were selected between 2 to 14 years of age.All patients got a single dose containing 25 mg of amoxicillin and 5 mg of clavulanic acid per kg of body weight as intravenous infusion over 2 min.The data acquired in the study showed mean plasma were 89.4 microgram of amoxicillin/ml and 19,5 picogram of clavulanic acid /ml with a terminal half lives of 1.2 and 0.8 h respectively.On the basis of study data obtained Schaad conclude that amoxicillin and clavulanic acid are pharmocokinetically compatible..Further they also suggested that intravenous administration of 25mg of amoxicillin and 5 mg of clavulanic acid per kg every 6h is a reasonable starting regimen in non-invasive childhood diseases caused by Haemophilus influenza, Staphylococcus aureus, Streptococcus spp, Neisseria spp, Branhamella catarrhalis and other susceptible organisms.

The concentrations of amoxicillin and clavulanic acid in gingivial cervicular fluid (GCF) was investigated by Tenenbaum etal(2007).Among 20 patients with rapidly progressive periodontis these concentrations were measured one hour after a dose of 500mg(1 tablet Augmentin) on day 0 and 1 h after the 10th intake on day 3.Periopapers® were introduced in 16 gingivial sites per subjects and time for the sampling of GCF and for the estimation of GCF volumes collected Periotron 6000® were used.For the determination of amoxicillin and clavulanic acid in microsamples(1-10µl) of GCF a HPLC method was developed.At day 0 the concentrations of amoxicillin and clavulanic acid were 14.05µg/ml and 0.40µg/ml respectively and 13.93µg/ml and 0.37µg/ml at day 3.Following the multiple administration of amoxicillin combined with clavulanic acid, effective levels above the MIC of some susceptible periodontal anaerobes (P.intermedia)involved in destructive periodontal diseases were achieved.

A new pharmacokinetically enhanced formulation developed by Thomas etal(2005) and actually it was designed to overcome infections caused by S.pneumoniae including penicillin resistant (PRSP,penicillin MIC≥2mg/l) isolates and those with elevated amoxicillin/clavulanic acid MICs,although maintaining coverage of β-lactamase producing pathogens.In adult patients with CAP a grouped efficacy analysis of four randomized(1:1) and one non-comparative clinical trials

of amoxicillin/clavulanate ,2000/125 mg twice daily was conducted along with conventional amoxicillin/clavulanate formulation as comparator agents.Amoxicillin/clavulanate 2000/125mg showed an efficacy(eradication of initial pathogen or clinical cure in patients for whom no repeat culture was performed) of 92.3%(274/297) and those of comparators 85.2%(46/54 P=0.11) at followup days(days 16-39).Twenty four out of 25 PRSP infected patients were successfully treated with amoxicillin/clavulanate 2000/125 mg.Some of the patients had withdraw from studies but as a whole both amoxicillin/clavulanate and comparators were well tolerated.

The pharmacokinetic parameters of amoxicillin and clavulanic acid were calculated and the parameters that may affect their observed differences in absorption were identified by Tom B etal(2003).An open,randomized,two-treatment,two period,two sequence,crossover Phase I,four different bioequivalence studies were conducted to obtained data from plasma concentration time curve.The co-amoxiclave formulations used were tablet 250/125,500/125 and 875/125mg or 10ml of an oral suspension 250/62.5mg/5ml.The available data for evaluation were from 144 subjects and 288 drug administration.The clavulanic acid AUC data ranged from 1.5 to 8mg.h/L varied by factor of 5 after administration of 125mg clavulanic acid dose(as potassium clavulanate).The absorption of amoxicillin and the demographic factor had no interaction with absorption of clavulanic acid and the reason for large variability in the absorption of clavulanic acid were unknown.Tom B etal concluded that after oral administration the absorption of clavulanic acid is highly variable and could vary up to five folds range between patients.

A reversed phase high performance liquid chromatography method was used by Tan L etal(1997) to develope a rapid and sensitive assay for amoxicillin in human plasma.After adding internal standard (tinidazole) plasma samples were prepared for analysis followed by protein precipitation with HClO4.Mobile phase consisted of 0.033 mol/l phosphate buffer(pH 7.2)methanol mixture(85:15) with a YWG C18H37 column as stationary phase and UV detection as 229nm.The data showed linear calibration curve in the range of 0.2µ.ml-l to 20.0µ.ml-l with gamma > 0.999 and > 86.7% plasma analytical recovery of amoxicillin.Within day and between day the relative standard deviations were <5.48% and <8.29% respectively.The peak levels of amoxicillin in plasma averaged 6.88 ± 2.25µg.ml-l at 84.4±21.1 min following oral administration of 500mg in human volunteers along with average half life time for amoxicillin was 62.8±14.6 min.

A simple,rapid and accurate method for simultaneous determination of amoxicillin and clavulanic acid was developed by Tai-Li Tsou etal(1997) using HPLC with β-cyclodextrin stationary phase.The method involved the use of tetraethylammonium acetate (TEAA) as an additive reagent in the mobile phase containing methanol-buffer solution,35:65 v/v(pH 4.5) detected at 225 nm and chromatogram within 12 min.The internal standard method for linearity and precision have been obtained with the recoveries ranged from 99.25 to 105.63% for amoxicillin and it was 99.50 to 101.64 for clavulanic acid.The method was considered convenient and reproducible for analysis of these two components in different dosage forms.

The concentrations of amoxicillin and clavulanic acid were investigated by Tenebaum etal(1997) in gingivial crevicular fluid(GCF).The plasma concentration were measured in 20 patients with rapidly progressive periodontis 1hr after a dose of 500mg(1 tablet augmentin R) on day 0 and 1 hr after 10th intake on day 3.Periopapers(R) were introduced in 16 gingivial site per subjects and time for sampling of GCF and then the GCF volumes obtained were estimated with the application of Periotron 6000(R).For the determination of amoxicillin and clavulanic acid in microsamples(1 -10 micro/l) of GCF a HPLC method has been developed.The resulting concentration of amoxicillin and clavulanic acid were 14.05(micro)g ml-l and 0.40(micro)g ml-l at day0,13.93(micro)g ml-l and 0.37(micro)g ml-1 at day 3.After multiple administration of amoxicillin/clavulanic acid ,effective levels of amoxicillin and clavulanic acid above the minimal inhibitory concentrations of some susceptible periodontal anaerobes (P.intermedia) responsible for causing destructive periodontal disease were achieved.

The antibacterial spectrum of amoxicillin enhanced by clavulanic acid by rendering most beta- lactamase producing isolates susceptible to the drug, updated by Todd PA etal(1990).Amoxicillin/clavulanic acid was clinically and bacteriologically superior to amoxicillin alone in clinical trials and also effective equally to other comparative agents like orally administered cephalosporins,cotrimoxazole,doxycycline and bacampicillin in the treatment of adults and children with the most common forms of infections came acrossed in general practice UTI, upper and lower respiratory tract infections,otorhinolaryngological infections and skin and soft tissue infections.The combination was also proved effective for the treatment of uncomplicated gonorrhea.chancroid and gynaecological infections.The effectiveness of

amoxicillin was restricted by beta-lactamase production but the combination of clavulanic acid with amoxicillin clearly extended the usefulness of a tried and proven first line antibacterial agent.

The relative bioavailability of amoxicillin/clavulanic acid dispersive tablets was studied by an unknown author in 2007.In a randomized crossover study a single oral dispersive tablet containing 125mg clavulanic acid and 500mg amoxicillin and Anqi tablets were administered to 18 healthy male volunteers and simultaneously plasma concentrations of amoxicillin and clavulanic acid were determined with HPLC.The resulting pharmacokinetic data of dispersive tablet and Anqi tablet for clavulanic acid were : T max(0.89±0.25)h and (1.01±0.30)h; C max(1.92±0.64)mg.L-1 and (1.49±0.65)mg.L-1;AUC 0→∞(4.347±1.542)mg.h.L-1 and (3.395±1.468)mg.h.L-1 and for amoxicillin the pharmacokinetic parameters were: T max(1.15±0.41)h and (1.34±0.40)h;C max(5.81±1.40)mg.L-1 and (4.60±1.37)mg.L-1;AUC 0→∞(13.472±3.114)mg.h.L-1 and (11.937±2.735)mg.h.L-1.The comparision showed significantly shorter clavulanic acid T max of clavulanic acid /amoxicillin dispersive tablet and significantly greater AUC 0→∞ of clavulanic acid /amoxicillin dispersive tablet than the control Anqi tablet respectively while the comparision did not show any marked difference between amoxicillin T max of both tablets.Hence it was concluded that the bioavailability of clavulanic acid /amoxicillin dispersive tablet is superior to anqi tablet.

The pharmacokinetic parameters of a parentral formulation of amoxicillin-clavulanic acid were determined by Urs Schaad etal(1963).The ratio in formulation was kept 5:1 for amoxicillin and clavulanic acid respectively.For study 12 pediatric patients were selected between 2 to 14 years of age.All patients got a single dose containing 25 mg of amoxicillin and 5 mg of clavulanic acid per kg of body weight as intravenous infusion over 2 min.The data acquired in the study showed mean plasma were 89.4 microgram of amoxicillin/ml and 19,5 picogram of clavulanic acid /ml with a terminal half lives of 1.2 and 0.8 h respectively.On the basis of study data obtained Schaad conclude that amoxicillin and clavulanic acid are pharmocokinetically compatible..Further they also suggested that intravenous administration of 25mg of amoxicillin and 5 mg of clavulanic acid per kg every 6h is a reasonable starting regimen in non-invasive childhood diseases caused by Haemophilus influenza, Staphylococcus aureus, Streptococcus spp, Neisseria spp, Branhamella catarrhalis and other susceptible organisms.

The extrapolation of the rate constant values were investigated by Vahadat etal(2007) to the frozen state from the liquid state data indicated marked acceleration of the rates of amoxicillin and clavulanate degradation for the pH values.For clavulanate the highest acceleration in rate was recorded as 15.0 folds and the lowest value was 4.6 fold for amoxicillin at -7.3 ºC in the hydrochloric acid system and the rate constant values obtained were interpreted by means of concentration model(Pincock,R.E,Kiovsky,T.E,1966.Kinetics of reaction in frozen solution.J.Chem.Edu.43,358-360),phase-temperature relationship of the solutes,buffer catalysis,pH change and polymerization reactions.In order to provide adequate explanation of the experimental results a kinetic model was deduced for the hydrochloric acid system.Sodium chloride was used as stabilizer for maintaining constant ionic strength(µ=0.5) in this system.When sodium chloride was included in the hydrochloric acid system the shelf life of amoxicillin was increased from 2.2 to 58.7 h at -7.3ºC.

A pharmacokinetic study was performed by Weitschies etal(2008) with simultaneous imaging of in vivo behavior of ER tablets by magnetic marker monitoring(MMM).The amoxicillin AUC was significantly lower (1854±280 µg min/ml) under fasting condition than after intake at the beginning of breakfast(2452±354µg min/ml) ar after breakfast (2605±446µg min/ml).In contrast clavulanic acid AUC was well comparable after tablet intake under fasting condition and at the beginning of breakfast(191±46 and 189±44µg min/ml).It was suggested by localization data that the reduced bioavailability of amoxicillin under fasting conditions was due to early gastric emptying in combination with poor absorption from deeper parts of small intestine.The reason for the decreased bioavailability of clavulanic acid after tablet intake following the meal was due to prolonged gastric residence of clavulanic acid which was caused by intragastric tablet deposition in the proximal stomach.

In human plasma for simultaneous determination of amoxicillin and ambroxol a rapid,simple and sensitive LC-MS/MS method was developed by Wen A etal(2008) and used clenbuterol as internal standard(IS).Precipitation of plasma samples were carried out with methanol with Lichrosper C- 18 column(150 x 4.6mm,dp 5 microm) used for separation.The mobile phase consisted of methanol(contained 0.2% formic acid) and water(contained 0.2% formic acid) used by gradient elution at a flow rate of 1.0 mL/min.Detection was performed using electrospray ionization in +ve ion multiple reaction monitoring (MRM) mode by monitoring the ion transition from m/z 365.9-

348.99 (amoxicillin), m/z 378.9--- >263.6(ambroxol) and m/z 277.0--- >203.0(IS).The linearity of calibration curves were observed in the concentration range of 520000 ng/ml for amoxicillin and 1-200 ng/ml for ambroxol with the intra and inter run precisions of <9% and the accuracies of 100±7%.In healthy chinese volunteers the method was validated and applied to pharmacokinetic studies of compound amoxicillin and ambroxol hydrochloride tablets.

The continuous infusion(CI) of ticarcillin/clavulanate is a potential therapeutic improvement over conventional intermittent dosing,it was concluded by Wendy etal(2005) because the major pharmacodynamic(PD) predictor of efficacy of β-lactam is the time that free drug levels exceed the MIC.The study involved a time period of 6 year retrospective arm evaluating efficacy and safety of CI ticarcillin-clavulanate in the home treatment of serious infections and a prospective arm additionally evaluating pharmacokinetic(PK) and pharmacodynamic(PD).Steady-state serum ticarcillin and clavulanate levels and MIC testing of significant pathogens were performed in the prospective arm.A continuous infusion dose of 9.3-12.4 g/day and mean CI duration of 18 days was administered to 112 patients(median age, 56 years).Those infections which were treated were include osteomyelitis(50 patients),septic arthritis(6), cellulitis(17),pulmonary infections(12),febrile neutropenia(7),vascular infections(7),intra-abdominal infection(2) and Gram-negative endocarditis(2) and out of 112 patients 91 patients means 81% were cured, 14(13%) had partial response and 7(6%) failed therapy.PICC line complications occurred among nine patients and five patients suffered from adverse drug events.Only four patients had sufficient data for a full PK/PD evaluation(both serum steady-state drug levels and ticarcillin and clavulanate MICs from a bacteriological isolate) although 18 patients had prospective PK/PD assessment.It was so difficult to obtain data in home based patients, particularly as serum clavulanate levels were found to deteriorate rapidly on storage.Free drug levels exceeding the MIC of the pathogen were found in three of four patients with matched PK/PD assessment..Hence it was found that the home CI of ticarcillin/clavulanate was safe,effective, convenient and practical therapy and had a therapeutic advance over traditional intermittent dosing when used in home setting.

Wibawa JI etal(2002) developed a rapid ,selective and sensitive HPLC assay for routine analysis of amoxicillin in rat plasma,gastric juice aspirate and gastric tissue which is applicable to low concentration of amoxicillin(<1 µg ml(-1)) or small sample volumes.A fluorescent product was formed by conversion of amoxicillin via an internal rearrangement which was subsequently

recovered by liquid-liquid extraction.The mobile phase consisted of methanol-water(55:45, v/v) and used with a Kromasil ODS 3 microm(150x 3.2 mm I.D) column which was maintained at 40ºC.At an lambda(ex) of 365nm and an lambda(em) of 445nm the fluorimetric detection was done.The data showed the LOQ for amoxicillin were 0.1µg ml(-1) for gastric juice aspirate(500 microL),0.5µgml(-1) for plasma(50 microL) and 0.075µg g(-1)for gastric juice(250mg).The inter and intra-day RSDs was less than 19% with linearity upto at least 15µg ml(-1) in gastric juice aspirate,upto 200 µg ml(-1)in plasma and upto 100µg g(-1) in gastric juice.For pharmacokinetic studies in individual rats the assay was applied to the measurement of amoxicillin in rat plasma,gastric juice aspirate and gastric tissue. Weber DJ,etal(1984) claimed the marketed availability of amoxicillin/clavulanic acid combination in 2:1 and 4:1 fixed ratio dosage forms.The evidence suggested that clavulanic acid ,a potent inhibitor of many beta lactamase enzymes increased the spectrum of amoxicillin in vitro and in vivo at achievable serum concentrations and at achievable urine levels against many beta lactamase producing strains.After oral administration both amoxicillin and clavulanic acid were well absorbed,reached peak serum level in 40-120 min and have similar half lives of 45 to 90 min.It was further predicted by Weber DJ etal that the combination suitable for the treatment of complicated UTIs,otitis media,sinusitis and respiratory tract infections but precise recommendation for its use needed to await further clinical trials that compared amoxicillin/clavulanic acid to alternative therapies.

The pharmacokinetic parameters of 500mg amoxicillin,125mg potassium clavulanate and 625 mg of their combination(augmentin) after oral administration were determined by Witkowski etal(1982).The study was randomized crossover involving 10 healthy volunteers.Amoxicillin showed absolute bioavailability(AUC oral/AUCi.v)of 0.70+/-0.12 and the mean maximum serum concentration of 6.5+/-1.6 mg/l after administration alone and 6.5+/-1.4 mg/l in combination while respective values for potassium clavulanate were 3.4+/-1.4 mg/l and 2.8+/-1.1 mg/l.there was no significant difference observed between pharmacokinetic parameters of both when administered alone or in combination.When administered alone the AUC for amoxicillin was 19.5+/-5.4 h x mg/l and 23.2 +/-10.6 h x mg/l in combination.For potassium clavulanate the respective value were 7.8+/-3.2 h x mg/l and 7.3+/- 2.0 h x mg/l.

Yoon KH etal(2004) using terbutaline as internal standard developed a simple,fast and sensitive HPLC-mass spectrometric(MS) method for simultaneous determination of amoxicillin and clavulanic acid in human plasma.Plasma proteins were precipitated with acetonitrile and on C8 reversed phase column with formic acid-water-acetonitrile(2:1000:100) the analytes were separated.These analytes were detected by electrospray ionization(ESI) mass spectrometry in negative selected ion monitoring(SIM) mode.In clinical studies the method was validated and successfully applied to analysis.The data showed five times lower than that of published HPLCUV method limit of quantification i.e 0.12 µg/ml for amoxicillin and 0.062µg/ml for clavulanic acid respectively.

ANTIMICROBIAL ACTIVITY: Anguilar etal (1997) studied the effect on Staphylococcus aureus viability and beta-lactamase activity of concentrations that simulated those in human serum with a combined dose of 875mg amoxicillin and 125mg of clavulanic acid in an in vitro pharmacodynamic model.A significant decrease in the initial inoculum of >90% six hours pre-exposure to concentration of amoxicillin- clavulanic acid combination which was higher than amoxicillin-clavulanic acid MIC.A significant decrease in beta-lactamase activity versus those of control even from 6h,when concentrations were sub-inhibitory was observed during study.

Brumfitt W and Hamilton-Miller J M(1984) evaluated that amoxicillin plus clavulanic acid (250+125 mg) was used every 8h for 7 days in fourty-four patients(43 female, 1 male)with a history of recurrent UTIs.The data showed 84% microbiological cure rate 1 week after end of treatment and 67% 1 month later with 20% mild side-effects.It was therefore resolved that the combination of amoxicillin and clavulanic acid considered as first line drug in patients with recurrent UTI.

In a Spanish hospital Elisenda Miró,etal(2002) analyzed over a 3 year period that almost 7% of 7,252 non-duplicated clinical isolates of E.coli showed reduced susceptibility to amoxicillinclavulanate.This was due to overproduction of Inhibitor –resistant β-lactamases.From these 7%, 0.37% produced the IRTs TEM-30,TEM-31,TEM-33,TEM-34,TEM-37,TEM-40,TEM- 51 and TEM-54.Class C β-lactamases overproducers were 5.3% while 0.8% were TEM-1 overproducer probably.Enzyme OXA-30 produced by 0.18%, on the other hand 0.15% overexpressed SHV-1 and 0.03% produced a PSE-1 enzyme.

In an invitro study Fuchs P.C et al(1983) compared augmentin(combination of two parts amoxicillin plus one part clavulanic acid) ampicillin or amoxicillin or both for its inhibitory and bactericidal activities against selected clinical isolates.They also determined regression analysis and tentative disk diffusion susceptibility break point.Three quality control organisms were used for disk diffusion test to find out provisional quality control limits.The susceptibility of augmentin was similar for all methicillin-susceptible staphylococci and Haemophilus influenza isolates but

MIC for β-lactamase producing strains of both groups was four folds higher than those for enzyme- negative strains.Augmentin also exhibit significant activity against K.pneumoniae,Citrobacter spp,Proteus vulgaris and about one third E-coli as compare to ampicillin.The interpretive disk diffusion susceptibility breakpoint for augmentin on the basis of regression and error rate bound analyses were susceptible greater than or equal to 18mm, resistant less than or equal to 13mm(Gram-negative bacilli), and susceptible greater than or equal to 20mm(Staphylococci and H.influenzae).

Gyssens etal(2011) evaluated safety and effectiveness of two regimens in a sequential manner i.e. Intravenous to oral regimen in a prospective ,randomized, double dummy, double blind, multicentre design study. During study moxifloxacin iv/oral versus piperacillin/tazobactam(TZP) iv followed by oral amoxicillin/clavulanate(AMC)regimen was used. According to complicated skin and skin structure infections(cSSSI) subtype /diagnosis(major abcess, diabetic foot infection, wound infection), surgical intervention and severity of illness the patients ≥18 years had been prospectively stratified and on the basis of predetermined criteria diagnosis and disease severity was classified and documented by frequent photograph with the help of autonomous data review committee. For 7-21 days randomized patients (total no. of patients were 813)received either 400mg moxifloxacin iv once daily followed by 400mg of moxifloxacin orally once daily or 4.0/0.5g of TZP iv thrice daily followed by 875/125 mg of AMC orally twice daily. The data review committee assessed the efficacy variable at test of cure(TOC) for per protocol (PP) population and clinical efficacy. The result of the data showed that the clinical success rate at TOC were similar for both regimens and were well tolerated. It was therefore concluded that once daily iv/oral moxifloxacin monotherapy considered non-inferior clinically and bacteriologically to iv TZP thrice followed by oral AMC twice daily in cSSSI patients.

Irene Mangin etal(2012) in human colonic microbiota assessed the possible modification due to amoxicillin-clavulanic acid(AMC) treatment on total bacteria and on Bifidobacterium species balance.An AMC dose of 875/125 mg twice a day for 5 days was given to eighteen healthy volunteers(19-36 years old).Before and after antibiotic exposure fecal samples were obtained and total bacteria and bifidobacteria quantified using PCR after DNA extraction.The concentration of total bacteria as well as bifidobacterium was significantly reduced at the end of AMC exposure as compared to before AMC exposure.In addition the number of Bifidobacterium was also reduced per sample significantly.Therefore it was concluded that a common antibiotic treatment may qualitatively alter the colonic microbiota.

In United State tertiary care hospital from october 1998 to December 1999 Keith S.Kaye etal(2004) analyzed the enzymatic basis and molecular epidemiology of amoxicillin-clavulanate resistant E- coli isolated and were screened for ampicillin-sulbactam resistance. Among total no. of 283 isolates which were resistant to ampicillin-sulbactam 69 were also resistant to amoxicillin- clavulanate by disk diffusion testing(zone diameter ≤ 13mm) and they were further tested by agar dilution,pulsed field gel electrophoresis, PCR analysis and isoelectric focusing. The cohort included 78% female with the mean age of 52 years.From isolates 12 were nosocomial and 57 were community acquired and it was found from results that 67 isolates were non-susceptible and

two were susceptible to amoxicillin-clavulanate by agar dilution testing although they were resistant by disk diffusion testing.Only 8 were resistant to ceftazidime and 37 were piperacillin/tazobactam resistant.The PCR assay showed β-lactamases distribution and found TEM type alone in 52 isolates AmpC type in four isolates(two contained CMY-2).Two isolates showed TEM type and CMY-2 while OXA-type found in one isolate.Also TEM type and SHV type found in one and another with TEM type with an unidentified enzyme.Two extended spectrum β- lactamase producing isolates identified from TEM-type enzymes but two isolates with inhibitor resistant TEM(IRT) enzymes (one with TEM-34[IRT-6] and other with a novel enzyme[TEM- 122]).These results were considered more interesting. In a prospective ,double blind,randomized,phase III comparative trial Mark.A.Malangoni etal(2006) compared the safety and efficacy of standard antimicrobial regimen of IV piperacillin/tazobactam followed by PO amoxicillin/clavulanate with that of sequential intravenous(IV) to oral PO moxifloxacin for the treatment of complicated intra-abdominal infections in adults.It was concluded that single IV/PO moxifloxacin at once daily therapy was as effective as that of standard IV Piperacillin/tazobactam/PO amoxicillin-clavulanate multiple daily therapy for the treatment of complicated intra-abdominal infections.

Mastour S. Al-Ghamdi etal(1999) focused on sharp worldwide increase in bacterial resistance against antimicrobial agents.They observed in both nosocomial and community acquired pathogens.It was concluded that this rise not only complicated the treatment pattern but also increase the cost of management.Therefore both at local and national level pattern of reistance to antimicrobial agents should be regularly investigated.

In an in vitro pharmacodynamic model Prieto etal(1998) studied the effect of concentrations simulated in human serum after single IV dose of amoxicillin(2g), amoxicillin-clavulanic acid(2g and 0.2 g) or vancomycin(0.5g) on the viability and β-lactamase activity of two isogenic heteroresistant Staphylococcus aureus.During study at concentration which was simulated to those in human serum over time the invitro effect of co-amoxiclav,amoxicillin and vancomycin versus control was assessed.The effect was observed on the viability of total population and the resistant sub-population of two isogenic strains of MRSA plus the effect on the β-lactamase activity of the corresponding strains.It was concluded that despite vancomycin amino-penicillinβ- lactamase inhibitor may be considered for empirical treatment.

Robert Wilson etal(2012) compared amoxicillin/clavulanic acid with that of moxifloxacin for treatment of acute exacerbations of chronic obstructive pulmonary disease(AECOPD) in a multiregional ,randomized,double blind non-inferiority out patient study.Clinical failure eight weeks post therapy in per-protocol population was considered primary end point.It was concluded that both moxifloxacillin and amoxicillin/clavulanic acid were effective and well tolerated in the treatment of out patients with AECOPD.

Schaper N.C etal(2013) reterospectively compared the safety and efficacy of two antibiotic regimens i.e. IV/PO moxifloxacin and IV Piperacillin/tazobactam followed by PO amoxicillin/clavulanic acid in patients with diabetic foot infections.No significant difference was

found between the demographic characteristics in either treatment group.Both treatment groups showed similar bacteriological success rate.Therefore safety and efficacy profile of moxifloxacin was concluded favorable and as an alternative antibiotic therapy for the management of diabetic foot infection.

Stephane Bronner etal(2002) investigated the potential bactericidal activity of amoxicillinclavulanate against β-lactamase producing E-coli strains to evaluate the extent to which enzyme production affects the activity.A 30 min IV infusion of amoxicillin-clavulanate was given to 6 adult yucatan miniature Pigs.In serum sample they determined pharmacokinetic parameters and compared them from human.The miniature pig showed a good model for pharmacodynamic study of amoxicillin-clavulanate.The strain’s level of β-lactamase production influenced the sensitivity of E-coli to the combination of amoxicillin plus clavulanate.In the study it was concluded that amoxicillin-clavulanate at concentration similar to those seen in human plasma after administration of 2.2g iv had bactericidal potential against low level penicillinase producing and intermediate level penicillinase producing E-coli strains.

Velerie Berry etal(1998) determined comparative antibacterial efficacy of three macrolides i.e. erythromycin,clarithromycin and azithromycin against Streptococcus pneumonia and Haemophilus influenza.They used amoxicillin-clavulanate as the active control.Amoxicillinclavulanate showed rapid antibacterial effect while macrolide twice of their MICs and at a concentrations attained in humans were bacteriostatic or number of viable S.pneumoniae reduced slowly in vitro. Azithromycin showed bactericidal activity against Hemophilus influenza whereas erythromycin.clarithromycin and clarithromycin plus 14 hydroxy clarithromycin at twice their MICs showed slow reduction in bacterial numbers.In serum of human azithromycin was bacteriostatic while clarithromycin and erythromycin were ineffective.Clarithromycin and amoxicillin-clavulanate were highly effective in experimental respiratory tract infections in rats against S.pneumoniae while azithromycin was less effective.Similarly against H.influenzae amoxicillin-clavulanate was highly effective.Therefore it was concluded that amoxicillinclavulanate was effective in vitro and in vivo while clarithromycin and erythromycin were ineffective in vivo and in vitro against H.influenzae with unpredictable activity of azithromycin against both pathogens.

EXPERIMENTAL

4. EXPERIMENTAL 4.1. PHYSICO-CHEMICAL TESTS OF DIFFERENT CO-AMOXICLAV BRANDS. 4.1.1. WEIGHT VARIATION TEST:

The weight variation test of tablets carried out using analytical balance (Sartorious, Germany) and results were interpreted graphically by preparing quality control chart in software Microsoft Excel™ 2007.

4.1.1.1. Method:

Twenty tablets of each brand were randomly selected and their individual weight were taken.Then the average mass was determined.

4.1.1.2. Limit/Tolerance: Not more than two of the individual masses deviate from the average mass by more than the percentage deviation shows in the table below(BP 2007):

Average Mass Percentage Deviation

Pharmaceutical Form 80 mg or less 10

More than 80 and less than 7.5 250mg

Tablet Uncoated and Film coated

More than 250mg 5

4.1.2 THICKNESS VARIATION TEST:

4.1.2.1. METHOD:

The thickness variation test of twenty tablets of each brand carried out using vernier caliper(Seikobrand,China) and results were interpreted graphically by preparing quality control chart in software Microsoft Excel™ 2007.

4.1.2.2. Limit/Tolerance: The average thickness of twenty tablets must be within ±5% thickness range.

4.1.3. HARDNESS TEST: 4.1.3.1. METHOD:

Twenty tablets of each brand were taken randomly and their hardness was determined using Fugiwara Scisakusho tablet hardness tester(Ogawa Seiki Co Ltd,Tokyo.Japan).Graphically the results were analyzed by preparing quality control charts in MS.Excell 2007.

4.1.3.2. Limit/Tolerance: For twenty tablets the average hardness must be greator than 5kg.

4.1.4. DISINTEGRATION TEST: 4.1.4.1. METHOD:

Disintegration test was performed according to USP32-NF27 procedure for uncoated and film coated tablets in USP Basket rack assembly(Erweka ZT2,Heusenstamm,Germany).One tablet was placed in each of six tube of the basket.,perforated disk was added and assembly was operated for 30 min at 37±2®C in water as an immersion fluid.

4.1.4.2. Limit/Tolerance: The test conducted three times.Out of eighteen tablets sixteen should pass this test.

4.1.5. DISSOLUTION TEST: 4.1.5.1. CHEMICALS AND REAGENTS:

1. Co-amoxiclav tablets(purchased from local market as shown in table 1) 2. Co-amoxiclav standard (provided by Central drug laboratory Karachi) 3. Deionized distilled water.

4.1.5.2. SPECTROPHOTOMETERIC CONDITION:

The dissolution of amoxicillin/clavulanic acid tablets were carried out according to USP procedure(United State Pharmacopea 28,2005) under the following conditions and absorbance was taken on Shimadzu,1601 UVPC.

4.1.5.3. PROCEDURE:

Preparation of Standard:

0.0025%w/v solution of Amoxicillin standard and 0.00125% w/v solution of Clavulanic acid standard was prepared in 100ml distilled water.

Medium: Water:900ml

Appratus 2: 75rpm, place six tablet(containing 250mg amoxicillin and 125 mg of clavulanic acid) in each vessel containing 900ml of distilled water. Time: 30 minutes.

Sampling:

After 30 min 10 mlspecimen was withdrawn from a zone midway between the surface of the dissolution medium and the top of the rotating paddle,not less then 1cm from vessel wall.The specimen was filtered then 4.5 ml of specimen was diluted in 50 ml distilled water and absorbance was measured.

4.1.5.4. Analysis of Specimen:

Total content of amoxicillin and clavulanic acid dissolved from tablet was calculated by comparing the absorbance of the sample to the absorbance of standard in UV-VIS spectrophotometer at 228nm for amoxicillin and 272nm for clavulanic acid respectively using the following formula(BP-1998). % Amoxicillin/Clavulanic acid

dissolved = Absorbance of the sample/Absorbance of standard x 100

4.1.5.5. Interpretation of Result:

The requirements were met of the quantities of the active ingredients dissolved from the unit tested.The quantity Q is the amount of dissolved active ingredients specified in the official monograph.Not less than 85%(Q) of the labeled amount of amoxicillin and not less than 80%(Q) of the labeled amount of clavulanic acid are dissolved in 30 minutes.

4.1.6. CONTENT ASSAY FOR CO-AMOXICLAV TABLETS BY HPLC: 4.1.6.1. CHEMICALS AND REAGENTS:

1. Hplc gradeMethanol (Merck,Damstabt,Germany) 2. Hplc grade Acetonitrile(Merck,Damstabt,Germany)

3. Disodium hydrogen phosphate(Merck,Damstabt,Germany) 4. Ortho-phosphoric acid(Merck,Damstabt,Germany) 5. Hplc grade Dicholro methane (Merck,Damstabt,Germany) 6. Co-Amoxiclav standard(obtained from central drug laboratory, Karachi) 7. Co-Amoxiclav tablets(different brands purchased from market) 8. Distilled water.

4.1.6.2. EQUIPMENTS AND GLASSWARES:

1. High Performance Liquid Chromatographic Pump(LC 10AT VP,Shimadzu Corp,Kyoto,Japan) 2. Spectrophotometeric Detector(SPD -10A, Shimadzu Corp,Kyoto,Japan) 3. Communication Bus Module(CBM 102, Shimadzu Corp,Kyoto,Japan) 4. HPLC column(Purospher star,Hibar, 250 x 4.6,5µ) 5. Chromatographic software(GC 10, Shimadzu Corp,Kyoto,Japan) 6. Analytical balance(Mettler Toledo,Scwerzenbach,Switzerland) 7. Ultra sonic bath(Clifton,Nickle Electro Ltd,somerset, England) 8. Filteration Assembly(sartorious,Germany) 9. Swinny Filteration Assembly(Micropore,England) 10. Microlitre syringe((Hamilton,England) 11. pH meter(Mettler Toledo,England) 12. Distillation Assembly(Hamitton,England) 13. Membrane filters(0.45µ pore size,47mm diameter,Micropore ,England) 14. Swinney filters(Micropore,England) 15. Vacuum pump(China) 16. Volumetric flask(50,100,500 and 1000ml-Pyrex.England) 17. Pipettes(Pyrex.England) 18. Conical flask(100,250,500 and 1000 ml.Pyrex.England) 19. Beakers(Pyrex,England) 20. Graduated cylinder(Pyrex.England) 21. Funnel(Pyrex.England)

4.1.6.3. METHOD: 4.1.6.3.1. HPLC METHOD:

Mobile Phase: 0.02M Na₂HPO₄:Methanol(90:10), pH 3.0 adjusted with ortho-phosphoric acid (Seyed Mohsen Foroutan etal,2007) Injection Volume: 20µl Wavelength of detection: 235 nm. Flow rate: 1.3 ml/min. 0.025% of reference standard solution of amoxicillin and clavulanic acid was prepared in mobile phase, filtered through swinney filteration assembly, injected and peak area was measured.

4.1.6.3.2. ASSAY METHOD:

Twenty tablets of co-amoxiclav were weighed and crushed into powder and then average weight was taken,dissolved,filtered and diluted in mobile phase upto 0.025% concentration.Samples were injected and the peak area was compared with the same concentration of co-amoxiclav reference standard solution.

4.1.6.4. LIMIT/TOLERANCE:

As for Co-Amoxiclav tablets in USP 28(2005) 90 -120%.

4.2. PHARMACOKINETIC EVALUATION OF CO-AMOXICLAV TABLET. 4.2.1. Objective:

In Pakistan Co-amoxiclav tablet of 375mg marketed by a multinational company GSK Pakistan and a local company Getz pharma.Physico-chemical and potency determination from both sources were analyzed.The main purpose of this study is to conduct pharmacokinetic study of Co- amoxiclav tablet in local population and to find out difference in the pharmacokinetic parameters with the previous reported data due to racial inconsistency.In the present study the methodology of bio-equivalence studies can be used to assess variation in the pharmacokinetic parameters in pharmacokinetic studies like drug-drug interactions.Co-amoxiclav is commonly prescribed antibiotic and antacid are also available as an OTC medication in Pakistan.The pharmacokinetic parameters of various antibiotics vary due to simultaneous administration of antacid.This may result due to poor absorption and bio-availability after oral administration reported in several studies.These studies strongly supported the need to conduct bio-availability studies of Co- amoxiclav alone and in combination with an antacid.

4.2.2. Selection criteria in the Pharmacokinetic study:

In the present study Co-amoxiclav 375mg film coated tablet containing 250 mg of amoxicillin and 125 mg of Clavulanic acid (AMCL 2) manufactured by GSK was selected as tested drug.The

concurrent formulation was syrup Mylanta(per 5 ml:Magnessium hydroxide 400 mg,aluminium hydroxide 400mg,simethicone 30mg).

4.2.3. Study location:

The sampling was carried out at Imam Clinic St-5 Block-1 North, Nazimabad, Karachi,SD,74700 Pakistan and analysis of samples conducted in the research lab of Department of Pharmaceutics, Faculty of Pharmacy,University of Karachi.

4.2.4. Study Design:

The study was a single dose,two treatment,two sequence cross over design in twelve healthy volunteers.An equal number of volunteers were assigned to each sequence.

4.2.5. Study design Principle:

The study covers not only to determine the pharmacokinetic parameters i.e. Cmax,Tmax , AUC,rate constant, volume of distribution,total clearance and half life of Co-amoxiclav in local population but also influence of an antacid on these parameters.These Pharmacokinetic parameters were then statistically compared using one way ANOVA test.

4.2.6. Volunteer Selection: i. The volunteers comprised of healthy male students of Faculty of Pharmacy, University of Karachi and Hamdard University. Biasness in the study was avoided and students gathered by displacing information on the notice board and giving them a free will to participate in the study. Their age group range between 21-30 years.The weight, height, medical history was taken prior to the study. ii. Complete physical examination,medical history, vital signs and other necessary tests needed for assessment were obtained.

4.2.7. Criteria 4.2.7.1. Inclusion criteria:

The subjects enrolled in this study will be member of community at large and must meet all of following criteria to be included in the study:

1. Healthy male having normal physical evaluation(12-lead ECG and vital sign) age between 21-30 yrs. 2. Normal laboratory evaluation(b/d hematology,chemistry and urinalysis).

3. Non-smoker

4. Capable of consent.

5. Able to swallow amoxicillin/clavulanic acid.

4.2.7.2. Exclusion criteria: Subjects to whom any of the following applies will be excluded from the study:

1. If subject has any clinically significant illness or heve any surgery within 4 weeks prior to dosing.

2. If during medical screening it will found that subject has any significant abnormality ar any abnormal lab test result.

3. Any other reason which in the opinion of investigator will prevent the subject from participating in the study.

4. If ECG abnormalities or vital sign abnormalities(systolic b.p lower than 90 or over 140 mmHg, diastolic b.p lower than 50 or over 90 mmHg or heart rate less than 50 or over 100 bpm)

5. History of significant alcohol abuse or drug abuse within one year prior to study.

6. Consumption of alcohol/xanthine containing food or beverages, grape fruit and orange juice.

7. History of allergic reaction to amoxicillin/clavulanic acid, penicillin or other related drugs.

8. Use of drug known to induce or inhibit hepatic drug metabolism within 30 days prior to administration of the study medication.

9. If subject used any investigational drug or participated in an investigational study within 30 days prior to dosing.

10. History or presence of any gastrointestinal pathology(e.g. chronic diarrhea, IBS, vomiting),having any liver or kidney disease or any other condition which may interfere with the absorption, distribution, metabolism or excretion of the drug.

11. History or presence of neurological ,endocrinal,cardiovascular,pulmonary,hematological,immunologic,psychiatric or metabolic disease. 12. If subject have taken any prescription medication within 14 days prior to the administration of study medication or any OTC product(including natural food supplements, vitamins,garlic as supplement)within 7 days prior to the administration of study medication Except topical products without systemic absorption.

13. Having difficulty to swallow study medication.

14. Smoking

15. Donation of plasma(500 ml) within 7 days prior to the drug administration.Donation or loss of whole blood(excluding the volume of blood that will be drawn during the screening procedure for this study) prior to administration of the study medication as follows:

 50ml to 599 ml of whole blood within 30 days.

 More than 499 ml of whole blood within 56days prior to the drug administrtion

4.2.7.3. Ethical Approval and Informed Consent:

The pharmacokinetic interaction study of amoxicillin/clavulanic acid and antacid did not face any ethical and practical difficulty but as healthy volunteers were involved in the study and blood samples was drawn therefore it required to be approved by ethical committee and a written informed consent obtained from volunteers.The study design was approved by the National Bioethics Committee , Ministry of Health, Government of Pakistan.Islamabad after critical ethical review and a written informed duly signed by volunteers has been taken.

4.2.7.4. Subject Code:

A subject code was assigned to each volunteer participated in the study. Each subject had given the right to withdraw from the study at any time without any reason.

4.2.7.5. Restriction:

Any type of beverages, tea, coffee and chocolate were not allowed to volunteers 48 hours prior to study till the last sample drawn. Moreover subjects were allowed to take food 10 hours during each of two trial period. The second period of study was conducted after two week washout period. For all volunteers in both trial periods the dietary regimen was kept similar.

4.2.7.6. Drug administration:

After an overnight fasting period 375 mg Co-amoxiclav tablet was administered orally with 250 ml water during each of two trial periods.

The administration plan was as follows: Treatment 1: 375mg Co-amoxiclav tablet were taken alone with water.

Treatment 2: 375 mg Co-amoxiclav tablet+10ml Mylanta syrup taken concomitantly with water.

4.2.8. Blood sample Collection and Plasma Separation: 4.2.8.1. Materials:

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1. Sterile disposable syringes (B.D). 2. Heparinized Centrifuge tubes. 3. Cotton wool. 4. Cannula with stopper. 5. Ethanol(Merck,Germany) 6. Microlitre Pippete(Vocal,England). 7. Refrigerator(LG).

4.2.8.2. Method:

In the morning of each study period cannula was inserted into the subject forearm and remained there till the last sample drawn. A blood sample of 7-10 ml was drawn at 0.5,1,1.5,2,3,4,6, and 8 hours. These samples were immediately transferred in heparanized centrifuged tubes and plasma was separated from each sample by centrifugation at 5000rpm for 5 min. Plasma samples were frozen immediately at –20⁰ C until assayed.

4.2.8.3. Analysis of Plasma Sample:

After modification and validation a high performance liquid chromatography method was used for determination of Co-amoxiclav tablet in plasma. As Antacids are locally acting agents and not expected to be absorbed systemically therefore the plasma concentration of Mylanta was not determined.

Details of the developed method for Co-amoxiclav are as follows:

4.2.8.4. Chemical/Reagents:

1. Methanol(Merck, Germany) 2. Acetonitrile (Merck, Germany) 3. Disodium hydrogen phosphate(Merck, Germany) 4. Dichloromethane(Merck, Germany) 5. Ortho-phosphoric acid(Merck, Germany) 6. Amoxicillin standard (obtained from CDL) 7. Clavulanic acid standard (obtained from CDL) 8. Co-amoxiclav tablet (purchased from market) 9. Deionized distilled water 10. Heparin

4.2.8.5. Apparatus & Equipment:

1. High Performance Liquid Chromatography Pump(LC 10A,Shimadzu ,Japan) 2. Spectrophotometeric Detector(SPD 10A,Shimadzu,Japan)

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3. CBM 102(Shimadzu, Japan) 4. pH meter (Mettler Toledo, England) 5. Centrifuge machine (Hereues, Germany) 6. Vortex mixer (England) 7. Ultrasonic Bath (Clifton, Somerset, England) 8. Filteration Assembly (Sartorious, Germany) 9. Swinney Filteration Assembly (Micropore, England) 10. Microlitre Syringe (Hamilton, Switzerland) 11. Membrane filter(0.45µ-47mm diameter, Micropore, England) 12. Swinney membrane filter (Micropore, England) 13. Distillation Plant (Hammitton Lab, England) 14. Vacuum(China) 15. Software GC 10 (Shimadzu, Japan)

4.2.8.6. Glasswares:

1. Beaker,Funnel, Volumeteric flask, Graduated cylinder, Pippettes (Pyrex, England) 2. Sterile vials for plasma. 3. Heparinized Centrifuged tubes.

4.2.9. Bioanalytical Method Validation: 4.2.9.1. Preparation of Mobile Phase:

Mobile Phase consisted of 10 volume of methanol and 90 volume of 0.02 M disodium hydrogen phosphate buffer with pH adjusted to 3.0 by phosphoric acid. Mobile phase was filtered and degassed before use (Seyed Mohsen Foroutan etal,2007).

4.2.9.2. Chromatographic Condition: An HPLC isocratic pump with UV-VIS detector was attached with RP 18e column (Hibar, 250 x 4.6cm).

4.2.9.3. Sample Preparation:

In a glass stoppered 15 ml centrifuge tube 0.75 ml of acetonitrile was added to 0.5ml of plasma. After mixing(30s) the mixture centrifuged for 10 min at 5000 x g. Then 2.5ml of dichloromethane was added to 300µl of supernatant. After mixing (30s) the mixture centrifuge for 10 min at 5000 x g. Then 20 µl of supernatant was injected into liquid chromatograph.

4.2.9.4. Wavelength of Detection:

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235 nm

4.2.9.5. Retention Time: 5-8 min for Clavulanic acid and 15-23 min for Amoxicillin.

4.3. Validation Parameters:

The above described method was also validated according to ICH guidelines. The fundamental parameters of validation were Specificity, linearity, accuracy, precision, sensitivity, reproducibility, stability and robustness. All these parameters were determined and validated.

4.4. System suitability parameters:

For good and accurate resolution and reproducibility of the presented method various suitability considerations including tailing factor, retention time, resolution, RSD% of retention time and peak areas were determined and were found within acceptable range.

4.4.1. Specificity :

The method was found to be specific for the determination of particular analyte.Specificity was determined by injecting the analytical placebo(containing all excepient of tablet except amoxicillin and clavulanic acid).

The interference by these excepients were determined by evaluating mixture of all excepient (placebo),standard solutions and commercial pharmaceutical preparations contained amoxicillin and clavulanic acid three times within the same chromatographic condition. Specificity of the method was confirmed by the chromatogram which did not show any interference peak.

4.4.2. Linearity:

The linearity of HPLC method was determined for amoxicillin and clavulanic acid.Ten dilutions of amoxicillin and eight dilutions of different concentrations were prepared then sample size of 20µl of each concentration was injected into HPLC.The detector response was measured at 235nm and the calibration plots(concentration versus peak area) were obtained using the linear regression method. The linearity data showed linearity over a concentration range of 0.01531.25 microgram/ml for amoxicillin and 0.121-15.6 microgram/ml for clavulanic acid. Correlation coefficient(R²),mean, SDEV,RSD%, % DEV and % accuracy were determined for both drugs. 4.4.3. Intra-Day and Inter-Day Accuracy and Precision:

Repeatable and intermediate precision of method was determined .During the same day with the same experimental condition repeatability of three determination(n=3) at the same concentration was calculated. The results of assay were compared and evaluated on five different days for intermediate precision(n=5). Statistacical parameters i.e. Mean, SDEV,RSD% and % DEV were determined for both drugs.

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4.4.4. Lower Limit of Quantification (LLOQ) and Limit of Detection (LOD):

LLOQ and LOD were determined by injecting three samples of eleven dilutions for Amoxicillin i.e. 3.9, 1.95, 0.97, 0.48,0.24, 0.121, 0.06,0.03,0.015,0.0075,0.0037 µg/ml and six dilutions for Clavulanic acid i.e.1.95, 0.97, 0.48, 0.24, 0.121, 0.06 µg/ml. Then Correlation coefficient (R²), mean, SDEV, %DEV for both drugs were determined. A calibration curve from sample data was drawn and then use to determine concentrations through back calculation, % accuracy and % precision was then calculated. The chromatogram of lower concentration were detectd and signal to noise (S/N) ratios were calculated to find out LLOQ and LOD.

4.4.5. Recovery:

By spiking drug –free human plasma (ten replicates for each standard) Co-amoxiclav recoveries (absolute and relative) from human plasma were determined. This had been done with known amounts of the drug to achieve amoxicillin concentrations of 0.06, 7.8 and 31.25 µg/ml and Clavulanic acid concentrations of 15.6, 3.9 and 0.24 µg/ml respectively. With the developed procedure the spiked samples were processed and evaluated. By comparing the concentrations obtained from the drug supplemented plasma with actual added amounts the relative (analytical) recovery was calculated.

The absolute (extraction) recovery was calculated by comparing the observed peak area obtained from the processed standard samples to direct injections of standard aqueous solutions prepared at concentrations which represented 100% recovery.

4.4.6. Stability:

The stability of amoxicillin/ clavulanic acid was assessed for spiked plasma samples stored at -20 ˚C over a period of one month. The plasma samples of Co-amoxiclav were analyzed immediately after preparation and at different days after storage over the study period.

4.4.7. Freeze and Thaw stability.

The freeze and thaw stability was determined by three freeze and thaw cycles(1, 15th and 30th day).Fifteen samples of each low ,medium and high concentrations i.e. 0.0037,3.9 and 31.25 µg/ml for amoxicillin and 0.06, 1.95 and 15.6 µg/ ml for clavulanic acid and were prepared and stored at -20˚C .Five Samples after thawing were allowed to stand on the bench top under room lighting till 2h had elapsed since their removal from the freezer. Same procedure was repeated at 15th and 30th day. The peak areas of these samples were compared with fresh stock prepared on the day of analysis and their DEV% were calculated.

4.4.8. Long Term Stability:

Ten samples of low, medium and high concentrations (i.e. 0.037, 3.9 and 31.25 µg/ml for amoxicillin and 0.06, 1.95 and 15.6 µg/ml for clavulanic acid ) were prepared and stored at -

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20˚C.Five samples of each concentration were analyzed at the end of 3rd week and the rest five at the end of 6th week then compared with freshly prepared samples and their DEV% were calculated.

4.5. Pharmacokinetic Analysis:

Various pharmacokinetic parameters were estimated after determination of drug concentration in volunteer’s plasma. These parameters were calculated by applying both compartmental and noncompartmental method of analysis. The results of Co-amoxiclav alone were taken as standard pharmacokinetic results reported in Pakistani population while the results of Co-amoxiclav + antacid ( aluminium hydroxide, magnesium hydroxide and simethicone combination) were used to estimate extent of interaction and bio-equivalence. All these parameters were determined using software Kinetica™ Ver 4.4.1.(Thermoelectron Corp.USA). This software commonly applied for both compartmental and non-compartmental analysis.The parameters determined were:

4.5.1. Compartmental:

Cmax

Tmax (observed and calculated)

Ka, A, B.

Alpha,Beta.

K₁₂,K₂₁,Kel.

T½ka,Tabs,T½α.

AUC0-t, AUC0-α.

4.5.2. Non-Compartmental:

ƛ. V , Vss, HVD.

AUClast. AUCextrapolated, AUCtotal,%AUCextrapolated.

AUMC, AUMClast, AUMCextrapolated, AUMCtotal.

MRT.

4.6. Statistical Analysis:

All pharmacokinetic parameters were statistically analyzed by two way ANOVA. The FDA guide lines followed and data analysis has been carried out for both non-transformed and logrithmly transformed form.

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The Latin square two way ANOVA in the pharmacokinetic software Kinetica considered to be most suitable preference by FDA to calculate all pharmacokinetic parameters based on two treatment, two sequence, cross-over study. The difference in the pharmacokinetic parameters considered significant if there is a probability of less than one in 20 times or 0.05 probability (P ≤ 0.05). The variance between different subjects (inter-subject variability), groups or treatments can be determined by ANOVA methods. Source Degree of Sum of Mean Square(MS) Fisher test(F) Probability freedom(df) square(SS) value(p) Period Number of SS MS period/MS * Periods period/df(period) error Subject(Seq) Num of SS MS subject/MS * subNum of subject/df(subject) error Seq Formulation Number of SS formulation/df MS * Formulations (formulation) formulation/MS -1 error Sequence (Number of SS MS * Seq)-1 sequence/d sequence/MS f (sequence) error Error df(total)-(df SS SS error/ df (total) subject + df (total)(SS period+ subject +SS ---- df period +SS sequence) sequence) Total Total number SS total/df(total) of data – 1 ----- The asterisk (*) indicate the level of significance i.e. α = 5% and every p value is compared with the level of significance .The conclusion after calculation can be drawn by software as follow:

 If p > 0.05 then the difference is not significant, so output the symbol NS.  If p < 0.05 then difference is significant represented by the symbol ***. The other parameters which were computed were geometric mean and geometric standard deviation, confidence interval, Log transformation and Non log transformation.

4.7. SERUM BACTERICIDAL TEST (SBT):

The guide lines of CLSI were followed to perform serum bactericidal test (SBT)(Lemmen etal,2004, Stein and Schooley, 2002).Serum bactericidal test can be determined by macrodilution method (1-2 ml in each test tube) and microdilution method (100 µl in each test tube).In the present study macrodilution method was chosen being more appropriate with high productivity.

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4.7.1. Materials Required:

4.7.1.a. Glass wares:

• Petri dish 100 x 15 mm.(Pyrex .England) • Test tubes 13 x 100 mm.(Pyrex. England) • Pipettes :Micropipettes, (Biohit, Henen.China) • Sterilized Swab and wire loop.

4.7.1.b. Media:

• Mueller Hinton Broth (MHB) (Oxoid.England) • Mueller Hinton Agar. (Oxoid .England) • Agar slants. • 0.5 McFarland turbidity Standard.

4.7.2. Equipments Required:

• Electrical type Autoclave (Shinghai. China) • Vortex mixer (Whirl mixer, England) • Incubator adjusted at 35 ± 1⁰C .

4.7.3. Criteria before Assesment:

4.7.3.1. Clinical isolate storage provision: The clinical isolates directly obtained from patients were stored at ₋20⁰C in trypticase soy broth and freezed(Nadler and Dowzicky, 2007). 4.7.3.2. Test tubes marking: Test tubes were organized and marked from number “1” to “10” and the dilution from tube no. 1 to tube no.8 mentioned as “1:2”, “1:4”, “1:8”,″1:16”, ″1:32”, ″1:64”, ″1:128” and “1:256” respectively. The tube no.9 marked as “Negative control” and tube no.10 as “Positive control”

4.7.3.3. Preparation of Inoculum: The inoculum of Staphylococcus aureus and Escherichia coli were prepared by touching 2-3 loops of culture in 5ml of Muller Hinton broth and then incubate for 3-4 hours at 35 ⁰C to observe turbidity. The turbidity was adjusted with 0.5Mc Farland standard and further diluted to obtained 7.5 x 10⁶ CFU/ml. 4.7.4. Procedure:

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1. The plasma samples containing peak concentrations were frozen at -20⁰C prior to SBT. 2. These samples were thaw and pooled i.e 1ml from each plasma sample collected in a beaker. 3. Then 1 ml from this pooled serum were taken and used for further dilutions. 4. 1 ml of Muellor Hinton broth was added from tube 1-10. 5. Then 1 ml pooled serum was added to tube one which will make total volume of 2 ml. 6. In order to prepared serial dilution transfer 1 ml from tube 2 through tube 9. Excess of 1 ml was discarded from tube 9 so that each tube has total volume of 1 ml. 7. Then 0.1 ml of 7.5 x 10⁶ CFU/ml added to each tube to obtain inoculums of 2 x 10⁵ CFU /ml Except tube 9 which was taken as negative control. During process any kind of agitation or shaking was avoided. 8. The tube 10 was taken as positive control containing 1 ml of broth and 0.1 ml of 7.5 x 10⁶ CFU/ml. 9. All of these tubes were incubated at 35⁰C for 24 hours. 10. Then after incubation inhibition was observed. The minimum concentration or dilution of serum that completely inhibits visible growth is called as Serum inhibitory concentration (SIC). 11. The formula used to convert dilution or concentration to titer was: titer = 1 /dilution. 12. From non-turbid tubes 0.1 ml were taken and streaked Muller Hinton Agar plates and incubated to observed colonies. For Staphylococcus aureus the incubation period is 24 hours and for other microbes extended upto 72 hours (Lorian 2005). 13. The lowest concentration or highest titer of serum produced 99.9% killing is called as Serum Bactericidal Concentration (SBC).Therefore if inoculum in each tube was 2 x 10⁵ CFU /ml then agar plate showed no more than 20 colonies from 0.1 ml sub culture for 99.9% killing.

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RESULTS

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5. RESULTS

In developing countries like Pakistan according to WHO the actual ratio of patients who received treatment following clinical guidelines in primary care is less than 40% in public sector and 30 % in private sector.The WHO also gave their estimation that unfortunately more than half of the medications are prescribed,sold or dispensed in-appropriately.The contributing factors include polypharmacy, overuse of antibiotics, failure to implement clinical guidelines for prescription leading to serious drug interactions and self –medication.Also one of the major issue in our country is that most of the pharmacies are running without a qualified Pharmacist. Therefore all medicines are in easy access of general public by laymen running their pharmacies without legal full- fillment.All these issues lead to severe harm for common people and developing serious consequences like antimicrobial resistance, adverse drug reactions and medication error and loss of resources.The appropriate and rational use of drug is much necessary to improve quality of health and medical care for patients in particular and community in general.

In present study all these issues were concerned and different brands of Co-amoxiclav were purchased from local market(detail given in table 2) and evaluated according pharmacopeal specifications.The table 3,4 ,5 and figure 4,5, and 6 showed details of weight variations,thickness variations and hardness variations of these tablets.The dissolution test and HPLC assay results shown in table 6 and 7.The HPLC method for Co-amoxiclav was validated according to FDA guideline provided for bio-analytical method validation.The details of linearity,accuracy and precision are given in table 8(a),8(b),9(a),9(b) and figure 7 and 8 respectively.The results of Intra- day and Inter-day precision and accuracy for Co-amoxiclav shown in table 10(a),10(b),11(a) and 11(b).The tables of 12(a), 12(b),13(a) and 13(b) presented the results of lower limit of quantification(LLOQ).The absolute and relative analytical recovery of Co-amoxiclav given away in table 14(a),14(b),15(a) and 15(b).The stability upon freeze and thaw and long term stability at 2 weeks and five weeks given in table 16(a),16(b),17(a),17(b),18(a) and 18(b).

In this crossover pharmacokinetic study twelve male healthy volunteers were included and out of twelve two volunteers withdraw during study(datails of volunteers participated in study given in table 19).The study protocol was approved by the National Bioethics Committee, Ministry of Health, Government of Pakistan.Islamabad and written informed consent was obtained from volunteers.The plasma concentration time profile and the compartmental and non-compartmental analysis of Co-amoxiclav alone were given in table 20,21(a),21(b),22(a) and 22(b) and in table 23,24(a),24(b),25(a) and 25(b) presented same parameters of Co-amoxiclav with simultaneous administration of antacid.The figure 9(a) and 9(b) showed the plasma conc. time plot of Coamoxiclav alone and figure 10(a) and 10(b) showed plasma conc.time plot of Co-amoxiclav + antacid.The mean plasma conc. of both were shown in figure 11.The chromatograms and pharmacokinetic analysis report generated by software “Kinetica version 4.4.1 of volunteers taken

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Co-amoxiclav alone or with antacid also included in separate section.The FDA guidelines followed for statistical comparision.The test of two way ANOVA applied for statistical comparision given in table ___ and ______.

Serum bactericidal test results against Gram-positive and Gram-negative isolates also presented in table ___.

TABLE 2 DETAILS OF DIFFERENT AMOXICILLIN/CLAVULANIC ACID PRODUCTS AVAILABLE IN PAKISTAN

NAME OF STRENGT BATCH DAT DAT RETAIL COMPANY NAME. PRODUCT H NO. E OF E OF PRICE(R (mg) MFG EXP. s) .

AMCLAV 250/125 B.009F7 02-12 08-13 75.00 Getz (AMCL1) 2 Pharma,kchi.Pakistan

AUGMENTI 250/125 2ATAP 04-12 04-14 82.00 GlaxoSmithKline.Pakis N tan (AMCL2)

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TABLE 3

WEIGHT VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN NO.OF TABLETS AMCL1(mg) AMCL2(mg) 1 746.15 667.23 2 747.10 664.12 3 748.17 658.67 4 745.29 670.12 5 745.35 671.29 6 748.14 663.17 7 741.19 669.10 8 740.23 672.20 9 741.30 655.17 10 746.49 656.35 11 749.16 655.49 12 741.54 651.54 13 749.45 661.45 14 745.29 665.27 15 745.23 672.33 16 749.18 670.19 17 742.15 654.29 18 742.27 662.17 19 748.18 670.14 20 743.33 671.10 MEAN 745.2 664.86 SD(±) 2.93 6.66 LIMIT +5% 782.45 698.103 LIMIT -5% 707.94 631.61

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FIGURE 5 WEIGHT VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN

Weight Variation Test of AMCL1

No.of tablets

Weight Variation Test of AMCL 2

No.of tablets

113

TABLE 4 THICKNESS VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN

______

Brands AMCL1 AMCL2

NO OF THICKNESS THICKNESS TABLETS 1 4.35 4.6 2 4.3 4.55 3 4.45 4.56 4 4.4 4.61 5 4.25 4.6 6 4.34 4.6 7 4.36 4.65 8 4.4 4.58 9 4.32 4.57 10 4.31 4.6 11 4.4 4.65 12 4.38 4.61 13 4.34 4.63 14 4.4 4.6 15 4.35 4.6 16 4.35 4.61 17 4.4 4.62 18 4.32 4.61 19 4.32 4.6 20 4.45 4.65 MEAN 4.35 4.60 SD(±) 0.049 0.026 LIMIT+5% 4.56 4.83 LIMIT -5% 4.13 4.37

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FIGURE 6 THICKNESS VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN

Thickness Variation Test of AMCL 1

No.of tablets

Thickness Variation of AMCL 2

No.of tablets

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TABLE 5 HARDNESS VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN

______

Brands AMCL1 AMCL2

NO OF TABLETS HARDNESS HARDNESS 1 18.1 15.95 2 18.51 14.45 3 14.91 12.29 4 18.42 15.27 5 17.65 15.18 6 17.2 14.33 7 15.54 14.1 8 18.21 15.17 9 18.17 13.3 10 17.35 12.85 11 15.49 15.65 12 16.23 13.45 13 18.12 15.54 14 17.67 12.16 15 16.12 15.49 16 18.29 14.3 17 17.14 14.29 18 17.1 14.16 19 17.33 14.19 20 17.45 15.14 MEAN 17.15 14.38 SD(±) 1.21 1.07

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FIGURE 7 HARDNESS VARIATION TEST OF AMOXICILLIN/CLAVULANIC ACID BRANDS AVAILABLE IN PAKISTAN Hardness Variation Test of AMCL 1

No.of tablets

Hardness Variation Test of AMCL 2

No.of tablets

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TABLE 6 DISSOLUTION OF DIFFERENT BRANDS OF AMOXICILLIN/CLAVULANIC ACID TABLET(250/125 mg) AVAILABLE IN PAKISTAN

PRODUC MEAN AVERAGE % PRODUC MEAN AVERAGE %DRU T NAME ABSO ABSORBAN DRUG T NAME ABSOR ABSORBANC G RBAN CE OF DISSO BANCE E OF DISSO E OF STANDARD LVED OF STANDARD LVED AMO (0.0025%) CLAVU (0.00125%) XICIL LANIC LIN ACID AT AT 228nm 272nm

AMCL1 0.80 111.1 COAM-1 0.045 112.5

0.72 0.04

AMCL2 0.74 102.7 COAM-2 0.039 97.5

SUM 1.54 213.8 0.084 210

MEAN 0.77 106.9 0.042 105

S.D 0.04 5.9 0.004 10.6

>> Not less than 85%(Q) of the labeled amount of amoxicillin and not less than 80%(Q) of the labeled amount of clavulanic acid are dissolved in 30 minutes(USP28).

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TABLE 7 HPLC ASSAY OF DIFFERENT BRANDS OF AMOXICILLIN/CLAVULANIC ACID TABLET(250/125 mg) AVAILABLE IN PAKISTAN

PRODUCT MEAN AVERAGE % PRODUCT MEAN AVERAGE % NAME PEAK PEAK LABELE NAME PEAK PEAK LABELE AREA AREA OF D AREA OF AREA OF D OF AMOXICI STRENG CLAVULA CLAVULAN STRENG AMOXIC LLN IN TH NIC ACID IC TH ILLIN STD SAMPLE ACID STD SAMPLE

AMCL1 2461152 106.1 AMCLAV 1125278 113.38

2318008 992430

AMCL2 2777781 119.8 AUGMEN 1091000 109.9 TIN

Mean 112.9 111.64

S.D 9.6 2.46

>> Amoxicillin and Clavulanate Potassium Tablets contain equivalent of not less than 90.0 % and not more than 120.0% of the labeled amount of amoxicillin and clavulanic acid(USP 28).

TABLE 8(a)_

______

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CALIBRATION CURVE OF AMOXICILLIN STD in Human Plasma___ (Linearity,Accuracy and Precision)

Conc(µg/ml) Peak Area Precision Accuracy

STD Sample1 Sample 2 Sample3 Mean S.D RSD% Dev%

31.25 394924 393452 395361 393789 394200 1018.90 0.25 -1.8_ 15.62 205620 206741 204873 206113 205909 950.56 0.46 0.14

7.81 115470 114321 116120 115834 115425 966.7 0.83 -0.03

3.90 68516 69481 67639 67821 68313 1015.0 1.48 -0.29

1.95 46997 46519 46810 46797 46708 164.3 0.35 -0.61

0.97 23504 23609 23476 23481 23522 75.3 0.32 0.07

0.48 17123 17221 17097 17243 17187 78.7 0.45 0.37 0.24 10150 10085 10268 10089 10147 104.5 1.03 -0.02

0.12 9598 9571 9586 9577 9578 7,54 0.07 -0.2

0.06 6384 6371 6269 6295 6311 53.0 0.83 -1.1

0.03 4330 4250 4385 4219 4284 88.2 2.06 -1.06

0.015 2865 2741 2773 2795 2769 27.15 0.98 -3.3

R² 0.996 0.996 0.997 0.996 0.996______-_____-______-

TABLE 8(b)_

Back Calculated Concentration of Amoxicillin Standard in Human Plasma__

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Conc(µg/ml) Back Calculated Conc. Precision Accuracy

Sample1 Sample 2 Sample3 Mean S.D RSD% Dev% 31.25 31.1 31.28 31.16 31.18 0.09 0.29 -0.22

15.62 15.70 15.56 15.65 15.63 0.07 0.45 0.06

7.81 7.73 7.85 7.83 7.80 0.06 0.82 -0.12

3.90 3.95 3.85 3.86 3.88 0.05 1.41 -0.51

1.95 1.93 1.94 1.94 1.93 0.005 0.29 -1.02

0.97 0.97 0.96 0.96 0.96 5.7 0.60 -1.03

0.48 0.482 0.479 0.483 0.481 0.002 0.43 0.20 0.24 0.238 0.242 0.238 0.239 0.003 0.96 -0.41

0.12 0.119 0.118 0.119 0.118 0.0004 0.48 -1.6

0.06 0.059 0.058 0.059 0.058 0.0005 0.99 -3.3

0.03 0.029 0.03 0.029 0.029 0.0005 1.99 -3.3 0.015

0.0143 0.0145 0.0146 0.0144 0.00015 1.06 -4.0

FIGURE 8

VALIDATION CHARTS OF AMOXICILLIN

LINEARITY, ACCURACY AND PRECISION

121

Sample 1 Peak Area Calibration

curve 450000 400000 y = 12398 x + 10878 350000 R² = 0.996 300000 250000 Sample 1 200000 150000 Linear ( Sample 1) 100000 50000 0 0 10 20 30 40

Conc.(micro-gram/ml)

Sample 2 Peak Area Calibration curve 500000 y = 12435 x + 10709 400000 R² = 0.997 300000 Sample 2 200000 Linear ( Sample 2) 100000

0 0 10 20 30 40

Conc(microgram/ml)

122

Sample 3 Peak area Calibartion curve 450000 400000 y = 12406 x + 10827 350000 R² = 0.996 300000 250000 Sample 3 200000 150000 Linear ( Sample 3) 100000 50000 0 0 10 20 30 40

Conc.(microgram/ml)

Calibration curve of Mean values 450000 400000 y = 12413 x + 10798 R² = 0.996 350000 300000 250000 Mean 200000 150000 Linear ( Mean ) 100000 50000 0 0 10 20 30 40

Conc.(microgram/ml)

TABLE 9(a)_

______

123

CALIBRATION CURVE OF CLAVULANIC ACID STD in Human_____ Plasma (Linearity, Accuracy and Precision)

Conc(µg/ml) Peak Area Precision Accuracy

STD Sample1 Sample 2 Sample3 Mean S.D RSD% Dev% 15.6 58457 58312 58211 58101 58208 105.5 0.18 -0.4

7.8 27929 27713 27815 27704 27744 61.6 0.22 -0.66 3.9 14614 14568 14519 14416 14501 77.5 0.53 -0.77

1.95 6029 5923 5887 5941 5917 27.4 0.46 -1.85

0.97 4653 4530 4586 4592 4569 34.1 0.74 -1.80

0.48 2826 2785 2754 2800 2779 23.4 0.84 -1.66

0.24 1720 1665 1699 1680 1681 17.03 1.01 -2.26

0.121 972 895 932 952 926.3 28.9 3.12 -2.05

R² 0.998 0.998 0.998 0.998 0.998 - - -

TABLE 9(b)_

Back Calculated Concentration of Clavulanic Acid Standard in Human___ Plasma

Conc(µg/ml) Back Calculated Conc. Precision Accuracy

Sample1 Sample 2 Sample3 Mean S.D RSD% Dev%

124

15.6 15.56 15.53 15.50 15.53 0.03 0.19 -0.44 7.8 7.73 7.76 7.73 7.74 0.01 0.22 -0.76 3.9 3.88 3.87 3.84 3.86 0.02 0.53 -1.02 1.95 1.91 1.90 1.92 1.91 0.01 0.52 -2.05 0.97 0.94 0.95 0.95 0.94 0.005 0.61 -3.09 0.48 0.47 0.46 0.47 0.46 0.005 1.25 -4.16 0.24 0.232 0.237 0.234 0.234 0.002 1.07 -2.5 0.121 0.111 0.116 0.118 0.115 0.003 3.13 -4.9

FIGURE 9

VALIDATION CHARTS OF CLAVULANIC ACID

125

60000 y = 3671 .x + 293.2 R² = 0.998 50000 40000 Sample 1 30000 Linear ( Sample 1) 20000 10000 0 0 5 10 15 20

LINEARITY, ACCURACY AND PRECISION Sample

1 Peak Area Calibration curve

70000

Conc(microgram/ml)

126

Sample2 Peak Area Calibration curve 70000

60000 y = 3666 .x + 314.5 R² = 0.998 50000 40000 Sample2 30000 Linear ( Sample 2) 20000 10000 0 0 5 10 15 20

Conc.(microgram/ml)

Sample3 Peak Area Calibration curve 70000

60000 y = 3656 .x + 325.6 R² = 0.998 50000 40000 Sample3 30000 Linear ( Sample 3) 20000

10000 0 0 5 10 15 20

Conc.(microgram/ml)

127

Calibration curve of Mean values 70000

60000 y = 3665 .x + 310.9 R² = 0.998 50000 40000 Mean 30000 Linear ( Mean ) 20000 10000 0 0 5 10 15 20

Conc(microgram/ml)

TABLE 10(a)

INTRA-DAY PRECISION AND ACCURACY OF AMOXICILLIN IN HUMAN PLASMA(n=3)

Nominal Conc Observed Conc Precision Accuracy (µg/ml) (µg/ml) Mean SD RSD% DEV%

31.25 32.1 30.5 30.07 30.08 1.06 3.4 -1.44 7.8 8.0 7.7 8.0 7.9 0.17 2.19 1.28 1.95 2.0 1.9 1.9 1.93 0.05 2.99 -1.02

0.06 0.055 0.06 0.06 0.058 0.002 4.9 -3.3

128

TABLE 10(b)_

INTRA-DAY PRECISION AND ACCURACY OF CLAVULANIC ACID IN HUMAN PLASMA(n=3)

Nominal Conc. Observed Conc. Precision Accuracy (µg/ml) (µg/ml) Mean SD RSD% DEV%

15.6 15.3 15.8 14.9 15.3 0.45 2.9 -1.92 3.9 3.85 3.9 3.87 3.87 0.02 0.65 -0.76 0.9 0.87 0.9 0.87 0.88 0.01 1.96 -2.2 0.24 0.24 0.24 0.235 0.238 0.002 1.21 -0.83

TABLE 11(a)

INTER-DAY PRECISION AND ACCURACY OF AMOXICILLIN IN HUMAN PLASMA(n=5)

Nominal Concentrations. (µg/ml)

Day 31.25 7.8 1.95 0.06 1 30.09 7.77 1.93 0.06

31.57 7.78 1.91 0.059

31.20 7.81 1.94 0.06

30.57 7.75 1.95 0.06

2 30.79 7.80 1.92 0.07

29.95 7.79 1.96 0.06

129

30.56 7.76 1.93 0.059

30.71 7.77 1.92 0.057

3 31.52 7.79 1.94 0.06

30.65 7.81 1.94 0.06

31.07 7.76 1.90 0.06

31.97 7.80 1.92 0.059

4 30.91 7.76 1.93 0.058

31.15 7.76 1.96 0.06

30.86 7.78 1.93 0.06

31.10 7.79 1.91 0.06

5 31.11 7.81 1.92 0.058 31.24 7.79 1.92 0.059 30.67 7.76 1.94 0.06

29.98 7.75 1.89 0.06

Mean 30.8 7.77 1.928 0.059

S.D 0.50 0.01 0.01 0.002

RSD% 1.64 0.002 0.92 4.16

DEV% -1.44 -0.38 -1.12 -1.7

130

TABLE 11(b)_

INTER-DAY PRECISION AND ACCURACY OF CLAVULANIC ACID IN HUMAN PLASMA(n=5)

Nominal Concentrations. (µg/ml)

Day 15.6 3.9 0.90 0.24

1 15.5 3.87 0.91 0.23 15.2 3.92 0.94 0.26

15.3 3.91 0.89 0.24

15.8 3.91 0.90 0.24

2 15.7 3.92 0.87 0.27

15.09 3.91 0.95 0.24

15.6 3.89 0.92 0.24

15.0 3.87 0.90 0.23

131

3 15.8 3.88 0.88 0.24

15.4 3.86 0.89 0.24

15.1 3.91 0.89 0.25

15.3 3.92 0.87 0.24

4 15.5 3.89 0.91 0.23

15.7 3.90 0.88 0.24

15.4 3.89 0.89 0.24

15.3 3.91 0.90 0.24

5 15.2 3.87 0.89 0.23

15.1 3.91 0.92 0.24 15.2 3.87 0.87 0.25

15.1 3.85 0.88 0.24

Mean 15.3 3.89 0.89 0.241

S.D 0.24 0.02 0.02 0.009

RSD% 1.6 0.55 2.4 3.99

DEV% -1.9 -0.25 -1.1 0.41

132

133

______

TABLE 12(a)

LOWER LIMIT OF QUANTIFICATION (LLOQ) FOR AMOXICILLIN

Conc(µg/ml) Peak Area Precision Accuracy

STD Sample1 Sample2 Sample3 Mean S.D RSD% Dev%

3.90 73919 72371 74123 72867 73120 903.05 1.23 -1.08

1.95 37337 36220 36591 38479 37096 1211.4 3.26 -0.64

0.97 18579 17901 18011 17861 17924 77.6 0.43 -3.5

0.48 9313 9264 9301 9297 9287 20.3 0.21 -0.27

0.24 4525 4498 4500 4471 4489 16.1 0.36 -0.79

0.12 2571 2432 2614 2489 2511 93.0 3.7 -2.3

0.06 1230 1190 1306 1251 1249 58.0 4.6 +1.54

0.03 6271 6167 6185 6299 6217 71.5 1.15 -0.86

0.015 3199 3179 3161 3147 3162 16.04 0.50 -1.15

0.0075 1580 1499 1568 1544 1537 35.0 2.27 -2.72 0.0037 774 752 770 767 763 9.64 1.26 -1.42

R² 0.994 0.994 0.994 0.993 0.994_____-______-______-__

134

TABLE 12(b)_

Back Calculated Concentration of Amoxicillin ______

Conc(µg/ml) Back Calculated Conc. Precision Accuracy

Sample1 Sample 2 Sample3 Mean S.D RSD% Dev%

3.90 3.81 3.91 3.84 3.85 0.05 1.33 -1.28

1.95 1.89 1.91 2.0 1.93 0.05 3.03 -1.02

0.97 0.93 0.94 0.93 0.93 0.005 0.62 -4.1

0.48 0.47 0.48 0.48 0.476 0.005 1.21 -0.83

0.24 0.23 0.23 0.23 0.23 0.000 0.00 -4.16

0.12 0.113 0.12 0.116 0.116 0.003 3.02 -3.3

0.06 0.058 0.063 0.061 0.0606 0.002 4.1 +1.1

0.03 0.029 0.028 0.03 0.029 0.001 3.4 -3.3

0.015 0.0149 0.0148 0.0147 0.0148 0.0001 0.67 -1.33

0.0075 0.0071 0.0074 0.0073 0.0072 0.0001 0.021 -4.0_

0.0037 0.0035 0.0036 0.0036 0.0035 0.00005 1.6 -5.4

135

______

TABLE 13(a)

LOWER LIMIT OF QUANTIFICATION (LLOQ) FOR CLAVULANIC ACID

Conc(µg/ml) Peak Area Precision Accuracy

STD Sample1 Sample 2 Sample3 Mean S.D RSD% Dev%

1.95 7858 7779 7832 7793 7801 27.4 0.35 -0.72

0.97 4821 4781 4768 4844 4797 40.6 0.84 -0.49

0.48 2946 3000 2897 2911 2936 55.8 1.9 -2.13

0.24 1995 1935 1889 1972 1932 41.5 2.15 -3.15 0.121 987 961 973 994 976 16.7 1.71 -1.11

0.06 683 671 689 675 678 9.45 1.39 -0.73

R² 0.983 0.982 0.986 0.983 0.984 - - -

136

TABLE 13(b)_

Back Calculated Concentration of Clavulanic Acid ______

Conc(µg/ml) Back Calculated Conc. Precision Accuracy

Sample1 Sample 2 Sample3 Mean S.D RSD% Dev%

1.95 1.93 1.94 1.93 1.93 0.005 0.29 -1.02 0.97 0.96 0.959 0.97 0.963 0.006 0.63 -0.72

0.48 0.48 0.472 0.474 0.475 0.004 0.87 -1.04

0.24 0.232 0.22 0.237 0.229 0.008 3.81 -1.29 0.121 0.117 0.119 0.121 0.119 0.002 1.68 -1.65

0.06 0.058 0.06 0.059 0.059 0.001 1.69 -1.66

TABLE 14(a)

137

______

ABSOLUTE ANALYTICAL RECOVERY OF AMOXICILLIN

NOMINAL CONC. PEAK AREA OF AMOXICILLIN IN PLASMA SAMPLE

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

31.25 413279 413149 413189 412739 413050 7.8 109790 109693 109758 109921 116321

0.06 6489 6465 6449 6478 6476

NOMINAL CONC. PEAK AREA OF AMOXICILLIN IN MOBILE PHASE

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

31.25 424312 424178 424199 423469 424106 7.8 115948 115786 115812 115945 125998

0.06 6595 6574 6529 6549 6591

NOMINAL CONC. % OF AMOXICILLIN RECOVERED

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 %MEAN±SD

31.25 97.3 97.4 97.4 97.4 97.3 97.36±0.05

7.8 94.6 94.7 94.8 94.8 92.3 94.24±1.08

0.06 98.3 98.3 98.7 98.9 98.2 98.48±0.30

138

TABLE 14(b)

______ABSOLUTE ANALYTICAL RECOVERY OF CLAVULANIC ACID

NOMINAL CONC. PEAK AREA OF CLAVULANIC ACID IN PLASMA SAMPLE

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

15.6 58423 58325 58465 58487 57898 3.9 15639 15667 15691 14970 15631 0.24 1890 1883 1865 1879 1785

NOMINAL CONC. PEAK AREA OF CLAVULANIC ACID IN MOBILE PHASE

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

15.6 59489 59302 59113 59657 58510 3.9 16000 16945 16741 15882 16420 0.24 1943 1967 1948 1960 1865

NOMINAL CONC. % OF CLAVULANIC ACID RECOVERED

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 %MEAN±SD

15.6 98.2 98.3 98.9 98.0 98.9 98.46±0.41

3.9 97.7 92.4 93.7 94.2 95.2 94.65±1.98

0.24 97.2 95.7 95.7 95.8 95.7 96.02±0.66

139

______

140

TABLE 15(a)

______RELATIVE ANALYTICAL RECOVERY OF AMOXICILLIN

NOMINAL CONC. OBSERVED CONCENTRATION OF AMOXICILLIN IN PLASMA SAMPLE

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

31.25 30.08 31.15 31.06 31.3 31.09 7.8 7.77 7.79 7.8 7.81 7.77

0.06 0.059 0.059 0.057 0.057 0.058

NOMINAL CONC. % OF AMOXICILLIN RECOVERED

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 MEAN ±SD DEV%

31.25 96.2 99.6 99.3 100.1 99.4 98.9±1.55 -1.1 7.8 99.6 99.8 100.0

100.1 99.6 99.8±0.22 -0.2 0.06 98.3 98.3 95.0 95.0 96.6 96.6±1.65 -3.4

141

TABLE 15(b) ______RELATIVE ANALYTICAL RECOVERY OF CLAVULANIC ACID

NOMINAL CONC. OBSERVED CONCENTRATION OF CLAVULANIC ACID IN PLASMA SAMPLE

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

15.6 15.5 15.2 15.1 15.4 15.2 3.9 3.85 3.89 3.9 3.87 3.91 0.24 0.23 0.24 0.235 0.24 0.23

NOMINAL CONC. % OF CLAVULANIC ACID RECOVERED

Conc.(µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 MEAN ±SD DEV%

15.6 99.3 97.4 96.7 98.7 97.4 97.9±1.06 -2.1 3.9 98.7 99.7 100.0 99.2 100.2 99.5±0.61 -0.5 0.24 95.8 100.0 97.9 100.0 95.8 97.9±2.1 -2.3

142

TABLE 16(a)

FREEZE AND THAW (F/T) STABILITY OF

AMOXICILLIN

NOMINAL LOW CONC: 0.0037 µg/ml

Observed conc F/T cycle 1 F/T cycle 2 F/T cycle 3

Sample 1 0.00369 0.00368 0.00371 Sample 2 0.00368 0.00370 0.00367 Sample 3 0.00369 0.00370 0.00368 Sample 4 0.00371 0.00372 0.00369 Sample 5 0.00370 0.00368 0.00369

NOMINAL MEDIUM CONC: 3.9 µg/ml

Observed conc. F/T cycle 1 F/T cycle 2 F/T cycle 3

Sample 1 3.87 3.89 3.87 Sample 2 3.89 3.88 3. 87 Sample 3 3.87 3.90 3.90 Sample 4 3.89 3.88 3.85 Sample 5 3.89 3.90 3.90

NOMINAL HIGH CONC: 31.25 µg/ml

Observed conc. F/T cycle 1 F/T cycle 2 F/T cycle 3 Sample 1 32.10 32.08 30.89

Sample 2 30.91 30.87 30.92

Sample 3 31.68 31.71 31.70

Sample 4 31.78 31.80 31.79

Sample 5 31.63 31.61 31.59

143

TABLE 16(b) SUMMARY OF AMOXICILLIN STABILITY IN HUMAN PLASMA

STABILITY NOMINAL CONC. MEAN OBSERVED CONC PRECISION ACCURACY (µg/ml) (µg/ml) (RSD%) (DEV%)

FREEZE/ THAW 0.0037 0.00369 0.36 -0.27

(n=3) 3.9 3.88 0.37 -0.51

31.25 31.51 1.27 0.83

TABLE 17(a)

FREEZE AND THAW (F/T) STABILITY OF

CLAVULANIC ACID

NOMINAL LOW CONC: 0.06 µg/ml

144

Observed conc. F/T cycle 1 F/T cycle 2 F/T cycle 3

Sample 1 0.059 0.060 0.058

Sample 2 0.058 0.061 0.061

Sample 3 0.059 0.058 0.059

Sample 4 0.057 0.059 0.058

Sample 5 0.059 0.060 0.061

NOMINAL MEDIUM CONC: 1.95 µg/ml

Observed conc. F/T cycle 1 F/T cycle 2 F/T cycle 3

Sample 1 1.95 1.96 1.95

Sample 2 1.94 1.95 1.96

Sample 3 1.94 1.93 1.94

Sample 4 1.95 1.96 1.96

Sample 5 1.93 1.93 1.94

NOMINAL HIGH CONC: 15.6 µg/ml

Observed conc. F/T cycle 1 F/T cycle 2 F/T cycle 3 Sample 1 15.57 15.56 15.58

Sample 2 15.60 15.59 15.61

Sample 3 15.60 15.61 15.59

Sample 4 15.58 15.59 15.58

Sample 5 15.57 15.60 15.55

145

TABLE 17(b)

SUMMARY OF CLAVULANIC ACID STABILITY IN HUMAN PLASMA

STABILITY NOMINAL CONC. MEAN OBSERVED CONC PRECISION ACCURACY (µg/ml) (µg/ml) (RSD%) (DEV%)

FREEZE/THAW 0.06 0.0591 2.22 -1.50

(n=3) 1.95 1.946 0.55 -0.20

15.6 15.58 0.10 -0.12

TABLE 18(a)

LONG TERM STABILITY OF AMOXICILLIN

NOMINAL LOW CONC: 0.0037 µg/ml

After 2 weeks After 5weeks

146

Sample 1 0.0036 0.00350

Sample 2 0.00365 0.00315

Sample 3 0.00369 0.00340

Sample 4 0.0037 0.00355

Sample 5 0.00368 0.00340

Mean 0.00366 0.0034

Precision RSD% 1.12 4.53

Accuracy DEV% -1.08 -8.1

NOMINAL MEDIUM CONC. 3.9 µg/ml

After 2 weeks After 5 weeks

Sample 1 3.85 3.65

Sample 2 3.87 3.64

Sample 3 3.87 3.54

Sample 4 3.88 3.51

Sample 5 3.87 3.48

Mean 3.86 3.56

Precision RSD% 0.29 2.16

Accuracy DEV% -1.02 -8.7

147

TABLE 18(a).Cont

LONG TERM STABILITY OF AMOXICILLIN

______NOMINAL HIGH CONC: 31.25 µg/ml

After 2 weeks After 5 weeks

Sample 1 31.15 26.65

Sample 2 31.17 25.01

Sample 3 30.98 29.58

Sample 4 31.19 30.76

Sample 5 31.20 29.98

Mean 31.11 28.39

Precision RSD% 0.29 8.63

Accuracy DEV% -0.448 -9.15

148

TABLE 18(b)

LONG TERM STABILITY OF CLAVULANIC ACID

NOMINAL LOW CONC: 0.06 µg/ml

After 2 weeks After 5 weeks

Sample 1 0.059 0.047

Sample 2 0.058 0.049

Sample 3 0.059 0.048

Sample 4 0.057 0.045

Sample 5 0.059 0.046

Mean 0.0584 0.047

Precision RSD% 1.53 3.36

Accuracy DEV% -2.66 21.66

NOMINAL MEDIUM CONC: 1.95 µg/ml

After 2 weeks After 5 weeks

Sample 1 1.942 1.870

Sample 2 1.938 1.621

149

Sample 3 1.948 1.804

Sample 4 1.945 1.518

Sample 5 1.947 1.424

Mean 1.944 1.64

Precision RSD% 0.20 11.4

Accuracy DEV% -0.30 -15.8

TABLE 18(b).Cont

LONG TERM STABILITY OF CLAVULANIC ACID

NOMINAL HIGH CONC: 15.6 µg/ml

After 2 weeks After 5 weeks

Sample 1 15.46 13.34

Sample 2 15.55 14.56

Sample 3 15.49 13.20

Sample 4 15.56 12.11

Sample 5 15.55 14.30

Mean 15.52 13.50

Precision RSD% 0.28 7.23

150

Accuracy DEV% -0.51 -13.46

TABLE 19

DETAILS OF THE VOLUNTEERS PARTICIPATED IN PHARMACOKINETIC STUDIES

S. Volunteer Sequence Age Weight Height Blood Pressure Blood Pressure Pulse No. code (yrs) (Kg) (ft.inch) (Phase I) (Phase II) 1 V1 AB 22 71 5’10” 110/70 110/80 68

2 V2 BA 20 90 5’11” 120/80 120/70 60

3 V3 AB 21 72 6’ 120/70 110/80 72

4 V4 BA 28 74 5’4” 120/80 100/70 60

151

5 V5 AB 28 56 5’7” 110/70 130/90 74

6 V6 BA 26 62 5’10” 130/80 110/80 88

7 V7 AB 23 55 5’7” 120/70 100/60 80

8 V8 BA 22 65 5’10” 110/80 120/80 64

9 V9 AB 26 81 5’10” 120/80 100/70 70

10 V10 BA 22 78 5’7” 130/70 120/80 72 A= Co-amoxiclav alone B= Co-amoxiclav +Antacid

TABLE 20

PLASMA CONCENTRATION TIME PROFILE OF CO-AMOXICLAV (250/125) TABLETS IN 10 HEALTHY VOLUNTEERS

AMOXICILLIN(250 mg) CONCENTRATION(µg/ml) Tim V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 Mean S.D e

0.5 4.5 4.44 4.02 4.12 4.08 4.11 4.18 4.31 4.25 4.64 4.26 0.20

1 5.9 5.4 5.62 5.21 5.12 5.67 5.66 5.25 5.33 5.49 5.46 0.24

1.5 6.48 6.36 6.72 6.54 6.49 6.43 6.38 6.43 6.49 6.38 6.47 0.10

2 8.9 8.91 9.07 8.78 8.89 8.92 8.74 8.86 8.93 8.84 8.88 0.09

3 6.87 6.51 6.05 6.11 6.23 6.28 6.35 6.32 6.34 6.41 6.34 0.22

152

4 4.76 4.65 4.45 4.17 4.23 4.24 4.14 4.18 4.42 4.51 4.37 0.21

6 2.22 2.45 2.67 2.35 2.15 2.17 2.22 2.25 2.42 2.38 2.32 0.15

8 1.32 1.39 1.23 1.43 1.54 1.55 1.43 1.46 1.47 1.56 1.43 0.10

CLAVULANIC ACID (125mg) CONCENTRATION(µg/ml) Time V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 Mean S.D

0.5 0.65 0.71 0.68 0.61 0.59 0.53 0.61 0.66 0.65 0.67 0.63 0.05

1 1.41 1.38 1.42 1.48 1.47 1.44 1.41 1.45 1.4 1.47 1.43 0.03

1.5 2.7 2.67 2.69 2.65 2.68 2.72 2.65 2.63 2.62 2.61 2.66 0.03

2 1.78 1.75 1.8 1.75 1.81 1.84 1.81 1.76 1.78 1.77 1.78 0.02

3 1.25 1.21 1.24 1.23 1.31 1.24 1.21 1.22 1.23 1.24 1.23 0.02

4 0.87 0.85 0.89 0.86 0.85 0.74 0.79 0.77 0.82 0.85 0.82 0.04

6 0.34 0.37 0.35 0.36 0.41 0.35 0.39 0.37 0.38 0.36 0.36 0.02

8 0.25 0.28 0.24 0.25 0.26 0.21 0.27 0.23 0.25 0.26 0.25 0.02

153

TABLE 21(a)

COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF AMOXICILLIN

Volun. Ka Kel Alph Cma Tmax AUC Vz Cl T½ Tabs T½ T½ Code a xcalc. calc. Ka alpha Kel

/hr /hr /hr µg/ml hr mg/L L L/hr hr hr Hr hr xhr

V1 0.553 0.524 0.514 7.553 1.856 38.13 19.86 6.555 1.25 6.263 1.322 1.302

V2 0.555 0.501 0.510 7.331 1.894 37.79 20.85 6.613 1.24 6.243 1.381 1.318

V3 0.576 0.500 0.480 7.342 1.859 37.20 20.64 6.718 1.20 6.007 1.385 1.485

V4 0.495 0.586 0.568 7.126 1.852 36.01 24.65 6.941 1.39 6.990 1.182 1.128

V5 0.563 0.510 0.501 7.130 1.864 36.17 22.54 6.911 1.23 6.155 1.357 1.375

V6 0.581 0.511 0.501 7.271 1.832 36.29 22.32 6.887 1.19 5.963 1.354 1.254

V7 0.582 0.513 0.511 7.226 1.826 35.94 22.03 6.955 1.18 5.948 1.348 1.384

V8 0.576 0.507 0.507 7.189 1.848 36.19 22.08 6.907 1.20 6.012 1.366 1.306

V9 0.573 0.492 0.429 7.261 1.879 37.20 23.37 6.719 1.20 6.045 1.406 1.363

V10 0.556 0.515 0.491 7.326 1.866 37.20 21.86 6.719 1.24 6.223 1.345 1.354

Mean 0.55 0.515 0.501 7.27 1.857 36.81 22.02 6.79 1.23 6.184 1.344 1.326 2 SD 0.026 0.026 0.03 0.124 0.019 0.79 1.37 0.145 0.06 0.306 0.061 0.093

RSD % 4.72 5.04 5.98 1.70 1.02 2.14 6.22 2.13 4.87 4.94 4.53 7.01

154

Min 0.495 0.492 0.429 7.126 1.826 35.94 19.86 6.555 1.18 5.948 1.182 1.128

Max 0.582 0.586 0.568 7.553 1.894 38.13 24.65 6.955 1.39 6.99 1.406 1.485

TABLE 21(b)

NON-COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF AMOXICILLIN

Volu AU MR Lz C T HV AU AU AU %AU AUM AUM AU Vss T½ n MC T max max D Cla Cex Ctot C C C MC Lz Code st tra al extra last extr Tot mg/ h l/h µg/ h H mg/ mg/l mg/l mg/l. mg/l. mg/l L h L. ml lxh xh xh (h)² (h)² . (h)² (h)² V1 141. 1.90 0.5 8.9 2 3.6 34.3 3.81 38.1 9.99 109.1 42.14 151. 25. 1.32 67 2 8 7 9 7 31 92 V2 143. 1.99 0.5 8.91 2 3.6 34.1 4.35 38.4 11.31 110.5 48.76 159. 26. 1.38 44 01 2 0 6 8 34 93 V3 138. 1.99 0.5 9.07 2 3.2 33.7 4.00 37.8 10.59 109.6 44.52 154. 26. 1.38 84 00 7 9 0 7 20 97 V4 134. 1.70 0.5 8.78 2 3.2 32.6 5.27 37.9 13.92 105.4 61.93 167. 29. 1.18 06 86 4 2 0 8 42 13 V5 135. 1.95 0.5 8.89 2 3.1 32.5 4.58 37.1 12.35 104.7 52.00 156. 28. 1.35 06 10 9 1 0 2 73 46 V6 133. 1.95 0.5 8.92 2 3.2 32.9 4.62 37.5 12.32 105.5 52.52 158. 28. 1.35 42 11 5 1 4 4 07 03 V7 131. 1.94 0.5 8.74 2 3.3 32.6 4.29 36.9 11.61 104.4 48.39 152. 27. 1.34 63 13 0 9 9 9 88 92 V8 134. 1.97 0.5 8.86 2 3.2 32.7 4.41 37.2 11.86 105.3 49.81 155. 28. 1.36 12 07 9 9 0 1 12 01 V9 140. 2.02 0.4 8.93 2 3.3 33.6 5.24 38.8 13.51 109.1 61.07 170. 28. 1.40 37 92 7 0 5 2 19 18 V10 139. 1.94 0.5 8.84 2 3.5 33.9 4.93 38.9 12.68 110.0 56.30 166. 27. 1.34 03 15 8 7 1 8 39 47 Mean 137. 1.93 0.5 8.88 2 3.3 33.3 4.55 37.8 12.01 107.4 51.74 159. 27. 1.34 16 16 7 3 9 1 16 70 SD 4.00 0.08 0.0 0.09 0.00 0.1 0.70 0.48 0.70 1.21 2.48 6.50 6.59 0.9 0.06 2 7 1

155

RSD 2.91 4.61 4.9 1.06 0.00 5.2 2.10 10.6 1.84 10.13 2.31 12.56 4.14 3.2 4.54 % 7 9 7 8 Min 131. 1.70 0.4 8.74 2 3.1 32.5 3.81 36.9 9.99 104.4 42.14 151. 25. 1.18 63 92 9 1 9 31 92 Max 143. 2.02 0.5 9.07 2 3.6 34.3 5.27 38.9 13.92 110.5 61.93 170. 29. 1.40 44 86 8 1 8 19 13

TABLE 22(a)

COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF CLAVULANIC ACID

Volu Ka Kel Alph Cmax Tma AUC Vz Cl T½Ka Tabs T½ T½ n. a calc. xcal Alpha Kel Code c. /hr /hr /hr µg/ml hr mg/L L L/h hr Hr hr Hr xhr r V1 0.703 0.570 0.470 1.852 1.57 7.98 44.4 15.6 0.98 4.92 1.21 1.11 7 6 V2 0.736 0.546 0.446 1.817 1.57 7.84 50.0 15.9 0.94 4.70 1.26 1.68 6 3 V3 0.706 0.569 0.469 1.862 1.57 8.01 46.1 15.5 0.98 4.90 1.21 1.17 4 9 V4 0.707 0.569 0.369 1.840 1.57 7.90 47.9 15.8 0.97 4.89 1.21 1.16 9 0 V5 0.692 0.559 0.459 1.866 1.60 8.18 47.0 15.2 1.00 5.00 1.23 1.38 4 8 V6 0.674 0.610 0.510 1.836 1.55 7.78 46.0 16.0 1.02 5.13 1.13 1.35 7 5 V7 0.706 0.569 0.469 1.827 1.57 7.85 49.9 15.9 0.98 4.90 1.21 1.16 0 0 V8 0.720 0.582 0.382 1.832 1.54 7.72 48.0 16.1 0.96 4.81 1.19 1.19 1 9 V9 0.707 0.566 0.466 1.823 1.57 7.85 48.4 15.9 0.97 4.89 1.22 1.22 1 0 V10 0.712 0.570 0.470 1.844 1.56 7.89 48.2 15.8 0.97 4.86 1.21 1.14 6 2

156

Mean 0.706 0.571 0.451 1.839 1.56 7.90 47.6 15.8 0.97 4.90 1.20 1.25 7 3 1 SD 0.016 0.016 0.043 0.016 0.01 0.13 1.74 0.25 0.02 0.11 0.03 0.17

RSD 2.29 2.88 9.53 0.88 1.00 1.64 3.65 1.61 2.21 2.28 2.74 13.8 % Min 0.674 0.546 0.369 1.817 1.54 7.72 44.4 15.2 0.94 4.70 1.13 1.11 7 8 Max 0.736 0.610 0.510 1.866 1.60 8.18 50.0 16.1 1.02 5.13 1.26 1.68 6 9

TABLE 22(b)

NON-COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF CLAVULANIC ACID

Volu AU MR Lz C T HV AU AUC AU %AU AUM AU AU Vss T½ n MC T Max ma D Clas extr C Cextr C MC MC Lz Code x t a Tota a Last extr tot l mg/ h l/h µg/ h h mg/l mg/l mg/l mg/l. mg/l mg/l. L h L. ml xh xh xh (h)² . (h)² (h)² (h)² V1 25.33 3.49 0.36 2.70 1.5 1.82 7.07 0.56 7.63 7.34 20.67 6.00 26.6 57. 1.21 7 26 V2 24.99 3.75 0.31 2.67 1.5 1.76 7.08 0.76 7.84 9.70 21.01 8.48 29.4 59. 1.26 9 89 V3 25.41 3.54 0.35 2.69 1.5 1.83 7.12 0.60 7.73 7.84 20.83 6.58 27.4 57. 1.21 2 29 V4 25.05 3.62 0.33 2.65 1.5 1.87 7.05 0.65 7.70 8.50 20.76 7.18 27.9 58. 1.21 4 79 V5 26.43 3.70 0.33 2.68 1.5 2.00 7.28 0.71 7.99 8.94 21.76 7.87 29.6 57. 1.23 4 91 V6 24.29 3.42 0.36 2.72 1.5 1.80 6.86 0.51 7.38 6.96 19.79 5.51 25.3 58. 1.13 0 02 V7 24.91 3.74 0.32 2.65 1.5 1.82 7.03 0.73 7.77 9.49 20.91 8.19 29.1 60. 1.21 0 21 V8 23.97 3.55 0.34 2.63 1.5 1.88 6.92 0.60 7.53 8.01 20.17 6.57 26.7 58. 1.19 5 96

157

V9 24.96 3.65 0.33 2.62 1.5 1.88 7.03 0.66 7.70 8.69 20.78 7.35 28.1 59. 1.22 4 32 V10 24.92 3.64 0.33 2.61 1.5 1.95 7.08 0.67 7.76 8.75 20.82 7.47 28.3 58. 1.21 0 73 Mean 25.02 3.61 0.33 2.66 1.5 1.86 7.05 0.64 7.70 8.42 20.75 7.12 27.8 58. 1.20 7 63 SD 0.65 0.10 0.01 0.03 0.0 0.07 0.11 0.07 0.16 0.88 0.51 0.95 1.37 1.0 0.03 1 RSD 2.63 3.00 4.98 1.35 0.0 3.85 1.59 12.2 2.16 10.50 2.48 13.3 4.93 1.7 2.74 % 2 8 3 Min 23.97 3.42 0.31 2.61 1.5 1.76 6.86 0.51 7.38 6.96 19.79 5.51 25.3 57. 1.13 0 26 Max 26.43 3.75 0.36 2.72 1.5 2.00 7.28 0.76 7.99 9.70 21.76 8.48 29.6 60. 1.26 4 21

TABLE 23

PLASMA CONCENTRATION TIME PROFILE OF CO-AMOXICLAV (250/125) TABLETS + ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS

AMOXICILLIN(250 mg) CONCENTRATION(µg/ml) Time V1b V2b V3b V4b V5b V6b V7b V8b V9b V10b Mean S.D 0.5 3.78 3.82 3.74 3.67 3.65 3.62 3.59 3.63 3.61 3.68 3.67 0.07 1 4.43 4.56 4.54 4.51 4.54 4.53 4.49 4.47 4.54 4.58 4.51 0.04 1.5 5.81 5.77 5.67 5.69 5.67 5.64 5.68 5.69 5.67 5.81 5.71 0.06 2 6.41 6.54 6.48 6.51 6.01 6.52 6.51 6.45 6.5 6.42 6.43 0.15 3 7.81 7.91 7.98 7.87 7.78 7.8 7.78 7.82 7.9 7.84 7.84 0.06 4 5.76 5.71 5.73 5.68 5.58 5.55 5.58 5.61 5.65 5.61 5.64 0.07 6 3.54 3.62 3.56 3.61 3.57 3.61 3.53 3.47 3.51 3.48 3.55 0.05 8 1.57 1.64 1.63 1.62 1.59 1.56 1.65 1.71 1.65 1.01 1.56 0.19

CLAVULANIC ACID (125mg) CONCENTRATION(µg/ml) Time V1b V2b V3b V4b V5b V6b V7b V8b V9b V10b Mean S.D 0.5 0.35 0.38 0.36 0.32 0.34 0.32 0.35 0.34 0.37 0.31 0.34 0.02 1 0.65 0.63 0.67 0.61 0.63 0.61 0.6 0.62 0.61 0.65 0.62 0.02 1.5 1.35 1.31 1.33 1.35 1.31 1.34 0.32 1.38 1.33 1.31 1.23 0.32 2 2.49 2.45 2.48 2.43 2.46 2.42 2.46 2.41 2.45 2.41 2.44 0.02

158

3 1.78 1.72 1.78 1.76 1.75 1.73 1.75 1.76 1.71 1.73 1.74 0.02 4 1.1 1.02 1.08 1.12 1.05 1.03 1.13 1.04 1.02 1.11 1.07 0.04 6 0.52 0.51 0.54 0.58 0.55 0.56 0.51 0.55 0.57 0.56 0.54 0.02 8 0.22 0.27 0.21 0.19 0.21 1.19 0.21 0.22 0.22 0.23 0.31 0.30

159

TABLE 24(a)

COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF AMOXICILLIN +ANTACID

Volu Ka Kel Alph Cma Tma AUC Vz Cl T½ Tabs T½ T½ n a xcalc xcalc Ka Alph Kel Code . . a /hr /hr /hr µg/m Hr mg/L L L/hr hr hr Hr Hr l xhr V1b 0.43 0.41 0.40 6.79 2.36 43.71 19.08 5.71 1.60 8.02 1.67 1.63

V2b 0.41 0.43 0.39 6.86 2.36 44.23 19.22 5.65 1.67 8.38 1.60 1.59

V3b 0.42 0.41 0.39 6.83 2.37 44.20 19.13 5.65 1.64 8.24 1.65 1.50

V4b 0.41 0.42 0.38 6.79 2.38 44.06 19.31 5.67 1.68 8.44 1.61 1.51

V5b 0.41 0.42 0.42 6.65 2.38 43.14 19.65 5.79 1.65 8.27 1.65 1.50

V6b 0.43 0.41 0.33 6.74 2.36 43.46 19.34 5.75 1.60 8.03 1.67 1.66

V7b 0.42 0.41 0.34 6.74 2.37 43.52 19.78 5.74 1.61 8.09 1.67 1.62

V8b 0.42 0.41 0.31 6.74 2.37 43.57 19.98 5.73 1.61 8.06 1.68 1.54

V9b 0.41 0.42 0.32 6.78 2.37 43.84 19.50 5.70 1.66 8.33 1.62 1.67

V10b 0.44 0.43 0.33 6.77 2.28 42.11 16.49 5.93 1.57 7.85 1.60 1.64

Mea 0.42 0.41 0.36 6.76 2.36 43.58 19.14 5.73 1.62 8.17 1.64 1.58 n SD 0.01 0.008 0.039 0.05 0.02 0.62 0.97 0.08 0.03 0.18 0.03 0.06

RSD 2.50 2.05 10.8 0.84 1.23 1.42 5.10 1.44 2.22 2.30 1.92 4.28 % Min 0.41 0.41 0.31 6.65 2.28 42.11 16.49 5.65 1.57 7.85 1.60 1.50

Max 0.44 0.43 0.42 6.86 2.38 44.23 19.98 5.93 1.68 8.44 1.68 1.67

TABLE 24(b)

160

NON-COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF AMOXICILLIN +ANTACID

Volu AU MR Lz C T HVD AU AU AU %AU AU AU AU Vss T½ n MC T Max ma C Cext C C MC MC MC Lz Code x Last ra Tota extra Las extr Tot l t mg/ h l/h µg/ h h mg/l mg/l mg/l mg/ mg/l mg/l. L h L. ml xh xh xh l. . (h)² (h)² (h)² (h)² V1b 206. 4.55 0.31 7.81 3 5.00 36.4 5.35 41.7 12.81 130 59.9 190. 27.2 1.6 8 2 7 .38 1 29 5 6 V2b 209. 4.61 0.30 7.91 3 5.02 36.8 5.71 42.5 13.43 132 64.4 196. 27.1 1.6 6 5 7 .20 6 66 2 8 V3b 210. 4.59 0.30 7.98 3 4.86 36.6 5.58 42.2 13.20 131 62.6 194. 27.1 1.6 4 7 5 .56 9 25 9 7 V4b 210. 4.61 0.30 7.87 3 4.96 36.5 5.64 42.2 13.36 131 63.5 194. 27.3 1.6 1 6 1 .42 2 95 5 8 V5b 205. 4.62 0.30 7.78 3 4.98 35.8 5.53 41.3 13.37 129 62.2 191. 27.9 1.6 7 4 7 .05 4 30 3 8 V6b 205. 4.58 0.31 7.8 3 4.98 36.2 5.42 41.6 13.02 129 60.8 190. 27.5 1.6 9 0 2 .82 3 66 0 7 V7b 206. 4.65 0.30 7.78 3 4.90 36.1 5.78 41.9 13.78 129 65.4 195. 27.7 1.6 6 6 5 .89 8 38 5 6 V8b 207. 4.69 0.29 7.82 3 4.83 36.1 5.99 42.1 14.20 129 68.1 198. 27.8 1.6 2 7 7 .99 2 11 5 6 V9b 208. 4.62 0.30 7.9 3 4.82 36.3 5.68 42.0 13.52 130 64.1 194. 27.5 1.6 3 6 5 .45 7 62 1 7 V10b 192. 4.03 0.39 7.84 3 4.86 35.3 5.01 38.3 7.85 123 31.7 154. 26.3 1.6 68 4 5 .14 2 86 1 0 Mean 206. 4.55 0.31 7.84 3 4.92 36.2 5.56 41.6 12.85 129 60.3 190. 27.3 1.6 32 5 4 .79 1 10 7 6 SD 5.09 0.18 0.02 0.06 0 0.07 0.43 0.26 1.15 1.79 2.5 10.3 12.6 0.46 0.0 2 1 4 2 RSD 2.46 4.13 9.24 0.83 0 1.52 1.19 4.80 2.77 14.00 1.9 17.1 6.65 1.71 1.4 % 4 0 2 Min 192. 4.03 0.29 7.78 3 4.82 35.3 5.01 38.3 7.85 123 31.7 154. 26.3 1.6 68 4 5 .14 2 86 1 0 Max 210. 4.69 0.39 7.98 3 5.02 36.8 5.99 42.5 14.20 132 68.1 198, 27.9 1.6 4 5 7 .20 2 11 3 8

161

TABLE 25(a)

COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF CLAVULANIC ACID+ANTACID

Volun Ka Kel Alpha Cmax Tmax AUC Vz Cl T½ Tabs T½ T½ Code calc. calc. Ka alpha Kel /hr /hr /hr µg/ml Hr mg/Lxh L L/hr hr hr Hr Hr r V1b 0.42 0.46 0.48 1.59 2.20 9.56 37.91 13.06 1.42 7.14 1.64 1.62 V2b 0.42 0.43 0.48 1.55 2.19 9.29 45.83 13.44 1.43 7.15 1.62 1.55 V3b 0.42 0.42 0.49 1.59 2.19 9.53 37.66 13.10 1.42 7.11 1.64 1.63 V4b 0.41 0.45 0.48 1.57 2.24 9.61 36.99 13.00 1.45 7.28 1.67 1.57 V5b 0.42 0.43 0.48 1.56 2.21 9.41 38.48 13.27 1.43 7.15 1.65 1.54 V6b 0.61 0.54 0.21 1.56 2.63 10.31 52.80 9.98 1.09 6.45 2.44 1.44 V7b 0.43 0.42 0.37 1.35 2.47 9.14 39.66 13.66 1.59 7.95 1.87 1.75 V8b 0.42 0.48 0.48 1.57 2.20 9.41 39.18 13.27 1.45 7.26 1.62 1.60 V9b 0.42 0.49 0.48 1.55 2.20 9.34 39.53 13.38 1.44 7.22 1.62 1.66 V10b 0.41 0.47 0.47 1.56 2.24 9.53 39.37 13.11 1.47 7.37 1.65 1.50 Mean 0.43 0.45 0.44 1.54 2.27 9.51 40.74 12.92 1.41 7.20 1.74 1.58 SD 0.06 0.03 0.08 0.06 0.14 0.31 4.88 1.05 0.12 0.36 0.25 0.08 RSD 1.41 8.40 20.15 4.54 6.59 3.30 11.99 8.16 8.93 5.02 14.72 5.50 % Min 0.41 0.42 0.21 1.35 2.19 9.14 36.99 9.98 1.09 6.45 1.62 1.44 Max 0.61 0.54 0.49 1.59 2.63 10.31 52.80 13.66 1.59 7.95 2.44 1.75

162

TABLE 25(b)

NON-COMPARTMENTAL ANALYSIS OF DIFFERENT PHARMACOKINETIC PARAMETERS OF CLAVULANIC ACID +ANTACID

Volu AU MR Lz C T HV AU AU AU %A AU AU AU Vss T½ n MC T max max D Clas Cex Ctot UC M MC MC Lz Code t tra al extra C Extr tot last mg/ h l/h µg/ H H mg/l mg/l mg/l mg/ mg/l mg/l L H L. ml xh xh xh l. . . (h)² (h)² (h)² (h)² V1b 42.4 2.37 0.42 2.49 2 2.31 7.57 0.55 8.12 6.79 25. 5.77 30.8 58.5 1.64 0 12 9 2 V2b 41.0 2.34 0.42 2.45 2 2.21 7.40 0.60 8.20 8.80 24. 7.86 33.6 62.3 1.62 6 73 0 5

V3b 42.1 2.37 0.42 2.48 2 2.29 7.56 0.53 8.10 6.62 25. 5.60 30.7 58.4 1.64 9 12 2 6 V4b 43.3 2.40 0.41 2.43 2 2.41 7.58 0.50 8.08 6.29 25. 5.29 30.7 58.7 1.67 6 44 3 0 V5b 41.9 2.38 0.41 2.46 2 2.24 7.46 0.54 8.00 6.81 24. 5.70 30.6 59.6 1.65 3 90 1 6 V6b 47.4 2.96 0.20 2.42 2 2.27 8.43 0.69 10.5 7.63 28. 5.37 33.1 63.8 2.44 5 2 78 0 3 V7b 45.7 2.70 0.36 2.46 2 2.09 6.97 0.50 7.48 6.76 24. 5.25 29.4 65.5 1.87 2 14 0 7 V8b 41.7 2.33 0.42 2.41 2 2.33 7.46 0.57 8.04 7.17 24. 6.07 31.0 59.9 1.62 4 94 2 0 V9b 41.4 2.34 0.42 2.45 2 2.21 7.44 0.58 8.03 7.31 24. 6.19 31.1 60.3 1.62 0 93 3 4 V10b 42.9 2.38 0.41 2.41 2 2.39 7.53 0.61 8.15 7.55 25. 6.50 31.9 60.1 1.65 9 44 4 0 Mean 43.0 2.45 0.39 2.44 2 2.27 7.54 0.56 8.27 7.17 25. 5.96 31.3 60.7 1.74 2 35 1 4 SD 2.03 0.20 0.06 0.02 0 0.09 0.35 0.05 0.81 0.71 1.2 0.78 1.24 2.40 0.25 5

163

RSD 4.73 8.45 17.7 1.14 0 4.14 4.76 10.2 9.85 9.91 4.9 13.1 3.97 3.96 14.7 % 6 4 6 1 2 Min 41.0 2.33 0.20 2.41 2 2.09 6.97 0.50 7.48 6.29 24. 5.29 29.4 58.4 1.62 6 14 0 6 Max 47.4 2.96 0.42 2.49 2 2.41 8.43 0.69 10.5 8.80 28. 7.86 33.6 65.5 2.44 5 2 78 0 7

FIGURE 10

PLASMA CONCENTRATION TIME PROFILE COMPARISON OFAMOXICILLIN(250mg) ALONE AND AMOXICILLIN(250mg) TABLET + ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS

r 9

) Amox(Alone) 6 Amox+Antacid Conc. ( µg/m l) 3

0 0 2 4 6 8 Time ( hr )

164

165

Amox(Alone)

Amox+Antacid

Amox(Alone) Amox+Antacid

Amox(Alone) Amox+Antaci d

166

Amox(Alone) Amox+Antacid

167

Amox(Alone) Amox+Antacid

168

FIGURE 11(a)

MEAN PLASMA CONCENTRATION TIME PROFILE OFAMOXICILLIN (250mg) TABLET ALONE IN 10 HEALTHY VOLUNTEERS

Mean Curve 9

Mean of 6 Plasma. Conc(µg /ml)

0 0 2 4 6 8 .Time(hr)

FIGURE 11(b)

MEAN PLASMA CONCENTRATION TIME PROFILE OFAMOXICILLIN TABLET ALONE(250mg) +ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS

Mean Curve 8

6 Mean of Plasm a. 4 Conc. µg/ml 2

0 0 2 4 6 8 Time (hr)

169

FIGURE 12

PLASMA CONCENTRATION TIME PROFILE COMPARISON OF CLAVULANIC ACID ALONE AND CLAVULANIC ACID(125mg) TABLETS + ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS

3 r Conc.( Clav.acid (Alone)

µg/ml) 2 Clav.acid+Antacid

0 0 2 4 6 8 Time(hr)

3 Clav.acid (Alone)

2 Clav.acid+Antacid Conc( µg/ml) 1

0 0 2 4 6 8 Time(hr)

Clav.acid (Alone) Clav.acid+Antacid

170

Clav.acid (Alone) Clav.acid+Antacid

2 Conc(µg/ml) Clav.acid (Alone) Clav.acid+Antacid 1

0 0 2 4 6 8 Time(hr)

2 Conc(µg/ml) Clav.acid (Alone)

1 Clav.acid+Antacid

0 0 2 4 6 8 Time(hr)

171

2 Clav.acid (Alone) Conc(µg/ml) Clav.acid+Antacid 1

0 0 2 4 6 8 Time(hr) 3

2 Conc(µg/ml) Clav.acid (Alone)

1 Clav.acid+Antaci

0 0 2 4 6 8 Time(hr)

2 Conc(µg/ml) Clav.acid (Alone)

1 Clav.acid+Antacid

0 0 2 4 6 8 Time(hr) 3

2 Clav.acid Conc(µg/ml) (Alone ) 1 Clav.acid+Anta 0 0 2 4 6 8 Time(hr)

FIGURE 13(a)

172

MEAN PLASMA CONCENTRATION TIME PROFILE OF CLAVULANIC ACID(125mg) TABLET ALONE IN 10 HEALTHY VOLUNTEERS

FIGURE 13(b)

MEAN PLASMA CONCENTRATION TIME PROFILE OF CLAVULANIC ACID TABLET ALONE(125mg) +ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS

173

STATISTATICAL ANALYSIS FOR LOG UNTRANSFORMED DATA

SPSS 16.0 data

TABLE 26 Comparison of Amoxicillin alone and Amoxicillin+ antacid Compartmental Pharmacokinetic Parameters H0:No significant interaction between Pharmacokinetic parameters of amoxicillin/clavulanic acid and antacid administered simultaneously.

HA: Significant interaction between Pharmacokinetic parameters of amoxicillin/clavulanic acid and antacid administered simultaneously. Variables Fcalc F tab Decision

174

Ka 251.53 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between apparent rate of absorption of amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. Alpha 1 121.702 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between distribution of amoxicillin in amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the distribution phase of amoxicillin when administered with amoxicillin/clavulanic acid. Ka T½ 305.917 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between absorption half life of amoxicillin in amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the absorption half life of amoxicillin when administered with amoxicillin/clavulanic acid. Alpha T½ 189.233 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between apparent rate of absorption of amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the distribution half life of amoxicillin when administered with amoxicillin/clavulanic acid. AUC 451.66 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between AUC of amoxicillin in amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the AUC of amoxicillin when administered with amoxicillin/clavulanic acid. Cl 398.75 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between clearance of amoxicillin in amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the clearance of amoxicillin when administered with amoxicillin/clavulanic acid.

175

Kel 121.7 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between rate of elimination of amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. T½ Kel 189.233 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between elimination half life of amoxicillin in amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the elimination half life of amoxicillin when administered with amoxicillin/clavulanic acid. Tabs 305.90 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between time for absorbance of amoxicillin in amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the absorbance time of amoxicillin when administered with amoxicillin/clavulanic acid. Tmax 2.11 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between amoxicillin in amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the time to reach maximum plasma concentration of amoxicillin when administered with amoxicillin/clavulanic acid. Vz 28.96 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between amoxicillin in amoxicillin/clavulanic acid when administered simultaneously with antacid and antacid alter the volume of distribution of amoxicillin when administered with amoxicillin/clavulanic acid.

176

TABLE 27 Comparison of Amoxicillin alone and Amoxicillin+ antacid Non- Compartmental Pharmacokinetic Parameters

H0:No significant interaction between Pharmacokinetic parameters of amoxicillin/clavulanic acid and antacid administered simultaneously.

HA: Significant interaction between Pharmacokinetic parameters of amoxicillin/clavulanic acid and antacid administered simultaneously. Variables Fcalc Ftab Decision AUClast 125.35 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between AUC last of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. %AUC 1.498 .237 As F calculated not significantly greater then F tabulated therefore we accept H0 means no significant interaction exist between AUC% of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. AUCextra 6.97 .017 As F calculated not significantly greater then F tabulated therefore we accept H0 means no significant interaction exist between AUCextra of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. AUCtotal 72.29 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between AUC total of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid.

177

HVD 640.46 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between half value duration of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. Lz 271.42 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is

a significant interaction exist between elimination rate constant of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. LzT½ 232.78 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between terminal half life of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. Cmax 865.98 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between maximum plasma concentartion of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. Tmax ------The time to reach plasma concentration increase from two to three hour along with significant difference in Cmax of amoxicillin in amoxicillin /clavulanic acid alone and with antacid indicate that due to interaction the Tmax of amoxicillin in amoxicillin/clavulanic acid become altered. MRT 1.58 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between mean residence time of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid.

178

Vss 1.021 .326 As F calculated not significantly greater then F tabulated therefore we accept H0 means no significant interaction exist between Vss of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. AUMC 1.13 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between AUMC of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. AUMCextra 4.94 .039 As F calculated not significantly greater then F tabulated therefore we accept H0 means no significant interaction exist between extrapolated AUMC of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. AUMClast 399.0 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between AUMC from time zero to time when last concentration observed of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid. AUMCtotal 47.07 .000 As F calculated is greater then F tabulated therefore we reject Ho and accept HA i.e. there is a significant interaction exist between AUMCtotal of amoxicillin in amoxicillin/clavulanic acid alone and amoxicillin present in amoxicillin/clavulanic acid when administered simultaneously with antacid.

179

TABLE 28 Comparison of Clavulanic acid alone and Clavulanic acid+ antacid Compartmental Pharmacokinetic Parameters

180

TABLE 29 Comparison of Clavulanic acid alone and Clavulanic acid+ antacid Non-Compartmental Pharmacokinetic Parameters

181

182

183

CHROMATOGRAMS

CHROMATOGRAM OF THE ASSAY OF AVAILABLE BRANDS OF CO-AMOXICLAV (AMOXICILLIN 250mg/CLAVULANIC ACID 125 mg) IN PAKISTAN. 1(a)

Clav.acid Amox

184

1(b)

Clav.acid Amox

185

2(a)

Amox

Clav.acid

2(b)

164

LINEARITY

Amoxicillin Conc.Ranges =31.25-0.015 µg/ml

Clavulanic acid Conc.Ranges =15.6-0.121 µg/ml

15.6µg / ml 31.2 5µg/ml

1.95µg/ ml 3.9µg/ml

165

0.97µg/ml 1.95µg/ml

0.48µg/ml 0.97µg/ml

0.24µg/ ml 0.48µg/ml

0.121µg /ml 0.24 µg/ml

0.121µg/ml

166

0.06µg /ml

0.03µg/ ml

0.015µg/ml

189

LLOQ(Amoxicillin)

Conc.Range=3.9-0.0037µg/ml

168

0.06 µg/ml

0.03µg/ml

0.015µg/ml

0.0075µg/ml

0.0037µg/ml

191

LLOQ(Clavulanic acid)

Conc.Range=1.95-0.06µg/ml

1.95µg/ml

0.97µg/ml

0.48µg/ml

170

0.24µg/ml

193

0.121µg/ml

0.06µg/ml

ABSOLUTE ANALYTICAL RECOVERY OF CO-AMOXICLAV IN PLASMA SAMPLES.

Amoxicillin Conc. =31.25,7.8 and 0.06 µg/ml

Clavulanic acid Conc. =15.6, 3.9 and 0.24 µg/ml

C.A.15.6 µg/ml Amox 31 .25µg/ ml

C.A.3.9µ g/ml Amox 7.8µg/ ml

Amox 0.06µg/m l

C.A. 0.24 µg/m l

195

172

ABSOLUTE ANALYTICAL RECOVERY OF CO-AMOXICLAV IN MOBILE PHASE.

Amoxicillin Conc. =31.25,7.8 and 0.06 µg/ml

Clavulanic acid Conc. =15.6, 3.9 and 0.24 µg/ml

Amox 31.25µg/ ml C.A.15.6 µg/ml

Amox 7.8µg/ml C.A.3.9µg/ml

Amox 0.06µg/ml

C.A.0.24µg/ml

197

HPLC CHROMATOGRAMS OF PLASMA SAMPLES OF CO- AMOXICLAV(TABLET 250 mg)ALONE IN 10 HEALTHY VOLUNTEERS.

VOLUNTEER 1(V1)

0.5 hr

1 hr

1.5 hr

2 hr

198

3 hr

4hr

6 hr

8 hr

VOLUNTEER 2(V2)

199

0.5 hr

1 hr

1.5 hr

2 hr

200

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 3(V3)

201

0.5 hr

1 hr

1.5 hr

2 hr

202

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 4(V4)

203

0.5 hr

1hr

1.5 hr

2 hr

204

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 5(V5)

205

0.5 hr

1 hr

1.5 hr

2 hr

206

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 6(V6)

207

0.5 hr

1 hr

1.5 hr

2 hr

208

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 7(V7)

209

0.5 hr

1 hr

1.5 hr

2 hr

210

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 8(V8)

211

0.5 hr

1 hr

1.5 hr

2 hr

212

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 9(V9)

213

0.5 hr

1 hr

1.5 hr

2 hr

214

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 10(V10)

215

0.5 hr

1 hr

1.5 hr

2 hr

216

3 hr

4 hr

6 hr

8 hr

HPLC CHROMATOGRAMS OF PLASMA SAMPLES OF CO-

217

AMOXICLAV(TABLET 250 mg)+ANTACID(10ml) IN 10 HEALTHY VOLUNTEERS.

VOLUNTEER 1(V1b)

0.5 hr

1 hr

1.5 hr

2 hr

218

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 2(V2b)

219

0.5 hr

1 hr

1.5 hr

2 hr

220

3 hr

4 hr

6 hr 6 h

8 hr

VOLUNTEER 3(V3b)

221

0.5 hr

1 hr

1.5 hr

2 hr

222

3 hr

4 hr

6 hr

8 hr

223

VOLUNTEER 4(V4b)

0.5 hr

1 hr

1.5 hr

2 hr

224

3 hr

4hr

6 hr

8 hr

VOLUNTEER 5(V5b)

225

0.5 hr

1 hr

1.5 hr

2 hr

226

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 6(V6b)

227

0.5 hr

1 hr

1.5 hr

2 hr

228

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 7(V7b)

229

0.5 hr

1 hr

1.5 hr

2 hr

230

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 8(V8b)

231

0.5 hr

1 hr

1.5 hr

2 hr

232

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 9(V9b)

233

0.5 hr

1 hr

1.5 hr

2 hr

234

3 hr

4 hr

6 hr

8 hr

VOLUNTEER 10(V10b)

235

0.5 hr

1 hr

1.5 hr

2 hr

236

3 hr

4 hr

6 hr

8 hr

237

DISCUSSION

7.DISCUSSION

238

239

240

241

242

243

244

245

246

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73) Tan L, Zhou JH, Luo N, Yuan YS(1997). Determination of amoxicillin in human plasma by high performance liquid chromatography and its pharmacokinetics. ActaPharmaceuticaSinica ,32(7):558‐60.

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260

APPENDIX Pharmacokinetic Analysis Software Generated Report (Kinetica 4.4.1 Report)

KINETICA REPORT FOR AMOXICILLIN

Kinetica version 4.4.1 ======

Methods subreport Date/Time: 1/8/2013 13:33

======

Method Name: MakeConcUnit

261

dim QteUnit as inputText dim VolumeUnit as inputText dim ConcUnit as OutputText sub MakeConcUnit()

ConcUnit = QteUnit + "/" + VolumeUnit end sub

Method Name: PkExFitDerive

' Secondary PK parameters '

First order Extravascular input

' Output parameters:

' Thalf_Ka, Tabs, Thalf_alpha, Thalf_beta, Thalf_gamma

' Thalf_Lz, Thalf_Kel, Vz, Cl dim Ka as InputNumber

dim A as InputNumber

dim Alpha as InputNumber

dim B as InputNumber

dim Beta as InputNumber

dim C as InputNumber

dim Gamma as InputNumber

dim Dose as InputNumber

dim Lz as InputNumber dim Kel as InputNumber

dim AUC as InputNumber

dim MRT as InputNumber

dim Thalf_Ka as OutputNumber

dim Tabs as OutputNumber

dim Thalf_alpha as OutputNumber

dim Thalf_beta as OutputNumber dim

Thalf_gamma as OutputNumber dim

262

Thalf_Lz as OutputNumber dim

Thalf_Kel as OutputNumber

dim Vz as OutputNumber

dim Cl as OutputNumber sub PkExFitDerive () Thalf_alpha_status = 3 'MISSING Thalf_beta_status = 3 Thalf_gamma_status = 3 Thalf_lz_status = 3 Thalf_kel_status = 3 Thalf_ka_status = 3 if Ka_status = 0 and Ka > 0 then Thalf_Ka = log(2)/Ka Tabs = 5 * Thalf_Ka Thalf_Ka_status = 0 Tabs_status = 0 end if

if Alpha_status = 0 and alpha > 0 then

Thalf_alpha = log(2)/Alpha

Thalf_alpha_status = 0

end if if Beta_status = 0 and Beta

> 0 then

Thalf_beta = log(2)/Beta

Thalf_beta_status = 0

end if

if Gamma_status = 0 and Gamma > 0 then

Thalf_gamma = log(2)/Gamma

Thalf_Gamma_status = 0

end if if Lz_status = 0 and Lz

> 0 then

Thalf_Lz = log(2)/Lz

Thalf_Lz_status = 0

263

end if if Kel_status = 0 and Kel

> 0 then

Thalf_Kel = log(2)/Kel

Thalf_Kel_status = 0

end if Cl_status = 3 if

AUC_status = 0 and AUC <> 0 then

Cl = dose / AUC

Cl_status = 0

end if

Vz_status = 3 if Lz_status =

0 and Lz > 0 then

Vz = CL / Lz

Vz_status = 0

end if end sub

Method Name: PkExFitDerive

' Secondary PK parameters '

First order Extravascular input

' Output parameters:

' Thalf_Ka, Tabs, Thalf_alpha, Thalf_beta, Thalf_gamma ' Thalf_Lz, Thalf_Kel, Vz, Cl dim Ka as InputNumber

dim A as InputNumber

dim Alpha as InputNumber

dim B as InputNumber

dim Beta as InputNumber

dim C as InputNumber

dim Gamma as InputNumber

dim Dose as InputNumber

264

dim Lz as InputNumber

dim Kel as InputNumber

dim AUC as InputNumber

dim MRT as InputNumber dim Thalf_Ka as OutputNumber

dim Tabs as OutputNumber

dim Thalf_alpha as OutputNumber

dim Thalf_beta as OutputNumber dim

Thalf_gamma as OutputNumber dim

Thalf_Lz as OutputNumber dim

Thalf_Kel as OutputNumber

dim Vz as OutputNumber

dim Cl as OutputNumber

sub PkExFitDerive ()

Thalf_alpha_status = 3 'MISSING

Thalf_beta_status = 3

Thalf_gamma_status = 3

Thalf_lz_status = 3

Thalf_kel_status = 3

Thalf_ka_status = 3 if

Ka_status = 0 and Ka > 0 then

Thalf_Ka = log(2)/Ka

Tabs = 5 * Thalf_Ka

Thalf_Ka_status = 0

Tabs_status = 0

end if if Alpha_status = 0 and alpha

> 0 then

265

Thalf_alpha = log(2)/Alpha

Thalf_alpha_status = 0

end if if Beta_status = 0 and Beta

> 0 then

Thalf_beta = log(2)/Beta

Thalf_beta_status = 0

end if

if Gamma_status = 0 and Gamma > 0 then

Thalf_gamma = log(2)/Gamma

Thalf_Gamma_status = 0

end if if Lz_status = 0 and Lz

> 0 then

Thalf_Lz = log(2)/Lz

Thalf_Lz_status = 0 end if if Kel_status = 0 and Kel >

0 then

Thalf_Kel = log(2)/Kel

Thalf_Kel_status = 0

end if

Cl_status = 3

if AUC_status = 0 and AUC <> 0 then

Cl = dose / AUC

Cl_status = 0

end if

Vz_status = 3 if Lz_status =

0 and Lz > 0 then

266

Vz = CL / Lz

Vz_status = 0

end if end sub

Dataset name: 1

------

Dataset name: 1

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 21.2535 Alpha 0.557117 Ka 0.52074

solution found in 2 iterations

Objective function: 3.48382

Akaike criteria: 17.2332

Schwartz criteria: 14.529 Parameter Mean S.D. C.V.(%) A 21.2535 119.031 560.054 Alpha 0.557117 3.07402 551.772 Ka 0.52074 2.85264 547.805

Dataset 1, standard deviation of some parameters is high Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999904 1 Ka -0.999852 -0.999668 1 Dataset 1, possible overparametrization

267

Very high correlation between parameters

Possible overparametrization

T C Ycalc Residuals Weighted Res. 0 0 0 0 0

0.5 4.5 4.22669 0.273314 0.273314 1 5.9 6.45686 -0.556855 -0.556855 1.5 6.48 7.39802 -0.918018 -0.918018 2 8.9 7.53476 1.36524 1.36524 3 6.87 6.59518 0.274825 0.274825 4 4.76 5.13191 -0.371909 -0.371909 6 2.22 2.62266 -0.402656 -0.402656 8 1.32 1.19191 0.128093 0.128093

Without weights With weights Sum -0.207961 -0.207961 Mean -0.0231068 -0.0231068 SumOfSquares 3.48382 3.48382 Std. dev. 0.659452 0.659452 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval),

curve may be approximated by : (with R = -0.997292)

C(T) = 17.5512 * exp(-0.329435*T) giving Lz = -B =

0.329435 and thalf = log(2)/Lz = 2.10405 and

ComputedCLast = C(8) = 1.25815 and AUCextra =

ComputedCLast/Lz = 3.81912

268

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.25815*8)/0.329435 + 1.25815/(0.329435*0.329435) = 42.1459

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 4.5 1.125 1.125 0.5625 0.5625 1 5.9 2.6 3.725 2.0375 2.6 1.5 6.48 3.095 6.82 3.905 6.505 2 8.9 3.845 10.665 6.88 13.385 3 6.87 7.84125 18.5063 19.4342 32.8192 4 4.76 5.75063 24.2569 19.9518 52.7709 6 2.22 6.66019 30.9171 32.4624 85.2333 8 1.32 3.46237 34.3794 23.9379 109.171

Dataset 1(Plasma) 9 Undetectable Outlier

Dataset 1(Plasma)

C(/)

0 0 2 4 6 8 T()

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular

Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting Method Name: MacroToMicro

Nb comp 1

269

Dataset name: 2

------

Dataset name: 2

------

Marquardt Fitting Model FitMacroExtravascular Parameter Initial Mean A 16.4196 Alpha 0.311722 Ka 0.894885 solution found in 13 iterations

Objective function: 4.22794

Akaike criteria: 18.9754

Schwartz criteria: 16.2713 Parameter Mean S.D. C.V.(%) A 18.8358 67.8207 360.063 Alpha 0.498256 1.84698 370.69 Ka 0.558622 2.08945 374.036 Dataset 2, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outlier

Correlation Matrix A Alpha Ka A 1 Alpha 0.999718 1 Ka -0.999577 -0.999071 1 Dataset 2, possible overparametrization

Very high correlation between parameters

Possible overparametrization

T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 4.44 4.03961 0.400392 0.400392 1 5.4 6.20397 -0.80397 -0.80397 1.5 6.36 7.14651 -0.786515 -0.786515 2 8.91 7.31812 1.59188 1.59188

270

3 6.51 6.47623 0.0337724 0.0337724 4 4.65 5.09594 -0.445936 -0.445936 6 2.45 2.66462 -0.214623 -0.214623 8 1.39 1.23999 0.150007 0.150007

Without weights With weights Sum -0.0749898 -0.0749898 Mean -0.0083322 -0.0083322 SumOfSquares 4.22794 4.22794 Std. dev. 0.726921 0.726921 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.999535)

C(T) = 16.4196 * exp(-0.311722*T) giving Lz = -B =

0.311722 and thalf = log(2)/Lz = 2.2236 and

ComputedCLast = C(8) = 1.35622 and AUCextra =

ComputedCLast/Lz = 4.35072

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.35622*8)/0.311722 + 1.35622/(0.311722*0.311722) = 48.7627

271

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

Dataset name: 3

------

Dataset name: 3

------

Marquardt Fitting

272

Model FitMacroExtravascular Parameter Initial Mean A 16.6198 Alpha 0.320259 Ka 1.04677 solution found in 13 iterations

Objective function: 4.14975

Akaike criteria: 18.8074

Schwartz criteria: 16.1033 Parameter Mean S.D. C.V.(%) A 21.0581 86.8702 412.525 Alpha 0.566316 2.28231 403.011 Ka 0.510327 2.03512 398.787 Dataset 3, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999774 1 Ka -0.999651 -0.999214 1 Dataset 3, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 4.02 4.10542 -0.0854196 -0.0854196 1 5.62 6.27386 -0.653863 -0.653863 1.5 6.72 7.19121 -0.471213 -0.471213 2 9.07 7.32732 1.74268 1.74268 3 6.05 6.41992 -0.369924 -0.369924 4 4.45 5.00121 -0.551213 -0.551213 6 2.67 2.56283 0.107172 0.107172 8 1.23 1.16859 0.0614098 0.0614098

Without weights With weights Sum -0.220371 -0.220371 Mean -0.0244856 -0.0244856

273

SumOfSquares 4.14975 4.14975 Std. dev. 0.719753 0.719753

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.996776)

C(T) = 16.6198 * exp(-0.320259*T) giving Lz = -B =

0.320259 and thalf = log(2)/Lz = 2.16433 and

ComputedCLast = C(8) = 1.28213 and AUCextra =

ComputedCLast/Lz = 4.00343

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.28213*8)/0.320259 + 1.28213/(0.320259*0.320259) = 44.528

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

0 0 0.5 4.02 1.005 1.005 0.5025 0.5025 1 5.62 2.41 3.415 1.9075 2.41 1.5 6.72 3.085 6.5 3.925 6.335 2 9.07 3.9475 10.4475 7.055 13.39 3 6.05 7.45837 17.9059 18.395 31.785 4 4.45 5.20911 23.115 18.0988 49.8837 6 2.67 6.96911 30.0841 34.2548 84.1385 8 1.23 3.71582 33.7999 25.5355 109.674

274

Dataset 3(Plasma) 10 Undetectable Outlier Dataset 3(Plasma) 8

0 0 2 4 6 8 T()

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular

Nb comp 1

Use lag time No

Weight 1

Plot curve Yes

Execute Fitting

------

Method Name: MacroToMicro

Nb comp 1

Dataset name: 4

------

Dataset name: 4

------

Marquardt Fitting

Model FitMacroExtravascula Parameter Initial Mean A 15.0322 Alpha 0.301241 Ka 1.06831

275

solution found in 9 iterations

Objective function: 4.35507

Akaike criteria: 19.2421

Schwartz criteria: 16.5379 Parameter Mean S.D. C.V.(%) A 18.6575 109.406 586.389 Alpha 0.519478 3.10401 597.525 Ka 0.559112 3.36019 600.987 Dataset 4, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999882 1 Ka -0.999821 -0.999607 1 Dataset 4, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 4.12 3.98312 0.13688 0.13688 1 5.21 6.08371 -0.873707 -0.873707 1.5 6.54 6.96929 -0.429292 -0.429292 2 8.78 7.09693 1.68307 1.68307 3 6.11 6.21 -0.0999966 -0.0999966 4 4.17 4.83077 -0.660769 -0.660769 6 2.35 2.46746 -0.11746 -0.11746 8 1.43 1.12088 0.309122 0.309122

Without weights With weights Sum -0.0521549 -0.0521549 Mean -0.00579499 -0.00579499 SumOfSquares 4.35507 4.35507 Std. dev. 0.737798 0.737798

276

Dataset 4(Plasma) 9 Undetectable Outli er

Dataset 4(Plasma)

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 4 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.999144)

C(T) = 12.0055 * exp(-0.26756*T) giving Lz = -B =

0.26756 and thalf = log(2)/Lz = 2.59062 and

ComputedCLast = C(8) = 1.41182 and AUCextra =

ComputedCLast/Lz = 5.27665

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.41182*8)/0.26756 + 1.41182/(0.26756*0.26756) = 61.9345

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

0 0 0.5 4.12 1.03 1.03 0.515 0.515 1 5.21 2.3325 3.3625 1.8175 2.3325 1.5 6.54 2.9375 6.3 3.755 6.0875

277

2 8.78 3.83 10.13 6.8425 12.93 3 6.11 7.36451 17.4945 18.1893 31.1193 4 4.17 5.07839 22.5729 17.6131 48.7324 6 2.35 6.34698 28.9199 31.1316 79.8639 8 1.43 3.70414 32.624 25.6236 105.488 ======

Dataset name: 5 ------

Dataset name: 5

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 14.9615 Alpha 0.298854 Ka 1.05484 solution found in 9 iterations

Objective function: 5.01876

Akaike criteria: 20.5186

Schwartz criteria: 17.8145 Parameter Mean S.D. C.V.(%) A 20.4547 93.1926 455.604 Alpha 0.565846 2.51681 444.786 Ka 0.508389 2.23706 440.03 Dataset 5, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka

278

A 1 Alpha 0.999761 1 Ka -0.999632 -0.99917 1 Dataset 5, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 4.08 3.97503 0.104967 0.104967 1 5.12 6.0783 -0.958298 -0.958298 1.5 6.49 6.97131 -0.481308 -0.481308 2 8.89 7.10762 1.78238 1.78238 3 6.23 6.23515 -0.00514531 -0.00514531 4 4.23 4.86336 -0.633355 -0.633355 6 2.15 2.49854 -0.348538 -0.348538 8 1.54 1.14224 0.397757 0.397757

Without weights With weights Sum -0.141536 -0.141536 Mean -0.0157262 -0.0157262 SumOfSquares 5.01876 5.01876 Std. dev. 0.791875 0.791875 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

279

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.989018)

C(T) = 14.9615 * exp(-0.298854*T) giving Lz = -B =

0.298854 and thalf = log(2)/Lz = 2.31935 and

ComputedCLast = C(8) = 1.36978 and AUCextra =

ComputedCLast/Lz = 4.58342

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.36978*8)/0.298854 + 1.36978/(0.298854*0.298854) = 52.004

Table of results

T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

1 2.3 3.32 1.79 2.3

280

1.5 3.845

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular

Nb comp 1

Use lag time No

Weight 1

Plot curve Yes

Execute Fitting

------

Method Name: MacroToMicro

Nb comp 1

281

Dataset name: 6

------

Dataset name: 6

------

Marquardt Fitting

Model FitMacroExtravascular

282

Parameter Initial Mean A 15.0141 Alpha 0.298337 Ka 1.02453 solution found in 9 iterations

Objective function: 4.32235

Akaike criteria: 19.1742

Schwartz criteria: 16.47 Parameter Mean S.D. C.V.(%) A 19.0888 117.761 616.91 Alpha 0.526746 3.30666 627.753 Ka 0.563834 3.55877 631.174 Dataset 6, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999898 1 Ka -0.999844 -0.999659 1 Dataset 6, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 4.11 4.0973 0.0127005 0.0127005 1 5.67 6.23933 -0.569333 -0.569333 1.5 6.43 7.12611 -0.696106 -0.696106 2 8.92 7.2348 1.6852 1.6852 3 6.28 6.29253 -0.0125339 -0.0125339 4 4.24 4.86544 -0.625439 -0.625439 6 2.17 2.45515 -0.285149 -0.285149

283

8 1.55 1.10174 0.448258 0.448258

Without weights With weights Sum -0.0423983 -0.0423983 Mean -0.00471093 -0.00471093 SumOfSquares 4.32235 4.32235 Std. dev. 0.73503 0.73503

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.989132)

C(T) = 15.0141 * exp(-0.298337*T) giving Lz = -B =

0.298337 and thalf = log(2)/Lz = 2.32337 and

ComputedCLast = C(8) = 1.38029 and AUCextra =

ComputedCLast/Lz = 4.62663

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.38029*8)/0.298337 + 1.38029/(0.298337*0.298337) = 52.5211

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

0 0 0.5 4.11 1.0275 1.0275 0.51375 0.51375 1 5.67 2.445 3.4725 1.93125 2.445 1.5 6.43 3.025 6.4975 3.82875 6.27375 2 8.92 3.8375 10.335 6.87125 13.145 3 6.28 7.52295 17.858 18.5878 31.7328

284

4 4.24 5.19339 23.0513 18.0073 49.7402 6 2.17 6.18062 29.232 30.2182 79.9583 8 1.55 3.6853 32.9173 25.5908 105.549

Dataset 6(Plasma) 9 Undetectable Outlier Dataset 6(Plasma)

0 0 2 4 6 8 T()

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

Dataset name: 7

------

Dataset name: 7

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 15.317 Alpha 0.306634 Ka 1.00191

285

solution found in 11 iterations Objective function: 3.85327

Akaike criteria: 18.1403

Schwartz criteria: 15.4361 Parameter Mean S.D. C.V.(%) A 18.4362 55.2768 299.827 Alpha 0.513006 1.58944 309.829 Ka 0.584433 1.8316 313.397 Dataset 7, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999621 1 Ka -0.99942 -0.998745 1 Dataset 7, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0

0.5 4.18 4.09494 0.0850633 0.0850633 1 5.66 6.22578 -0.565776 -0.565776 1.5 6.38 7.09982 -0.719818 -0.719818 2 8.74 7.19771 1.54229 1.54229 3 6.35 6.24369 0.106312 0.106312 4 4.14 4.81637 -0.676368 -0.676368 6 2.22 2.42126 -0.20126 -0.20126 8 1.43 1.08378 0.346217 0.346217

Without weights With weights Sum -0.0833424 -0.0833424 Mean -0.00926027 -0.00926027 SumOfSquares 3.85327 3.85327 Std. dev. 0.693947 0.693947 AUC * calculation

286

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.994083)

C(T) = 15.317 * exp(-0.306634*T) giving Lz = -B =

0.306634 and thalf = log(2)/Lz = 2.2605 and

ComputedCLast = C(8) = 1.31771 and AUCextra =

ComputedCLast/Lz = 4.29733

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.31771*8)/0.306634 + 1.31771/(0.306634*0.306634) = 48.3931

======

Hard Coded Method Output Variables

287

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 8

------

Dataset name: 8

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 15.3092 Alpha 0.304232 Ka 0.99443 solution found in 13 iterations Objective function: 4.77582

Akaike criteria: 20.0721

Schwartz criteria: 17.3679 Parameter Mean S.D. C.V.(%) A 20.8735 76.2159 365.132 Alpha 0.57715 2.04693 354.661 Ka 0.506956 1.77467 350.063 Dataset 8, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1

288

Alpha 0.99965 1 Ka -0.999456 -0.998773 1 Dataset 8, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 4.31 4.03509 0.274914 0.274914 1 5.25 6.15523 -0.905231 -0.905231 1.5 6.43 7.04274 -0.612741 -0.612741 2 8.86 7.1636 1.6964 1.6964 3 6.32 6.25543 0.0645672 0.0645672 4 4.18 4.85746 -0.677457 -0.677457 6 2.25 2.47433 -0.224335 -0.224335 8 1.46 1.12218 0.337821 0.337821

Without weights With weights Sum -0.0460582 -0.0460582 Mean -0.00511758 -0.00511758 SumOfSquares 4.77582 4.77582 Std. dev. 0.772624 0.772624 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.993844)

C(T) = 15.3092 * exp(-0.304232*T) giving Lz = -B =

0.304232 and thalf = log(2)/Lz = 2.27835 and

ComputedCLast = C(8) = 1.34259 and AUCextra =

ComputedCLast/Lz = 4.41306

289

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.34259*8)/0.304232 + 1.34259/(0.304232*0.304232) = 49.81

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 4.31 1.0775 1.0775 0.53875 0.53875 1 5.25 2.39 3.4675 1.85125 2.39 1.5 6.43 2.92 6.3875 3.72375 6.11375 2 8.86 3.8225 10.21 6.84125 12.955 3 6.32 7.51863 17.7286 18.5853 31.5403 4 4.18 5.17648 22.9051 17.9399 49.4802 6 2.25 6.23203 29.1371 30.5209 80.0011 8 1.46 3.65323 32.7904 25.3101 105.311

Dataset 8(Plasma) 9 Undetectable Outlier Dataset 8(Plasma)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

Method Name: MacroToMicro

Nb comp 1

======

290

Dataset name: 9

------

Dataset name: 9

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 15.559 Alpha 0.30138 Ka 0.985028 solution found in 13 iterations

Objective function: 4.39819

Akaike criteria: 19.3307

Schwartz criteria: 16.6266 Parameter Mean S.D. C.V.(%) A 20.9502 81.4844 388.944 Alpha 0.563576 2.13552 378.923 Ka 0.501913 1.88043 374.653 Dataset 9, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999723 1 Ka -0.999575 -0.999037 1 Dataset 9, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 4.25 4.02828 0.221723 0.221723 1 5.33 6.17329 -0.843293 -0.843293 1.5 6.49 7.09594 -0.605944 -0.605944 2 8.93 7.25079 1.67921 1.67921

291

3 6.34 6.38929 -0.049288 -0.049288 4 4.42 5.00616 -0.586157 -0.586157 6 2.42 2.59558 -0.175575 -0.175575 8 1.47 1.19772 0.272276 0.272276

Without weights With weights Sum -0.0870479 -0.0870479 Mean -0.00967199 -0.00967199 SumOfSquares 4.39819 4.39819 Std. dev. 0.741396 0.741396 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0

= 0

t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 4 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.99852)

C(T) = 13.062 * exp(-0.275219*T) giving Lz = -B =

0.275219 and thalf = log(2)/Lz = 2.51853 and

ComputedCLast = C(8) = 1.44477 and AUCextra =

ComputedCLast/Lz = 5.24953

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.44477*8)/0.275219 + 1.44477/(0.275219*0.275219) = 61.0702

Table of results

T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 4.25 1.0625 1.0625 0.53125 0.53125

292

1 5.33 2.395 3.4575 1.86375 2.395 1.5 6.49 2.955 6.4125 3.76625 6.16125 2 8.93 3.855 10.2675 6.89875 13.06 3 6.34 7.56121 17.8287 18.6876 31.7476 4 4.42 5.32241 23.1511 18.4688 50.2164 6 2.42 6.64041 29.7915 32.5394 82.7558 8 1.47 3.8114 33.6029 26.3644 109.12

Dataset 9(Plasma) 9 Undetectable Outlier Dataset 9(Plasma)

0 0 2 4 6 8 T()

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular

Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

Method Name: MacroToMicro

Nb comp 1

Dataset name: 10 ------

Dataset name: 10

------

Marquardt Fitting Model FitMacroExtravascular Parameter Initial Mean

293

A 15.2201 Alpha 0.29382 Ka 0.911915 solution found in 8 iterations

Objective function: 4.2763

Akaike criteria: 19.0778

Schwartz criteria: 16.3736 Parameter Mean S.D. C.V.(%) A 18.8574 70.6048 374.415 Alpha 0.506631 1.95061 385.015 Ka 0.56561 2.19733 388.489 Dataset 10, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999735 1 Ka -0.9996 -0.999124 1 Dataset 10, possible overparametrization

Very high correlation between parameters

Possible overparametrization

T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 4.64 4.07912 0.560876 0.560876 1 5.49 6.24061 -0.75061 -0.75061 1.5 6.38 7.1611 -0.781104 -0.781104 2 8.84 7.30486 1.53514 1.53514 3 6.41 6.41481 -0.00480778 -0.00480778 4 4.51 5.00874 -0.498742 -0.498742 6 2.38 2.57875 -0.198748 -0.198748 8 1.56 1.18151 0.378491 0.378491

Without weights With weights Sum 0.240498 0.240498 Mean 0.026722 0.026722

294

SumOfSquares 4.2763 4.2763 Std. dev. 0.730571 0.730571 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 4 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.99852)

C(T) = 13.062 * exp(-0.275219*T) giving Lz = -B =

0.275219 and thalf = log(2)/Lz = 2.51853 and

ComputedCLast = C(8) = 1.44477

and AUCextra = ComputedCLast/Lz = 5.24953

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.44477*8)/0.275219 + 1.44477/(0.275219*0.275219) = 61.0702

295

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular

Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

KINETICA REPORT FOR AMOXICILLIN + ANTACID

Dataset name: 1

------

296

Dataset name: 1

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 20.6248 Alpha 0.313544

Ka 0.742204 solution found in 11 iterations Objective

function: 2.92622

Akaike criteria: 15.6634

Schwartz criteria: 12.9592 Parameter Mean S.D. C.V.(%) A 18.2454 338.977 1857.87 Alpha 0.417036 7.80602 1871.78 Ka 0.427189 7.99105 1870.61 Dataset 1, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka

A 1

Alpha 0.999991 1

Ka -0.999988 -0.999971 1

Dataset 1, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 3.78 3.15562 0.624383 0.624383 1 4.43 5.1104 -0.680402 -0.680402

297

1.5 5.81 6.20709 -0.39709 -0.39709 2 6.41 6.70146 -0.291461 -0.291461 3 7.81 6.59096 1.21904 1.21904 4 5.76 5.76208 -0.00208251 -0.00208251 6 3.54 3.71591 -0.175914 -0.175914 8 1.57 2.13017 -0.56017 -0.56017

Without weights With weights Sum -0.263699 -0.263699 Mean -0.0292999 -0.0292999 SumOfSquares 2.92622 2.92622 Std. dev. 0.603997 0.603997 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.993168)

C(T) = 20.6248 * exp(-0.313544*T) giving Lz = -B =

0.313544 and thalf = log(2)/Lz = 2.21068 and

ComputedCLast = C(8) = 1.6789 and AUCextra =

ComputedCLast/Lz = 5.35459

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.6789*8)/0.313544 + 1.6789/(0.313544*0.313544) = 59.9143

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 3.78 0.945 0.945 0.4725 0.4725 1 4.43 2.0525 2.9975 1.58 2.0525 1.5 5.81 2.56 5.5575 3.28625 5.33875

298

2 6.41 3.055 8.6125 5.38375 10.7225 3 7.81 7.11 15.7225 18.125 28.8475 4 5.76 6.73307 22.4556 23.3952 52.2427 6 3.54 9.12059 31.5762 44.8658 97.1085 8 1.57 4.84594 36.4221 33.2721 130.381

Dataset 1(Plasma) 8 Undetectable Outlier

Dataset 1(Plas ma) 6

0 0 2 4 6 8 T()

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting Method Name: MacroToMicro

Nb comp 1

Dataset name: 2

------

Dataset name: 2

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 20.1184

299

Alpha 0.305477 Ka 0.750839 solution found in 11 iterations Objective function: 2.92507

Akaike criteria: 15.6599

Schwartz criteria: 12.9557 Parameter Mean S.D. C.V.(%) A 18.3952 276.806 1504.77 Alpha 0.415982 6.31542 1518.19 Ka 0.428363 6.49985 1517.37 Dataset 2, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999986 1 Ka -0.999982 -0.999957 1 Dataset 2, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 3.82 3.19017 0.629833 0.629833 1 4.56 5.16621 -0.606208 -0.606208 1.5 5.77 6.2747 -0.5047 -0.5047 2 6.54 6.77428 -0.234278 -0.234278 3 7.91 6.66225 1.24775 1.24775 4 5.71 5.82414 -0.114141 -0.114141 6 3.62 3.75564 -0.135642 -0.135642 8 1.64 2.15281 -0.512813 -0.512813

Without weights With weights Sum -0.230198 -0.230198 Mean -0.0255776 -0.0255776 SumOfSquares 2.92507 2.92507

300

Std. dev. 0.604068 0.604068 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0

= 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.992978)

C(T) = 20.1184 * exp(-0.305477*T) giving Lz = -B =

0.305477 and thalf = log(2)/Lz = 2.26906 and

ComputedCLast = C(8) = 1.74685 and AUCextra =

ComputedCLast/Lz = 5.71844

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.74685*8)/0.305477 + 1.74685/(0.305477*0.305477) = 64.4672

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 3.82 0.955 0.955 0.4775 0.4775 1 4.56 2.095 3.05 1.6175 2.095 1.5 5.77 2.5825 5.6325 3.30375 5.39875 2 6.54 3.0775 8.71 5.43375 10.8325 3 7.91 7.225 15.935 18.405 29.2375 4 5.71 6.75036 22.6854 23.4432 52.6807 6 3.62 9.1718 31.8572 45.1647 97.8454 8 1.64 5.0014 36.8586 34.3566 132.202

301

Dataset 2(Plasma) 8 Undetectable Outlier Dataset 2(Plasma) 6

0 0 2 4 6 8 T()

MacroToMicro: Division by zero ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 3 ------

Dataset name: 3

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 20.4517 Alpha 0.309108 Ka 0.741236 solution found in 11 iterations Objective function: 3.19922

Akaike criteria: 16.4662

Schwartz criteria: 13.762 Parameter Mean S.D. C.V.(%) A 18.256 272.913 1494.92

302

Alpha 0.413375 6.23815 1509.08 Ka 0.426489 6.43232 1508.2 Dataset 3, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999985 1 Ka -0.99998 -0.999951 1 Dataset 3, possible overparametrization

Very high correlation between parameters

Possible overparametrization

T C Ycalc Residuals Weighted Res. 0 0 0 0 0

0.5 3.74 3.15571 0.584287 0.584287 1 4.54 5.11614 -0.576144 -0.576144 1.5 5.67 6.22087 -0.550866 -0.550866 2 6.48 6.72369 -0.243694 -0.243694 3 7.98 6.62736 1.35264 1.35264 4 5.73 5.80667 -0.0766677 -0.0766677 6 3.56 3.76125 -0.20125 -0.20125 8 1.63 2.16576 -0.535757 -0.535757

Without weights With weights Sum -0.247453 -0.247453 Mean -0.0274948 -0.0274948 SumOfSquares 3.19922 3.19922 Std. dev. 0.631706 0.631706 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

303

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.994373)

C(T) = 20.4517 * exp(-0.309108*T) giving Lz = -B =

0.309108 and thalf = log(2)/Lz = 2.24241

and ComputedCLast = C(8) = 1.72496 and

AUCextra = ComputedCLast/Lz = 5.58044

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.72496*8)/0.309108 + 1.72496/(0.309108*0.309108) = 62.6969

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 3.74 0.935 0.935 0.4675 0.4675 1 4.54 2.07 3.005 1.6025 2.07 1.5 5.67 2.5525 5.5575 3.26125 5.33125 2 6.48 3.0375 8.595 5.36625 10.6975 3 7.98 7.23 15.825 18.45 29.1475 4 5.73 6.79301 22.618 23.5884 52.7359 6 3.56 9.11851 31.7365 44.8719 97.6078 8 1.63 4.94124 36.6778 33.9518 131.56

Dataset 3(Plas ma) 8 Undetectable Outlier Dataset 3(Plasma) 6

0 0 2 4 6 8 T()

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

304

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 4 ------

Dataset name: 4

------

Marquardt Fitting Model FitMacroExtravascular Parameter Initial Mean A 20.1184 Alpha 0.306686 Ka 0.755528 solution found in 9 iterations

Objective function: 2.84017

Akaike criteria: 15.3948

Schwartz criteria: 12.6906 Parameter Mean S.D. C.V.(%) A 18.9003 192.667 1019.39 Alpha 0.428931 4.32552 1008.44 Ka 0.41037 4.13182 1006.85 Dataset 4, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.99997 1

305

Ka -0.99996 -0.999903 1 Dataset 4, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 3.67 3.14406 0.525944 0.525944 1 4.51 5.09799 -0.58799 -0.58799 1.5 5.69 6.19972 -0.509721 -0.509721 2 6.51 6.70187 -0.191868 -0.191868 3 7.87 6.60795 1.26205 1.26205 4 5.68 5.79158 -0.111579 -0.111579 6 3.61 3.75413 -0.144129 -0.144129 8 1.62 2.16331 -0.54331 -0.54331

Without weights With weights Sum -0.300598 -0.300598 Mean -0.0333998 -0.0333998 SumOfSquares 2.84017 2.84017 Std. dev. 0.594782 0.594782

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.992505)

C(T) = 20.1184 * exp(-0.306686*T) giving Lz = -B =

0.306686 and thalf = log(2)/Lz = 2.26012 and

ComputedCLast = C(8) = 1.73005 and AUCextra =

ComputedCLast/Lz = 5.64111

306

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.73005*8)/0.306686 + 1.73005/(0.306686*0.306686) = 63.5226

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 3.67 0.9175 0.9175 0.45875 0.45875 1 4.51 2.045 2.9625 1.58625 2.045 1.5 5.69 2.55 5.5125 3.26125 5.30625 2 6.51 3.05 8.5625 5.38875 10.695 3 7.87 7.19 15.7525 18.315 29.01 4 5.68 6.71559 22.4681 23.3224 52.3324 6 3.61 9.13416 31.6023 44.9832 97.3156 8 1.62 4.96704 36.5693 34.113 131.429 Dataset 4(Plasma) 8 Undetectable Outli er Dataset 4(Plasma) 6

0 0 2 4 6 8 T()

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

Dataset name: 5 ------

Dataset name: 5

307

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 19.906 Alpha 0.307478 Ka 0.699509 solution found in 9 iterations

Objective function: 3.00881 Akaike criteria: 15.9139

Schwartz criteria: 13.2097

Parameter Mean S.D. C.V.(%) A 18.1221 2454.42 13543.8 Alpha 0.420032 56.8923 13544.8 Ka 0.418579 56.6364 13530.6 Dataset 5, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 1 1 Ka -1 -0.999999 1 Dataset 5, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 3.65 3.07542 0.574576 0.574576 1 4.54 4.98751 -0.447512 -0.447512 1.5 5.67 6.06631 -0.396306 -0.396306 2 6.01 6.55861 -0.548614 -0.548614 3 7.78 6.46847 1.31153 1.31153 4 5.58 5.67072 -0.0907245 -0.0907245

308

6 3.57 3.67728 -0.107279 -0.107279 8 1.59 2.11964 -0.529641 -0.529641

Without weights With weights Sum -0.233971 -0.233971 Mean -0.0259967 -0.0259967 SumOfSquares 3.00881 3.00881 Std. dev. 0.612651 0.612651 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.992001)

C(T) = 19.906 * exp(-0.307478*T) giving Lz = -B =

0.307478 and thalf = log(2)/Lz = 2.2543 and

ComputedCLast = C(8) = 1.70097 and AUCextra =

ComputedCLast/Lz = 5.53201

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.70097*8)/0.307478 + 1.70097/(0.307478*0.307478) = 62.2476

309

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 6

------

Dataset name: 6

------

310

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 20.1593 Alpha 0.310397 Ka 0.766306 solution found in 11 iterations Objective

function: 2.7457

Akaike criteria: 15.0903

Schwartz criteria: 12.3862 Parameter Mean S.D. C.V.(%) A 17.9645 185.212 1030.99 Alpha 0.413351 4.31445 1043.77 Ka 0.431105 4.49821 1043.41 Dataset 6, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka

A 1

Alpha 0.999972 1

Ka -0.999963 -0.999911 1 Dataset 6, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 3.62 3.13534 0.484663 0.484663 1 4.53 5.0773 -0.5473 -0.5473 1.5 5.64 6.1666 -0.526597 -0.526597 2 6.52 6.65746 -0.137461 -0.137461 3 7.8 6.54722 1.25278 1.25278 4 5.55 5.72354 -0.173536 -0.173536 6 3.61 3.69086 -0.0808558 -0.0808558

311

8 1.56 2.11584 -0.55584 -0.55584

Without weights With weights Sum -0.284148 -0.284148 Mean -0.031572 -0.031572 SumOfSquares 2.7457 2.7457 Std. dev. 0.584885 0.584885 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.990053)

C(T) = 20.1593 * exp(-0.310397*T) giving Lz = -B =

0.310397 and thalf = log(2)/Lz = 2.2331 and

ComputedCLast = C(8) = 1.68285 and AUCextra =

ComputedCLast/Lz = 5.42159

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.68285*8)/0.310397 + 1.68285/(0.310397*0.310397) = 60.8393

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 3.62 0.905 0.905 0.4525 0.4525 1 4.53 2.0375 2.9425 1.585 2.0375 1.5 5.64 2.5425 5.485 3.2475 5.285 2 6.52 3.04 8.525 5.375 10.66 3 7.8 7.16 15.685 18.22 28.88 4 5.55 6.61131 22.2963 22.9525 51.8325 6 3.61 9.02136 31.3177 44.4621 96.2946 8 1.56 4.88664 36.2043 33.531 129.826

312

Dataset 6(Plasma) 8 Undetectable Outlier Dataset 6(Plasma) 6

0 0 2 4 6 8 T()

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

Dataset name: 7 ------

Dataset name: 7

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 19.3795 Alpha 0.301145 Ka 0.774288 solution found in 11 iterations Objective function: 2.59281

Akaike criteria: 14.5747

Schwartz criteria: 11.8705

313

Parameter Mean S.D. C.V.(%) A 18.0321 228.847 1269.11 Alpha 0.414328 5.31104 1281.85 Ka 0.428384 5.48843 1281.2 Dataset 7, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999982 1 Ka -0.999977 -0.999944 1 Dataset 7, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 3.59 3.12863 0.461372 0.461372 1 4.49 5.06863 -0.578628 -0.578628 1.5 5.68 6.15871 -0.478713 -0.478713 2 6.51 6.65179 -0.141794 -0.141794 3 7.78 6.5472 1.2328 1.2328 4 5.58 5.72831 -0.148315 -0.148315 6 3.53 3.70003 -0.170027 -0.170027 8 1.65 2.12451 -0.474511 -0.474511

Without weights With weights Sum -0.297813 -0.297813 Mean -0.0330903 -0.0330903 SumOfSquares 2.59281 2.59281 Std. dev. 0.568216 0.568216 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

314

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.994266)

C(T) = 19.3795 * exp(-0.301145*T) giving Lz = -B =

0.301145 and thalf = log(2)/Lz = 2.30171 and

ComputedCLast = C(8) = 1.74204 and AUCextra =

ComputedCLast/Lz = 5.78473

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.74204*8)/0.301145 + 1.74204/(0.301145*0.301145) = 65.487

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 3.59 0.8975 0.8975 0.44875 0.44875 1 4.49 2.02 2.9175 1.57125 2.02 1.5 5.68 2.5425 5.46 3.2525 5.2725 2 6.51 3.0475 8.5075 5.385 10.6575 3 7.78 7.145 15.6525 18.18 28.8375 4 5.58 6.61918 22.2717 22.9841 51.8216 6 3.53 8.9541 31.2258 44.0895 95.9112 8 1.65 4.94397 36.1697 33.9871 129.898 Dataset 7(Plasma) 8 Undetectable Outli er

Dataset 7(Plasma) 6

0 0 2 4 6 8 T()

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

315

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 8

------

Dataset name: 8

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 19.0599 Alpha 0.296594 Ka 0.765129 solution found in 11 iterations Objective function: 2.72942

Akaike criteria: 15.0368

Schwartz criteria: 12.3326 Parameter Mean S.D. C.V.(%) A 17.9356 180.158 1004.48 Alpha 0.411602 4.18717 1017.29 Ka 0.429785 4.37043 1016.89 Dataset 8, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka

316

A 1 Alpha 0.999971 1 Ka -0.999961 -0.999907 1 Dataset 8, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 3.63 3.12309 0.506905 0.506905 1 4.47 5.06136 -0.591359 -0.591359 1.5 5.69 6.15196 -0.46196 -0.46196 2 6.45 6.64676 -0.196764 -0.196764 3 7.82 6.54676 1.27324 1.27324 4 5.61 5.73195 -0.121948 -0.121948 6 3.47 3.70769 -0.237689 -0.237689 8 1.71 2.13206 -0.422062 -0.422062

Without weights With weights Sum -0.251637 -0.251637 Mean -0.0279597 -0.0279597 SumOfSquares 2.72942 2.72942 Std. dev. 0.583351 0.583351 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1 AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.996698)

C(T) = 19.0599 * exp(-0.296594*T) giving Lz = -B =

0.296594 and thalf = log(2)/Lz = 2.33703 and

ComputedCLast = C(8) = 1.77684 and AUCextra =

ComputedCLast/Lz = 5.99083

317

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.77684*8)/0.296594 + 1.77684/(0.296594*0.296594) = 68.1255

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 3.63 0.9075 0.9075 0.45375 0.45375 1 4.47 2.025 2.9325 1.57125 2.025 1.5 5.69 2.54 5.4725 3.25125 5.27625 2 6.45 3.035 8.5075 5.35875 10.635 3 7.82 7.135 15.6425 18.18 28.815 4 5.61 6.65394 22.2964 23.105 51.92 6 3.47 8.90931 31.2058 43.836 95.7559 8 1.71 4.97413 36.1799 34.2371 129.993

Dataset 8(Plas ma) 8 Undetectable Outlier Dataset 8(Plasma) 6

0 0 2 4 6 8 T()

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular

Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

318

Dataset name: 9

------

Dataset name: 9

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 19.867 Alpha 0.304825 Ka 0.760513 solution found in 9 iterations

Objective function: 2.8595

Akaike criteria: 15.4558

Schwartz criteria: 12.7517 Parameter Mean S.D. C.V.(%) A 18.672 359.89 1927.43 Alpha 0.425821 8.16466 1917.39 Ka 0.416014 7.96598 1914.84 Dataset 9, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999992 1 Ka -0.999989 -0.999973 1 Dataset 9, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0 0.5 3.61 3.1468 0.463203 0.463203 1 4.54 5.09916 -0.559165 -0.559165 1.5 5.67 6.19714 -0.527142 -0.527142

319

2 6.5 6.69472 -0.194717 -0.194717 3 7.9 6.5922 1.3078 1.3078 4 5.65 5.77004 -0.120044 -0.120044 6 3.51 3.72993 -0.219934 -0.219934 8 1.65 2.14331 -0.493308 -0.493308

Without weights With weights Sum -0.343305 -0.343305 Mean -0.038145 -0.038145 SumOfSquares 2.8595 2.8595 Std. dev. 0.59649 0.59649 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.995254)

C(T) = 19.867 * exp(-0.304825*T) giving Lz = -B =

0.304825 and thalf = log(2)/Lz = 2.27392 and

ComputedCLast = C(8) = 1.73405 and AUCextra =

ComputedCLast/Lz = 5.68867

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.73405*8)/0.304825 + 1.73405/(0.304825*0.304825) = 64.1715

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 3.61 0.9025 0.9025 0.45125 0.45125 1 4.54 2.0375 2.94 1.58625 2.0375 1.5 5.67 2.5525 5.4925 3.26125 5.29875 2 6.5 3.0425 8.535 5.37625 10.675 3 7.9 7.2 15.735 18.35 29.025

320

4 5.65 6.71227 22.4473 23.3058 52.3308 6 3.51 8.99085 31.4381 44.2436 96.5744 8 1.65 4.92819 36.3663 33.8832 130.458

Dataset 9(Plas ma) 8 Undetectable Outlier Dataset 9(Plasma) 6

0 0 2 4 6 8 T()

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 10

------

Dataset name: 10

------

Marquardt Fitting

Model FitMacroExtravascular

Parameter Initial Mean A 28.0496 Alpha 0.395006 Ka 0.749961 solution found in 9 iterations

Objective function: 3.5417

321

Akaike criteria: 17.3815

Schwartz criteria: 14.6773 Parameter Mean S.D. C.V.(%) A 18.2396 465.716 2553.32 Alpha 0.433097 11.1238 2568.44 Ka 0.441267 11.3286 2567.29 Dataset 10, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999994 1 Ka -0.999992 -0.999981 1 Dataset 10, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0 0 0 0 0

0.5 3.68 3.23411 0.445889 0.445889 1 4.58 5.1982 -0.618199 -0.618199 1.5 5.81 6.26632 -0.456324 -0.456324 2 6.42 6.71461 -0.294611 -0.294611 3 7.84 6.50508 1.33492 1.33492 4 5.61 5.60188 0.00811783 0.00811783 6 3.48 3.50524 -0.025242 -0.025242 8 1.01 1.94966 -0.939661 -0.939661

Without weights With weights Sum -0.545108 -0.545108 Mean -0.0605675 -0.0605675 SumOfSquares 3.5417 3.5417 Std. dev. 0.662258 0.662258 AUC * calculation

322

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.975724)

C(T) = 28.0496 * exp(-0.395006*T) giving Lz = -B =

0.395006 and thalf = log(2)/Lz = 1.75478 and

ComputedCLast = C(8) = 1.18997 and AUCextra =

ComputedCLast/Lz = 3.01253

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (1.18997*8)/0.395006 + 1.18997/(0.395006*0.395006) = 31.7268

MacroToMicro: Division by zero

======

Hard Coded Method Output Variables

323

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting Method Name: MacroToMicro

Nb comp 1

======

KINETICA REPORT FOR CLAVULANIC ACID

Dataset name: 1

------

Dataset name: 1

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.55116 Alpha 0.570313 Ka 0.703719 solution found in 3 iterations

Objective function: 1.13695

Akaike criteria: 7.15514

Schwartz criteria: 4.45097 Parameter Mean S.D. C.V.(%) A 4.55116 17.0644 374.945 Alpha 0.570313 2.25263 394.981 Ka 0.703719 2.84796 404.701 Dataset 2, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka

324

A 1

Alpha 0.998944 1

Ka -0.998243 -0.996412 1 Dataset 1, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0

0.5 0.65 1.16479 -0.514785 -0.514785 1 1.41 1.69508 -0.285083 -0.285083 1.5 2.7 1.8508 0.849205 0.849205 2 1.78 1.79694 -0.016941 -0.016941 3 1.25 1.4308 -0.180802 -0.180802 4 0.87 1.01417 -0.144172 -0.144172 6 0.34 0.431807 -0.091807 -0.091807 8 0.25 0.164365 0.0856349 0.0856349

Without weights With weights Sum -0.298751 -0.298751 Mean -0.0331946 -0.0331946 SumOfSquares 1.13695 1.13695 Std. dev. 0.375339 0.375339 AUC * calculation

First point time = 0, t0 and c0 set to first point values

c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 1.5 to infinity (auto detected best interval),

curve may be approximated by : (with R = -0.984945)

C(T) = 3.93044 * exp(-0.368321*T) giving Lz = -B =

0.368321 and thalf = log(2)/Lz = 1.88191

325

and ComputedCLast = C(8) = 0.206425 and

AUCextra = ComputedCLast/Lz = 0.56045

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.206425*8)/0.368321 + 0.206425/(0.368321*0.368321) = 6.00523

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.65 0.1625 0.1625 0.08125 0.08125

1 1.41 0.515 0.6775 0.43375 0.515

1.5 2.7 1.0275 1.705 1.365 1.88

2 1.78 1.10407 2.80907 1.91302 3.79302

3 1.25 1.49942 4.3085 3.70448 7.4975

4 0.87 1.04855 5.35704 3.63832 11.1358

6 0.34 1.1282 6.48525 5.46689 16.6027

8 0.25 0.585395 7.07064 4.06781 20.6705

Dataset 1(Plasma) 3 Undetectable Outlier

Dataset 1(Plasma)

C(/)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

326

Method Name: FitMacroExtravascular

Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 2

------

Dataset name: 2

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.28566 Alpha 0.546419 Ka 0.736728 solution found in 2 iterations

Objective function: 1.07548

Akaike criteria: 6.65493

Schwartz criteria: 3.95077 Parameter Mean S.D. C.V.(%) A 4.28566 10.9576 255.681 Alpha 0.546419 1.50558 275.535 Ka 0.736728 2.10246 285.378 Dataset 2, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix

327

A Alpha Ka A 1 Alpha 0.997829 1 Ka -0.996409 -0.992779 1 Dataset 2, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.71 1.14589 -0.43589 -0.43589 1 1.38 1.66475 -0.284746 -0.284746 1.5 2.67 1.81527 0.854728 0.854728 2 1.75 1.7608 -0.010797 -0.010797 3 1.21 1.40098 -0.190982 -0.190982 4 0.85 0.993786 -0.143786 -0.143786 6 0.37 0.425625 -0.0556246 -0.0556246 8 0.28 0.163878 0.116122 0.116122

Without weights With weights Sum -0.150976 -0.150976 Mean -0.0167752 -0.0167752 SumOfSquares 1.07548 1.07548 Std. dev. 0.366222 0.366222 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.985111)

C(T) = 3.08909 * exp(-0.318212*T) giving Lz = -B =

0.318212 and thalf = log(2)/Lz = 2.17826 and

328

ComputedCLast = C(8) = 0.242241 and AUCextra =

ComputedCLast/Lz = 0.761258

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.242241*8)/0.318212 + 0.242241/(0.318212*0.318212) = 8.48236

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.71 0.1775 0.1775 0.08875 0.08875

1 1.38 0.5225 0.7 0.43375 0.5225

1.5 2.67 1.0125 1.7125 1.34625 1.86875

2 1.75 1.08885 2.80135 1.88638 3.75513

3 1.21 1.46343 4.26479 3.61368 7.36882

4 0.85 1.01943 5.28421 3.53806 10.9069

6 0.37 1.15422 6.43843 5.6129 16.5198

8 0.28 0.645825 7.08425 4.49081 21.0106

Dataset 2(Plasma) 3 Undetectable Outlier

Dataset 2(Plasma)

C(/)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

329

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 3

------

Dataset name: 3

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.56057 Alpha 0.569113 Ka 0.706836 solution found in 3 iterations

Objective function: 1.07867

Akaike criteria: 6.6816

Schwartz criteria: 3.97744 Parameter Mean S.D. C.V.(%) A 4.56057 16.0436 351.79 Alpha 0.569113 2.11255 371.2 Ka 0.706836 2.69055 380.647 Dataset 3, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1

330

Alpha 0.998876 1 Ka -0.998129 -0.996186 1 Dataset 3, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.68 1.17182 -0.491815 -0.491815 1 1.42 1.70456 -0.284559 -0.284559 1.5 2.69 1.86036 0.829637 0.829637 2 1.8 1.80552 -0.00551906 -0.00551906 3 1.24 1.4366 -0.196604 -0.196604 4 0.89 1.01765 -0.127654 -0.127654 6 0.35 0.432875 -0.0828752 -0.0828752 8 0.24 0.164674 0.0753257 0.0753257

Without weights With weights Sum -0.284064 -0.284064 Mean -0.0315627 -0.0315627 SumOfSquares 1.07867 1.07867 Std. dev. 0.365669 0.365669 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.989997)

C(T) = 3.50002 * exp(-0.350192*T) giving Lz = -B =

0.350192 and thalf = log(2)/Lz = 1.97934 and

ComputedCLast = C(8) = 0.21251 and AUCextra =

ComputedCLast/Lz = 0.606838

331

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.21251*8)/0.350192 + 0.21251/(0.350192*0.350192) = 6.58758

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.68 0.17 0.17 0.085 0.085

1 1.42 0.525 0.695 0.44 0.525

1.5 2.69 1.0275 1.7225 1.36375 1.88875

2 1.8 1.10764 2.83014 1.91988 3.80863

3 1.24 1.50265 4.33279 3.71006 7.51869

4 0.89 1.05534 5.38813 3.66459 11.1833

6 0.35 1.1572 6.54533 5.60855 16.7918

8 0.24 0.583099 7.12843 4.04512 20.837

Dataset 3(Plasma) 3 Undetectable Outlier

Dataset 3(Plasma)

C(/)

0 0 2 4 6 8 T()

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular

332

Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1 ======

Dataset name: 4

------

Dataset name: 4

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.50573 Alpha 0.56976 Ka 0.707923 solution found in 3 iterations

Objective function: 1.07131

Akaike criteria: 6.61991

Schwartz criteria: 3.91575 Parameter Mean S.D. C.V.(%) A 4.50573 15.9538 354.078 Alpha 0.56976 2.12891 373.65 Ka 0.707923 2.71272 383.194 Dataset 4, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka

A 1

333

Alpha 0.998871 1

Ka -0.99812 -0.99617 1

Dataset 4, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.61 1.159 -0.549003 -0.549003 1 1.48 1.6852 -0.205198 -0.205198 1.5 2.65 1.83845 0.811553 0.811553 2 1.75 1.78349 -0.0334912 -0.0334912 3 1.23 1.41788 -0.187883 -0.187883 4 0.86 1.00356 -0.143557 -0.143557 6 0.36 0.426182 -0.0661817 -0.0661817 8 0.25 0.161869 0.0881308 0.0881308

Without weights With weights Sum -0.28563 -0.28563 Mean -0.0317367 -0.0317367 SumOfSquares 1.07131 1.07131 Std. dev. 0.36439 0.36439 AUC * calculation

First point time = 0, t0 and c0 set to first point values

c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval),

curve may be approximated by : (with R = -0.990144)

C(T) = 3.30668 * exp(-0.337907*T) giving Lz = -B =

0.337907 and thalf = log(2)/Lz = 2.0513 and

334

ComputedCLast = C(8) = 0.221505 and AUCextra =

ComputedCLast/Lz = 0.65552

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.221505*8)/0.337907 + 0.221505/(0.337907*0.337907) = 7.1841

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.61 0.1525 0.1525 0.07625 0.07625

1 1.48 0.5225 0.675 0.44625 0.5225

1.5 2.65 1.0325 1.7075 1.36375 1.88625

2 1.75 1.08448 2.79198 1.87915 3.7654

3 1.23 1.47475 4.26674 3.64364 7.40904

4 0.86 1.03399 5.30073 3.5882 10.9972

6 0.36 1.14833 6.44906 5.57706 16.5743

8 0.25 0.60333 7.05239 4.18672 20.761

Dataset 4(Plasma) 3 Undetectable Outlier Dataset 4(Plasma)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

335

------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 5

------

Dataset name: 5

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.5771 Alpha 0.559534 Ka 0.692473 solution found in 3 iterations

Objective function: 1.116

Akaike criteria: 6.98774

Schwartz criteria: 4.28358 Parameter Mean S.D. C.V.(%) A 4.5771 16.6526 363.825 Alpha 0.559534 2.14629 383.584 Ka 0.692473 2.72107 392.95 Dataset 5, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.998915 1 Ka -0.998216 -0.996338 1 Dataset 5, possible overparametrization

Very high correlation between parameters

Possible overparametrization

336

T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0

0.5 0.59 1.15907 -0.569066 -0.569066 1 1.47 1.69607 -0.226067 -0.226067 1.5 2.68 1.86208 0.817918 0.817918 2 1.81 1.81787 -0.00786543 -0.00786543 3 1.31 1.46345 -0.153454 -0.153454 4 0.85 1.04876 -0.198765 -0.198765 6 0.41 0.456433 -0.0464329 -0.0464329 8 0.26 0.177583 0.0824172 0.0824172

Without weights With weights Sum -0.301315 -0.301315 Mean -0.0334794 -0.0334794 SumOfSquares 1.116 1.116 Std. dev. 0.371805 0.371805 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.994441)

C(T) = 3.38801 * exp(-0.332132*T) giving Lz = -B =

0.332132 and thalf = log(2)/Lz = 2.08696 and

ComputedCLast = C(8) = 0.237683 and AUCextra =

ComputedCLast/Lz = 0.715628

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.237683*8)/0.332132 + 0.237683/(0.332132*0.332132) = 7.87967

Table of results T C AUC AUCcum AUMC AUMCcum

337

h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.59 0.1475 0.1475 0.07375 0.07375

1 1.47 0.515 0.6625 0.44125 0.515

1.5 2.68 1.0375 1.7 1.3725 1.8875

2 1.81 1.10831 2.80831 1.92146 3.80896

3 1.31 1.54655 4.35486 3.82479 7.63375

4 0.85 1.06347 5.41833 3.68393 11.3177

6 0.41 1.207 6.62533 5.88963 17.2073

8 0.26 0.658652 7.28399 4.56074 21.768

Dataset 5(Plasma) 3 Undetectable Outli er

Dataset 5(Plasma)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

338

Dataset name: 6

------

Dataset name: 6

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.75176 Alpha 0.610273 Ka 0.674757 solution found in 2 iterations

Objective function: 1.34089

Akaike criteria: 8.64001

Schwartz criteria: 5.93585

Parameter Mean S.D. C.V.(%) A 4.75176 42.0676 885.305 Alpha 0.610273 5.53872 907.581 Ka 0.674757 6.19332 917.86 Dataset 6, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka

A 1

Alpha 0.999764 1

Ka -0.9996 -0.999174 1

Dataset 6, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res.

339

0 0 0 0 0 0.5 0.53 1.16271 -0.632708 -0.632708 1 1.44 1.6867 -0.246696 -0.246696 1.5 2.72 1.83528 0.884722 0.884722 2 1.84 1.77522 0.0647809 0.0647809 3 1.24 1.40178 -0.161776 -0.161776 4 0.74 0.984244 -0.244244 -0.244244 6 0.35 0.409841 -0.0598407 -0.0598407 8 0.21 0.151905 0.058095 0.058095

Without weights With weights Sum -0.337668 -0.337668 Mean -0.0375187 -0.0375187 SumOfSquares 1.34089 1.34089 Std. dev. 0.407465 0.407465 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.99318)

C(T) = 3.57132 * exp(-0.367451*T) giving Lz = -B

= 0.367451 and thalf = log(2)/Lz = 1.88637 and

ComputedCLast = C(8) = 0.188875 and AUCextra =

ComputedCLast/Lz = 0.514013

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.188875*8)/0.367451 + 0.188875/(0.367451*0.367451) = 5.51097

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

340

0 0

0.5 0.53 0.1325 0.1325 0.06625 0.06625

1 1.44 0.4925 0.625 0.42625 0.4925

1.5 2.72 1.04 1.665 1.38 1.8725

2 1.84 1.1257 2.7907 1.9517 3.8242

3 1.24 1.52032 4.31102 3.75093 7.57512

4 0.74 0.968586 5.27961 3.34857 10.9237

6 0.35 1.04178 6.32139 5.08011 16.0038

8 0.21 0.548132 6.86952 3.79046 19.7943

Dataset 6(Plasma) 3 Undetectable Outli er

Dataset 6(Plasma)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

Method Name: MacroToMicro

Nb comp 1

======

341

Dataset name: 7

------

Dataset name: 7

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.47919 Alpha 0.569902 Ka 0.706677 solution found in 3 iterations

Objective function: 1.13795

Akaike criteria: 7.16306

Schwartz criteria: 4.4589

Parameter Mean S.D. C.V.(%) A 4.47919 16.621 371.071 Alpha 0.569902 2.2305 391.384 Ka 0.706677 2.83571 401.274 Dataset 7, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.998893 1 Ka -0.998156 -0.996241 1 Dataset 7, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.61 1.15046 -0.540463 -0.540463 1 1.41 1.67322 -0.263222 -0.263222 1.5 2.65 1.82585 0.824151 0.824151

342

2 1.81 1.77171 0.0382907 0.0382907 3 1.21 1.40918 -0.199182 -0.199182 4 0.79 0.997839 -0.207839 -0.207839 6 0.39 0.424089 -0.0340885 -0.0340885 8 0.27 0.161182 0.108818 0.108818

Without weights With weights Sum -0.273534 -0.273534 Mean -0.0303927 -0.0303927 SumOfSquares 1.13795 1.13795 Std. dev. 0.375772 0.375772 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.988603)

C(T) = 3.13242 * exp(-0.32225*T) giving Lz = -B =

0.32225 and thalf = log(2)/Lz = 2.15096 and

ComputedCLast = C(8) = 0.237832 and AUCextra =

ComputedCLast/Lz = 0.738035

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.237832*8)/0.32225 + 0.237832/(0.32225*0.32225) = 8.19454

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.61 0.1525 0.1525 0.07625 0.07625

1 1.41 0.505 0.6575 0.42875 0.505

343

1.5 2.65 1.015 1.6725 1.34625 1.85125

2 1.81 1.10169 2.77419 1.9105 3.76175

3 1.21 1.48992 4.26411 3.67493 7.43668

4 0.79 0.985123 5.24923 3.41304 10.8497

6 0.39 1.13333 6.38256 5.5344 16.3841

8 0.27 0.652662 7.03522 4.52872 20.9128

Dataset 7(Plasma) 3 Undetectable Outlier

Dataset 7(Plasma)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 8

------

Dataset name: 8

------

344

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.49643 Alpha 0.58239 Ka 0.720173 solution found in 3 iterations

Objective function: 1.02898

Akaike criteria: 6.25711

Schwartz criteria: 3.55294

Parameter Mean S.D. C.V.(%) A 4.49643 16.0348 356.613 Alpha 0.58239 2.18888 375.844 Ka 0.720173 2.77588 385.446 Dataset 8, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.998921 1 Ka -0.998178 -0.996309 1 Dataset 8, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.66 1.16933 -0.509328 -0.509328 1 1.45 1.68966 -0.239663 -0.239663 1.5 2.63 1.83188 0.798122 0.798122 2 1.76 1.76609 -0.00608586 -0.00608586 3 1.22 1.38666 -0.166657 -0.166657 4 0.77 0.969293 -0.199293 -0.199293 6 0.37 0.401481 -0.031481 -0.031481 8 0.23 0.148723 0.0812771 0.0812771

345

Without weights With weights Sum -0.273109 -0.273109 Mean -0.0303455 -0.0303455 SumOfSquares 1.02898 1.02898 Std. dev. 0.357192 0.357192 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.993875)

C(T) = 3.31331 * exp(-0.34569*T) giving Lz = -B =

0.34569 and thalf = log(2)/Lz = 2.00511 and

ComputedCLast = C(8) = 0.208551 and AUCextra =

ComputedCLast/Lz = 0.603289

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.208551*8)/0.34569 + 0.208551/(0.34569*0.34569) = 6.57148

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.66 0.165 0.165 0.0825 0.0825

1 1.45 0.5275 0.6925 0.445 0.5275

1.5 2.63 1.02 1.7125 1.34875 1.87625

2 1.76 1.08298 2.79548 1.87714 3.75339

3 1.22 1.47355 4.26902 3.63897 7.39235

346

4 0.77 0.977803 5.24683 3.38494 10.7773

6 0.37 1.09157 6.3384 5.32571 16.103

8 0.23 0.588948 6.92735 4.07615 20.1791

Dataset 8(Plasma) 3 Undetectable Outlier

Dataset 8(Plasma)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 9

------

Dataset name: 9

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.45441 Alpha 0.566873

347

Ka 0.707584 solution found in 3 iterations

Objective function: 1.0297

Akaike criteria: 6.26337

Schwartz criteria: 3.55921

Parameter Mean S.D. C.V.(%) A 4.45441 15.2672 342.744 Alpha 0.566873 2.05274 362.115 Ka 0.707584 2.62895 371.54 Dataset 9, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.998822 1 Ka -0.998042 -0.996011 1 Dataset 9, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.65 1.14619 -0.496186 -0.496186 1 1.4 1.66794 -0.267943 -0.267943 1.5 2.62 1.82116 0.798842 0.798842 2 1.78 1.76824 0.0117638 0.0117638 3 1.23 1.40823 -0.178232 -0.178232 4 0.82 0.998541 -0.178541 -0.178541 6 0.38 0.425669 -0.0456693 -0.0456693 8 0.25 0.162327 0.0876727 0.0876727

Without weights With weights Sum -0.268293 -0.268293 Mean -0.0298103 -0.0298103 SumOfSquares 1.0297 1.0297 Std. dev. 0.357369 0.357369

348

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.992695)

C(T) = 3.28288 * exp(-0.335317*T) giving Lz = -B =

0.335317 and thalf = log(2)/Lz = 2.06714 and

ComputedCLast = C(8) = 0.224513 and AUCextra =

ComputedCLast/Lz = 0.669555

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.224513*8)/0.335317 + 0.224513/(0.335317*0.335317) = 7.35322

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.65 0.1625 0.1625 0.08125 0.08125

1 1.4 0.5125 0.675 0.43125 0.5125

1.5 2.62 1.005 1.68 1.3325 1.845

2 1.78 1.0865 2.7665 1.88393 3.72893

3 1.23 1.4881 4.2546 3.67452 7.40344

4 0.82 1.01118 5.26579 3.50507 10.9085

6 0.38 1.14415 6.40993 5.57549 16.484

8 0.25 0.620954 7.03089 4.30347 20.7875

349

Dataset 9(Plasma) 3 Undetectable Outli er

Dataset 9(Plasma)

0 0 2 4 6 8 T() ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 10

------

Dataset name: 10

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 4.50594 Alpha 0.570599 Ka 0.71248 solution found in 2 iterations

Objective function: 0.949976

Akaike criteria: 5.53813

Schwartz criteria: 2.83397

350

Parameter Mean S.D. C.V.(%) A 4.50594 14.634 324.772 Alpha 0.570599 1.958 343.148 Ka 0.71248 2.50912 352.168 Dataset 10, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.998818 1 Ka -0.998027 -0.995988 1 Dataset 10, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0

0.5 0.67 1.16496 -0.494959 -0.494959 1 1.47 1.69163 -0.221629 -0.221629 1.5 2.61 1.84307 0.766925 0.766925 2 1.77 1.78571 -0.015706 -0.015706 3 1.24 1.41612 -0.176125 -0.176125 4 0.85 0.999914 -0.149914 -0.149914 6 0.36 0.42271 -0.0627097 -0.0627097 8 0.26 0.159875 0.100125 0.100125

Without weights With weights Sum -0.253991 -0.253991 Mean -0.0282212 -0.0282212 SumOfSquares 0.949976 0.949976 Std. dev. 0.343294 0.343294 AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

351

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.987709)

C(T) = 3.27375 * exp(-0.333689*T) giving Lz = -B =

0.333689 and thalf = log(2)/Lz = 2.07723 and

ComputedCLast = C(8) = 0.226826 and AUCextra =

ComputedCLast/Lz = 0.679754

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.226826*8)/0.333689 + 0.226826/(0.333689*0.333689) = 7.47512

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0

0.5 0.67 0.1675 0.1675 0.08375 0.08375

1 1.47 0.535 0.7025 0.45125 0.535

1.5 2.61 1.02 1.7225 1.34625 1.88125

2 1.77 1.08144 2.80394 1.87507 3.75632

3 1.24 1.48932 4.29326 3.67922 7.43553

4 0.85 1.03276 5.32601 3.58222 11.0178

6 0.36 1.14069 6.4667 5.54207 16.5598

8 0.26 0.614586 7.08128 4.26883 20.8287

352

Dataset 10(Plasma) 3 Undetectable Outlier

Dataset 10(Plasma)

0 0 2 4 6 8 T()

======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

KINETICA REPORT FOR CLAVULANIC ACID + ANTACID

Dataset name: 1

------

Dataset name: 1

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 1.75004 Alpha 0.484169 Ka 0.424784 solution found in 20 iterations Objective function: 1.59432

353

Akaike criteria: 10.198

Schwartz criteria: 7.49385 Parameter Mean S.D. C.V.(%) A 4.0376 36.9279 914.601 Alpha 0.421915 4.00732 949.794 Ka 0.485293 4.63191 954.455 Dataset 1, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999647 1 Ka -0.999512 -0.998882 1 Dataset 1, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.35 0.780938 -0.430938 -0.430938 1 0.65 1.24509 -0.595094 -0.595094 1.5 1.35 1.48897 -0.138968 -0.138968 2 2.49 1.5829 0.907104 0.907104 3 1.78 1.50976 0.270235 0.270235 4 1.1 1.28044 -0.180439 -0.180439 6 0.52 0.777868 -0.257868 -0.257868 8 0.22 0.420608 -0.200608 -0.200608

Without weights With weights Sum -0.626576 -0.626576 Mean -0.0696195 -0.0696195 SumOfSquares 1.59432 1.59432 Std. dev. 0.440269 0.440269

354

Dataset 1(Plasma) 2.5 Undetectable Outlier

Dataset 1 Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0

= 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.999362)

C(T) = 5.75486 * exp(-0.405829*T) giving Lz = -B =

0.405829 and thalf = log(2)/Lz = 1.70798 and

ComputedCLast = C(8) = 0.223893 and AUCextra =

ComputedCLast/Lz = 0.551692

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.223893*8)/0.405829 + 0.223893/(0.405829*0.405829) = 5.77296

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

355

0 0 0.5 0.35 0.0875 0.0875 0.04375 0.04375 1 0.65 0.25 0.3375 0.20625 0.25 1.5 1.35 0.5 0.8375 0.66875 0.91875 2 2.49 0.96 1.7975 1.75125 2.67 3 1.78 2.11518 3.91268 5.22889 7.89889 4 1.1 1.41283 5.32551 4.88846 12.7873 6 0.52 1.54824 6.87375 7.54966 20.337 8 0.22 0.697511 7.57126 4.78379 25.1208 Marquardt Fitting

Model FitMicroExtravascular Parameter Initial Mean Volume 30.959 Kel 0.421915 Ka 0.485293 solution found in 3 iterations

Objective function: 1.59432

Akaike criteria: 9.73157

Schwartz criteria: 6.85073 Parameter Mean S.D. C.V.(%) Volume 30.959 310.487 1002.9 Kel 0.421915 4.3898 1040.45 Ka 0.485293 5.074 1045.55 Dataset 1, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.999647 1 Ka 0.999512 -0.998882 1 Dataset 1, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res.

356

0.5 0.35 0.780938 -0.430938 -0.430938 1 0.65 1.24509 -0.595094 -0.595094 1.5 1.35 1.48897 -0.138968 -0.138968 2 2.49 1.5829 0.907104 0.907104 3 1.78 1.50976 0.270235 0.270235 4 1.1 1.28044 -0.180439 -0.180439 6 0.52 0.777868 -0.257868 -0.257868 8 0.22 0.420608 -0.200608 -0.200608

Without weights With weights Sum -0.626576 -0.626576 Mean -0.078322 -0.078322 SumOfSquares 1.59432 1.59432 Std. dev. 0.469839 0.469839 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 2

------

Dataset name: 2

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 1.33941 Alpha 0.488626 Ka 0.423126

357

solution found in 22 iterations Objective function: 1.52319

Akaike criteria: 9.78725

Schwartz criteria: 7.08309 Parameter Mean S.D. C.V.(%) A 3.95828 39.4438 996.488 Alpha 0.425623 4.39105 1031.68 Ka 0.484122 5.01747 1036.4 Dataset 2, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.9997 1 Ka -0.999585 -0.999048 1 Dataset 2, possible overparametrization

Very high correlation between parameters

Possible overparametrization

T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.38 0.76326 -0.38326 -0.38326 1 0.63 1.21611 -0.586115 -0.586115 1.5 1.31 1.45335 -0.143345 -0.143345 2 2.45 1.54398 0.90602 0.90602 3 1.72 1.4706 0.249403 0.249403 4 1.02 1.24542 -0.225423 -0.225423 6 0.51 0.754305 -0.244305 -0.244305 8 0.27 0.406553 -0.136553 -0.136553

Without weights With weights Sum -0.563578 -0.563578 Mean -0.0626197 -0.0626197 SumOfSquares 1.52319 1.52319

358

Std. dev. 0.431262 0.431262

Dataset 2 (Plasma) 2.5 Undetectable Outlier

Dataset 2 (Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 4 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.999692)

C(T) = 3.8168 * exp(-0.332284*T) giving Lz = -B =

0.332284 and thalf = log(2)/Lz = 2.08601 and

ComputedCLast = C(8) = 0.26744 and AUCextra =

ComputedCLast/Lz = 0.804854

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.26744*8)/0.332284 + 0.26744/(0.332284*0.332284) = 8.86102

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

359

0 0 0.5 0.38 0.095 0.095 0.0475 0.0475 1 0.63 0.2525 0.3475 0.205 0.2525 1.5 1.31 0.485 0.8325 0.64875 0.90125 2 2.45 0.94 1.7725 1.71625 2.6175 3 1.72 2.06352 3.83602 5.0981 7.7156 4 1.02 1.33966 5.17568 4.63073 12.3463 6 0.51 1.47155 6.64723 7.18909 19.5354 8 0.27 0.75473 7.40196 5.20365 24.7391 Marquardt Fitting

Model FitMicroExtravascular Parameter Initial Mean Volume 31.5794 Kel 0.425623 Ka 0.484122 solution found in 3 iterations

Objective function: 1.52319

Akaike criteria: 9.36645

Schwartz criteria: 6.48561 Parameter Mean S.D. C.V.(%) Volume 31.5794 345.065 1092.69 Kel 0.425623 4.81015 1130.14 Ka 0.484122 5.49636 1135.32 Dataset 2, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.9997 1 Ka 0.999585 -0.999048 1 Dataset 2, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res.

360

0.5 0.38 0.76326 -0.38326 -0.38326 1 0.63 1.21611 -0.586115 -0.586115 1.5 1.31 1.45335 -0.143345 -0.143345 2 2.45 1.54398 0.90602 0.90602 3 1.72 1.4706 0.249403 0.249403 4 1.02 1.24542 -0.225423 -0.225423 6 0.51 0.754305 -0.244305 -0.244305 8 0.27 0.406553 -0.136553 -0.136553

Without weights With weights Sum -0.563578 -0.563578 Mean -0.0704472 -0.0704472 SumOfSquares 1.52319 1.52319 Std. dev. 0.460355 0.460355 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 3

------

Dataset name: 3

------

Marquardt Fitting Model FitMacroExtravascular Parameter Initial Mean A 1.76044 Alpha 0.490094 Ka 0.420866 solution found in 20 iterations

361

Objective function: 1.55239

Akaike criteria: 9.95814

Schwartz criteria: 7.25397 Parameter Mean S.D. C.V.(%) A 4.02045 35.2086 875.736 Alpha 0.421647 3.83887 910.448 Ka 0.487053 4.45727 915.15

Dataset 3, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999624 1 Ka -0.99948 -0.998811 1 Dataset 3, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.36 0.780153 -0.420153 -0.420153 1 0.67 1.24339 -0.573389 -0.573389 1.5 1.33 1.48639 -0.156395 -0.156395 2 2.48 1.5796 0.900399 0.900399 3 1.78 1.50558 0.274419 0.274419 4 1.08 1.27603 -0.196035 -0.196035 6 0.54 0.774205 -0.234205 -0.234205 8 0.21 0.41813 -0.20813 -0.20813

362

Without weights With weights Sum -0.613488 -0.613488 Mean -0.0681654 -0.0681654 SumOfSquares 1.55239 1.55239 Std. dev. 0.434535 0.434535 Dataset 3(Plasma) 2.5 Undetectable Outlier

Dataset 3 Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.998249)

C(T) = 5.815 * exp(-0.409444*T) giving Lz = -B =

0.409444 and thalf = log(2)/Lz = 1.6929 and

ComputedCLast = C(8) = 0.219784 and AUCextra =

ComputedCLast/Lz = 0.536788

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.219784*8)/0.409444 + 0.219784/(0.409444*0.409444) = 5.60532

Table of results

363

T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 0.36 0.09 0.09 0.045 0.045 1 0.67 0.2575 0.3475 0.2125 0.2575 1.5 1.33 0.5 0.8475 0.66625 0.92375 2 2.48 0.9525 1.8 1.73875 2.6625 3 1.78 2.11069 3.91069 5.2185 7.881 4 1.08 1.40097 5.31166 4.84532 12.7263 6 0.54 1.55811 6.86977 7.61198 20.3383 8 0.21 0.698811 7.56859 4.78328 25.1216 Marquardt Fitting

Model FitMicroExtravascular Parameter Initial Mean Volume 31.091 Kel 0.421647 Ka 0.487053 solution found in 3 iterations

Objective function: 1.55239

Akaike criteria: 9.51834

Schwartz criteria: 6.63751 Parameter Mean S.D. C.V.(%) Volume 31.091 298.561 960.28 Kel 0.421647 4.20528 997.346 Ka 0.487053 4.88269 1002.5 Dataset 3, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.999624 1 Ka 0.99948 -0.998811 1 Dataset 3, possible overparametrization

Very high correlation between parameters

364

Possible overparametrization T C Ycalc Residuals Weighted Res. 0.5 0.36 0.780153 -0.420153 -0.420153 1 0.67 1.24339 -0.573389 -0.573389 1.5 1.33 1.48639 -0.156395 -0.156395 2 2.48 1.5796 0.900399 0.900399 3 1.78 1.50558 0.274419 0.274419 4 1.08 1.27603 -0.196035 -0.196035 6 0.54 0.774205 -0.234205 -0.234205 8 0.21 0.41813 -0.20813 -0.20813

Without weights With weights Sum -0.613488 -0.613488 Mean -0.0766861 -0.0766861 SumOfSquares 1.55239 1.55239 Std. dev. 0.463733 0.463733 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

Method Name: MacroToMicro

Nb comp 1

======

Dataset name:4

------

Dataset name: 4

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean

365

A 1.79353 Alpha 0.480644 Ka 0.412336 solution found in 20 iterations

Objective function: 1.53077

Akaike criteria: 9.83191

Schwartz criteria: 7.12775 Parameter Mean S.D. C.V.(%) A 3.98791 37.7423 946.417 Alpha 0.414956 4.07621 982.323 Ka 0.475722 4.69115 986.112 Dataset 4, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999673 1 Ka -0.999552 -0.998968 1 Dataset 4, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.32 0.759245 -0.439245 -0.439245 1 0.61 1.21551 -0.605508 -0.605508 1.5 1.35 1.45958 -0.109582 -0.109582 2 2.43 1.55805 0.871955 0.871955 3 1.76 1.49829 0.261706 0.261706 4 1.12 1.28113 -0.161134 -0.161134 6 0.58 0.791049 -0.211049 -0.211049 8 0.19 0.434701 -0.244701 -0.244701

Without weights With weights Sum -0.637559 -0.637559 Mean -0.0708399 -0.0708399

366

SumOfSquares 1.53077 1.53077 Std. dev. 0.430929 0.430929

Dataset 4(Plasma) 2.5 Undetectable Outlier

Dataset 4 (Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.9943)

C(T) = 6.01065 * exp(-0.417678*T) giving Lz = -

B = 0.417678 and thalf = log(2)/Lz = 1.65953 and ComputedCLast = C(8) = 0.212697 and

AUCextra = ComputedCLast/Lz = 0.509236

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.212697*8)/0.417678 + 0.212697/(0.417678*0.417678) = 5.2931

Table of results T C AUC AUCcum AUMC AUMCcum

367

h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 0.32 0.08 0.08 0.04 0.04 1 0.61 0.2325 0.3125 0.1925 0.2325 1.5 1.35 0.49 0.8025 0.65875 0.89125 2 2.43 0.945 1.7475 1.72125 2.6125 3 1.76 2.07702 3.82452 5.13681 7.74931 4 1.12 1.41598 5.2405 4.90276 12.6521 6 0.58 1.6412 6.88169 8.02728 20.6794 8 0.19 0.698922 7.58062 4.76508 25.4444 Marquardt Fitting Model FitMicroExtravascular Parameter Initial Mean Volume 31.3447 Kel 0.414956 Ka 0.475722 solution found in 3 iterations

Objective function: 1.53077

Akaike criteria: 9.40614

Schwartz criteria: 6.5253 Parameter Mean S.D. C.V.(%) Volume 31.3447 325.291 1037.78 Kel 0.414956 4.46526 1076.08 Ka 0.475722 5.1389 1080.23 Dataset 4, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.999673 1

368

Ka 0.999552 -0.998968 1 Dataset 4, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0.5 0.32 0.759245 -0.439245 -0.439245 1 0.61 1.21551 -0.605508 -0.605508 1.5 1.35 1.45958 -0.109582 -0.109582 2 2.43 1.55805 0.871955 0.871955 3 1.76 1.49829 0.261706 0.261706 4 1.12 1.28113 -0.161134 -0.161134 6 0.58 0.791049 -0.211049 -0.211049 8 0.19 0.434701 -0.244701 -0.244701

Without weights With weights Sum -0.637559 -0.637559 Mean -0.0796949 -0.0796949 SumOfSquares 1.53077 1.53077 Std. dev. 0.459807 0.459807 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting

Method Name: MacroToMicro

Nb comp 1

======

Dataset name:5

------

Dataset name: 5

------

369

Parameter Mean S.D. C.V.(%) A 3.94715 35.6629 903.511 Alpha 0.419069 3.937 939.465 Ka 0.484121 4.57021 944.023 Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 1.73545 Alpha 0.482641 Ka 0.422245 solution found in 20 iterations Objective function: 1.5785

Akaike criteria: 10.1083

Schwartz criteria: 7.40409

Dataset 5, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999627 1 Ka -0.999486 -0.998821 1 Dataset 5, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.34 0.762366 -0.422366 -0.422366 1 0.63 1.21671 -0.586714 -0.586714 1.5 1.31 1.45651 -0.146508 -0.146508

370

2 2.46 1.54997 0.910031 0.910031 3 1.75 1.4814 0.268601 0.268601 4 1.05 1.25899 -0.208987 -0.208987 6 0.55 0.768052 -0.218052 -0.218052 8 0.21 0.417077 -0.207077 -0.207077

Without weights With weights Sum -0.611073 -0.611073 Mean -0.067897 -0.067897 SumOfSquares 1.5785 1.5785 Std. dev. 0.438322 0.438322

Dataset 5(Plasma) 2.5 Undetectable Outlier

Dataset 5 (Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.997509)

C(T) = 5.67146 * exp(-0.405528*T) giving Lz = -B =

371

0.405528 and thalf = log(2)/Lz = 1.70925 and

ComputedCLast = C(8) = 0.22118 and AUCextra =

ComputedCLast/Lz = 0.545412

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.22118*8)/0.405528 + 0.22118/(0.405528*0.405528) = 5.70824

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 0.34 0.085 0.085 0.0425 0.0425 1 0.63 0.2425 0.3275 0.2 0.2425 1.5 1.31 0.485 0.8125 0.64875 0.89125 2 2.46 0.9425 1.755 1.72125 2.6125 3 1.75 2.08489 3.83989 5.15317 7.76567 4 1.05 1.37033 5.21022 4.73808 12.5037 6 0.55 1.54649 6.75671 7.56691 20.0707 8 0.21 0.706265 7.46297 4.83224 24.9029 Marquardt Fitting Model FitMicroExtravascular Parameter Initial Mean Volume 31.6684 Kel 0.419069 Ka 0.484121 solution found in 3 iterations

Objective function: 1.5785

Akaike criteria: 9.65178

Schwartz criteria: 6.77095 Parameter Mean S.D. C.V.(%) Volume 31.6684 313.751 990.737 Kel 0.419069 4.31277 1029.13 Ka 0.484121 5.00642 1034.13 Dataset 5, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

372

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.999627 1 Ka 0.999486 -0.998821 1 Dataset 5, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0.5 0.34 0.762366 -0.422366 -0.422366 1 0.63 1.21671 -0.586714 -0.586714 1.5 1.31 1.45651 -0.146508 -0.146508 2 2.46 1.54997 0.910031 0.910031 3 1.75 1.4814 0.268601 0.268601 4 1.05 1.25899 -0.208987 -0.208987 6 0.55 0.768052 -0.218052 -0.218052 8 0.21 0.417077 -0.207077 -0.207077

Without weights With weights Sum -0.611073 -0.611073 Mean -0.0763841 -0.0763841 SumOfSquares 1.5785 1.5785 Std. dev. 0.467794 0.467794 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

373

Parameter Mean S.D. C.V.(%) A 2.52107 3.40321 134.991 Alpha 0.201483 0.405996 201.504 Ka 0.635614 1.18739 186.81 Dataset name: 6

------

Dataset name: 6

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 0.887798 Alpha 0.215805 Ka 0.611183 solution found in 7 iterations

Objective function: 1.90795

Akaike criteria: 11.8143

Schwartz criteria: 9.1101

Dataset 6, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.983016 1 Ka -0.976856 -0.95271 1

T C Plasma C calc Plasma Residuals Plasma Weighted Res.

374

0 0 0 0 0 0.5 0.32 0.6512 -0.3312 -0.3312 1 0.61 1.0627 -0.452699 -0.452699 1.5 1.34 1.30574 0.0342609 0.0342609 2 2.42 1.43159 0.988409 0.988409 3 1.73 1.46842 0.261576 0.261576 4 1.03 1.35832 -0.328324 -0.328324 6 0.56 1.02044 -0.460443 -0.460443 8 1.19 0.713588 0.476412 0.476412

Without weights With weights Sum 0.187993 0.187993 Mean 0.0208881 0.0208881 SumOfSquares 1.90795 1.90795 Std. dev. 0.487856 0.487856

Dataset 6(Plasma) 2.5 Undetectable Outlier

Dataset 6 (Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

375

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.639932)

C(T) = 2.42977 * exp(-0.147105*T) giving Lz = -B =

0.147105 and thalf = log(2)/Lz = 4.71193 and

ComputedCLast = C(8) = 0.748983 and AUCextra =

ComputedCLast/Lz = 5.09149

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.748983*8)/0.147105 + 0.748983/(0.147105*0.147105) = 75.3433

Table of results T C AUC AUCcum AUMC AUMCcum h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 0.32 0.08 0.08 0.04 0.04 1 0.61 0.2325 0.3125 0.1925 0.2325 1.5 1.34 0.4875 0.8 0.655 0.8875 2 2.42 0.94 1.74 1.7125 2.6 3 1.73 2.05574 3.79574 5.08195 7.68195 4 1.03 1.34989 5.14562 4.66652 12.3485 6 0.56 1.54256 6.68818 7.55709 19.9056 8 1.19 1.75 8.43818 12.88 32.7856 Marquardt Fitting Model FitMicroExtravascular Parameter Initial Mean Volume 49.5821 Kel 0.201483 Ka 0.635614 solution found in 5 iterations

Objective function: 1.87866

Akaike criteria: 11.0445

Schwartz criteria: 8.16365 Parameter Mean S.D. C.V.(%) Volume 46.6369 66.8125 143.261

376

Kel 0.201342 0.42823 212.688 Ka 0.623878 1.22541 196.418 Dataset 6, standard deviation of some parameters is high

Standard deviation of some parameters is high

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.983885 1 Ka 0.978115 -0.954994 1

T C Ycalc Residuals Weighted Res. 0.5 0.32 0.681491 -0.361491 -0.361491 1 0.61 1.11509 -0.505094 -0.505094 1.5 1.34 1.37349 -0.0334872 -0.0334872 2 2.42 1.50927 0.910726 0.910726 3 1.73 1.55423 0.175771 0.175771 4 1.03 1.44237 -0.412371 -0.412371 6 0.56 1.0887 -0.528704 -0.528704 8 1.19 0.763558 0.426442 0.426442

Without weights With weights Sum -0.328209 -0.328209 Mean -0.0410261 -0.0410261 SumOfSquares 1.87866 1.87866 Std. dev. 0.516195 0.516195 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

377

======

Dataset name: 7

------

Dataset name: 7

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 1.82184 Alpha 0.377469 Ka 0.431329 solution found in 8 iterations

Objective function: 2.64239

Akaike criteria: 14.7452

Schwartz criteria: 12.041 Parameter Mean S.D. C.V.(%) A 3.38046 44.6137 1319.76 Alpha 0.369613 5.11725 1384.49 Ka 0.435885 6.02257 1381.69 Dataset 7, standard deviation of some parameters is too big for a good fit

378

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.9996 1 Ka -0.999478 -0.998761 1 Dataset 7, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.35 0.602394 -0.252394 -0.252394 1 0.6 0.985178 -0.385178 -0.385178 1.5 0.32 1.20851 -0.888508 -0.888508 2 2.46 1.31787 1.14213 1.14213 3 1.75 1.32266 0.42734 0.42734 4 1.13 1.18041 -0.0504056 -0.0504056 6 0.51 0.794119 -0.284119 -0.284119 8 0.21 0.475575 -0.265575 -0.265575

Without weights With weights Sum -0.556706 -0.556706 Mean -0.0618562 -0.0618562 SumOfSquares 2.64239 2.64239 Std. dev. 0.570959 0.570959

379

Dataset 7(Plasma) 2.5 Undetectable Outlier

Dataset 7 (Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values

c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 3 to infinity (auto detected best interval),

curve may be approximated by : (with R = -0.999732)

C(T) = 6.1832 * exp(-0.420929*T) giving Lz = -B =

0.420929 and thalf = log(2)/Lz = 1.64671 and

ComputedCLast = C(8) = 0.213185 and AUCextra =

ComputedCLast/Lz = 0.506464

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.213185*8)/0.420929 + 0.213185/(0.420929*0.420929) = 5.25492

Table of results T C AUC AUCcum AUMC AUMCcum / h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

380

0 0 0.5 0.35 0.0875 0.0875 0.04375 0.04375 1 0.6 0.2375 0.325 0.19375 0.2375 1.5 0.32 0.222714 0.547714 0.272597 0.510097 2 2.46 0.695 1.24271 1.35 1.8601 3 1.75 2.08489 3.3276 5.15317 7.01327 4 1.13 1.41747 4.74508 4.90965 11.9229 6 0.51 1.55865 6.30372 7.58871 19.5116 8 0.21 0.676206 6.97993 4.63473 24.1464 Marquardt Fitting Model FitMicroExtravascular Parameter Initial Mean Volume 36.9773 Kel 0.369613 Ka 0.435885 solution found in 3 iterations

Objective function: 2.64239

Akaike criteria: 13.7735

Schwartz criteria: 10.8926 Parameter Mean S.D. C.V.(%) Volume 36.9773 535.122 1447.17 Kel 0.369613 5.60567 1516.63 Ka 0.435885 6.5974 1513.57 Dataset 7, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.9996 1 Ka 0.999478 -0.998761 1 Dataset 7, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res. 0.5 0.35 0.602394 -0.252394 -0.252394

381

1 0.6 0.985178 -0.385178 -0.385178 1.5 0.32 1.20851 -0.888508 -0.888508 2 2.46 1.31787 1.14213 1.14213 3 1.75 1.32266 0.42734 0.42734 4 1.13 1.18041 -0.0504056 -0.0504056 6 0.51 0.794119 -0.284119 -0.284119 8 0.21 0.475575 -0.265575 -0.265575

Without weights With weights Sum -0.556706 -0.556706 Mean -0.0695882 -0.0695882 SumOfSquares 2.64239 2.64239 Std. dev. 0.609877 0.609877 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 8

------

Dataset name: 8

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 1.69859 Alpha 0.482222 Ka 0.42454

382

solution found in 22 iterations

Objective function: 1.49038 Akaike criteria: 9.59127 Schwartz criteria:

6.8871

Parameter Mean S.D. C.V.(%) A 4.02835 46.9972 1166.66 Alpha 0.427827 5.14064 1201.57 Ka 0.477069 5.75261 1205.82 Dataset 8 standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999788 1 Ka -0.999707 -0.999324 1 Dataset 8, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.34 0.766376 -0.426376 -0.426376 1 0.62 1.22252 -0.602522 -0.602522 1.5 1.38 1.4627 -0.0826995 -0.0826995 2 2.41 1.55569 0.854311 0.854311 3 1.76 1.48504 0.274961 0.274961 4 1.04 1.26034 -0.220341 -0.220341 6 0.55 0.766416 -0.216416 -0.216416 8 0.22 0.414609 -0.194609 -0.194609

Without weights With weights Sum -0.613691 -0.613691 Mean -0.0681879 -0.0681879 SumOfSquares 1.49038 1.49038

383

Std. dev. 0.425519 0.425519

Dataset 8(Plasma) 2.5 Undetectable Outlier

Dataset 8 (Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.997584)

C(T) = 5.46624 * exp(-0.396555*T) giving Lz = -B =

0.396555 and thalf = log(2)/Lz = 1.74792 and

ComputedCLast = C(8) = 0.229043 and AUCextra =

ComputedCLast/Lz = 0.577582

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.229043*8)/0.396555 + 0.229043/(0.396555*0.396555) = 6.07715

Table of results T C AUC AUCcum AUMC AUMCcum

384

/ h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

0 0 0.5 0.34 0.085 0.085 0.0425 0.0425 1 0.62 0.24 0.325 0.1975 0.24 1.5 1.38 0.5 0.825 0.6725 0.9125 2 2.41 0.9475 1.7725 1.7225 2.635 3 1.76 2.068 3.8405 5.11593 7.75093 4 1.04 1.36858 5.20908 4.7303 12.4812 6 0.55 1.53832 6.7474 7.52937 20.0106 8 0.22 0.720295 7.4677 4.93358 24.9442 Marquardt Fitting Model FitMicroExtravascular Parameter Initial Mean Volume 31.0301 Kel 0.427827 Ka 0.477069 solution found in 3 iterations

Objective function: 1.49038

Akaike criteria: 9.19224

Schwartz criteria: 6.3114 Parameter Mean S.D. C.V.(%) Volume 31.0301 396.964 1279.29 Kel 0.427827 5.63129 1316.25 Ka 0.477069 6.30167 1320.91 Dataset 8, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.999788 1 Ka 0.999707 -0.999324 1 Dataset 8, possible overparametrization

Very high correlation between parameters

385

Possible overparametrization T C Ycalc Residuals Weighted Res. 0.5 0.34 0.766376 -0.426376 -0.426376 1 0.62 1.22252 -0.602522 -0.602522 1.5 1.38 1.4627 -0.0826995 -0.0826995 2 2.41 1.55569 0.854311 0.854311 3 1.76 1.48504 0.274961 0.274961 4 1.04 1.26034 -0.220341 -0.220341 6 0.55 0.766416 -0.216416 -0.216416 8 0.22 0.414609 -0.194609 -0.194609

Without weights With weights Sum -0.613691 -0.613691 Mean -0.0767114 -0.0767114 SumOfSquares 1.49038 1.49038 Std. dev. 0.454077 0.454077 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name: 9

------

Dataset name: 9

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 1.6865

386

Alpha 0.483574 Ka 0.423892 solution found in 22 iterations

Objective function: 1.53457 Akaike criteria: 9.85425 Schwartz criteria:

7.15009

Parameter Mean S.D. C.V.(%) A 3.98064 43.7673 1099.5 Alpha 0.426154 4.83724 1135.09 Ka 0.479461 5.46375 1139.56 Dataset 9, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999751 1 Ka -0.999656 -0.999209 1 Dataset 9, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.37 0.760963 -0.390963 -0.390963 1 0.61 1.21368 -0.603685 -0.603685 1.5 1.33 1.4519 -0.121896 -0.121896 2 2.45 1.54397 0.906032 0.906032 3 1.71 1.47345 0.236552 0.236552 4 1.02 1.25021 -0.230205 -0.230205 6 0.57 0.759969 -0.189969 -0.189969 8 0.22 0.411024 -0.191024 -0.191024

Without weights With weights Sum -0.585157 -0.585157 Mean -0.0650175 -0.0650175

387

SumOfSquares 1.53457 1.53457 Std. dev. 0.432511 0.432511

Dataset 9(Plasma) 2.5 Undetectable Outlier

Dataset 9 (Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.996553)

C(T) = 5.40053 * exp(-0.393754*T) giving Lz = -B =

0.393754 and thalf = log(2)/Lz = 1.76035 and

ComputedCLast = C(8) = 0.231416 and AUCextra =

ComputedCLast/Lz = 0.587717

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.231416*8)/0.393754 + 0.231416/(0.393754*0.393754) = 6.19434

Table of results T C AUC AUCcum AUMC AUMCcum

388

/ h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)²

0 0 0.5 0.37 0.0925 0.0925 0.04625 0.04625 1 0.61 0.245 0.3375 0.19875 0.245 1.5 1.33 0.485 0.8225 0.65125 0.89625 2 2.45 0.945 1.7675 1.72375 2.62 3 1.71 2.05787 3.82537 5.08315 7.70315 4 1.02 1.33542 5.16079 4.61673 12.3199 6 0.57 1.5466 6.70739 7.58384 19.9037 8 0.22 0.7352877.44268 5.03207 24.9358 Marquardt Fitting Model FitMicroExtravascular Parameter Initial Mean Volume 31.402 Kel 0.426154 Ka 0.479461 solution found in 3 iterations

Objective function: 1.53457

Akaike criteria: 9.426

Schwartz criteria: 6.54516 Parameter Mean S.D. C.V.(%) Volume 31.402 378.598 1205.65 Kel 0.426154 5.29894 1243.43 Ka 0.479461 5.98524 1248.32 Dataset 9, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.999751 1 Ka 0.999656 -0.999209 1 Dataset 9, possible overparametrization

Very high correlation between parameters

389

Possible overparametrization T C Ycalc Residuals Weighted Res. 0.5 0.37 0.760963 -0.390963 -0.390963 1 0.61 1.21368 -0.603685 -0.603685 1.5 1.33 1.4519 -0.121896 -0.121896 2 2.45 1.54397 0.906032 0.906032 3 1.71 1.47345 0.236552 0.236552 4 1.02 1.25021 -0.230205 -0.230205 6 0.57 0.759969 -0.189969 -0.189969 8 0.22 0.411024 -0.191024 -0.191024

Without weights With weights Sum -0.585157 -0.585157 Mean -0.0731447 -0.0731447 SumOfSquares 1.53457 1.53457 Std. dev. 0.461638 0.461638 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

======

Dataset name:10

------

Dataset name: 21

------

Marquardt Fitting

Model FitMacroExtravascular Parameter Initial Mean A 1.688

390

Alpha 0.479552 Ka 0.412469 solution found in 22 iterations

Objective function: 1.44701 Akaike criteria: 9.32548 Schwartz criteria:

6.62132

Parameter Mean S.D. C.V.(%) A 4.00306 45.7029 1141.7 Alpha 0.419975 4.94349 1177.09 Ka 0.469658 5.54467 1180.58 Dataset 10, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix A Alpha Ka A 1 Alpha 0.999782 1 Ka -0.999701 -0.999309 1 Dataset 10, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Plasma C calc Plasma Residuals Plasma Weighted Res. 0 0 0 0 0 0.5 0.31 0.752599 -0.442599 -0.442599 1 0.65 1.20514 -0.555137 -0.555137 1.5 1.31 1.44741 -0.137415 -0.137415 2 2.41 1.54532 0.864677 0.864677 3 1.73 1.48646 0.243542 0.243542 4 1.11 1.27123 -0.161225 -0.161225 6 0.56 0.784953 -0.224953 -0.224953 8 0.23 0.431189 -0.201189 -0.201189

Without weights With weights Sum -0.614299 -0.614299 Mean -0.0682555 -0.0682555

391

SumOfSquares 1.44701 1.44701 Std. dev. 0.419088 0.419088

Dataset 10(Plasma) 2.5 Undetectable Outlier

Dataset 10 (Plasma) 2

1.5 C(/)

0.5

0 0 2 4 6 8 T()

AUC * calculation

First point time = 0, t0 and c0 set to first point values c0 = 0 t0 = 0

Data starts with a 0 time value, no need to apply AUC0 option

The AUC was computed using the Log Linear Method, trapezoidal when Cn > Cn-1

AUC log calculation start after user defined t = 0

AUCtot = AUClast+ CLast/Lz

From 2 to infinity (auto detected best interval), curve may be approximated by : (with R = -0.998743)

C(T) = 5.40866 * exp(-0.389452*T) giving Lz = -B =

0.389452 and thalf = log(2)/Lz = 1.7798 and

ComputedCLast = C(8) = 0.239881 and AUCextra =

ComputedCLast/Lz = 0.615944

and AUMCextra = (ComputedCLast*TLast)/Lz + ComputedCLast/(Lz²) = (0.239881*8)/0.389452 + 0.239881/(0.389452*0.389452) = 6.50912

Table of results T C AUC AUCcum AUMC AUMCcum

392

h µg/ml mg/L*h mg/L*h mg/L*(h)² mg/L*(h)² 0 0 0.5 0.31 0.0775 0.0775 0.03875 0.03875 1 0.65 0.24 0.3175 0.20125 0.24 1.5 1.31 0.49 0.8075 0.65375 0.89375 2 2.41 0.93 1.7375 1.69625 2.59 3 1.73 2.05125 3.78875 5.07156 7.66156 4 1.11 1.39715 5.1859 4.83852 12.5001 6 0.56 1.60777 6.79366 7.85692 20.357 8 0.23 0.741692 7.53536 5.08327 25.4403 Marquardt Fitting Model FitMicroExtravascular Parameter Initial Mean Volume 31.2261 Kel 0.419975 Ka 0.469658 solution found in 3 iterations

Objective function: 1.44701

Akaike criteria: 8.95598

Schwartz criteria: 6.07515 Parameter Mean S.D. C.V.(%) Volume 31.2261 390.926 1251.92 Kel 0.419975 5.41532 1289.44 Ka 0.469658 6.07388 1293.26 Dataset 10, standard deviation of some parameters is too big for a good fit

Standard deviation of some parameters is too big for a good fit

The model may not be the good one

NB : check data for possible outliers

Correlation Matrix Volume Kel Ka Volume 1 Kel -0.999782 1 Ka 0.999701 -0.999309 1 Dataset 10, possible overparametrization

Very high correlation between parameters

Possible overparametrization T C Ycalc Residuals Weighted Res.

393

0.5 0.31 0.752599 -0.442599 -0.442599 1 0.65 1.20514 -0.555137 -0.555137 1.5 1.31 1.44741 -0.137415 -0.137415 2 2.41 1.54532 0.864677 0.864677 3 1.73 1.48646 0.243542 0.243542 4 1.11 1.27123 -0.161225 -0.161225 6 0.56 0.784953 -0.224953 -0.224953 8 0.23 0.431189 -0.201189 -0.201189

Without weights With weights Sum -0.614299 -0.614299 Mean -0.0767874 -0.0767874 SumOfSquares 1.44701 1.44701 Std. dev. 0.447188 0.447188 ======

Hard Coded Method Output Variables

Method Name: FitMacroExtravascular Nb comp 1 Use lag time No Weight 1 Plot curve Yes Execute Fitting ------

Method Name: MacroToMicro

Nb comp 1

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394

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