á1090ñIN VIVO BIOEQUIVALENCE GUIDANCES1
This chapter is divided into two sections that provide guidances related to the general conduct of bioequivalence tests and to bioavailability protocols for specific drugs.These statements were prepared by the Food and Drug Administration,Office of Generic Drugs (FDA,OGD),Division of Bioequivalence,and presented herein.For general background information on related issues,refer to In Vitro and In Vivo Evaluation of Dosage Forms á1088ñ.

DRUG PROTOCOLS

Alprazolam Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Alprazolam is widely used in the management of anxiety disorders,panic disorder,social phobia,and depression.It is a triazolo analog of the 1,4-benzodiazepine class of compounds acting on the central nervous system (CNS).The exact mechanism of action of alprazolam is unknown;however,published literature indicates that this class of drugs exerts effects by binding to stereospecific receptors at several sites within the central nervous system.The drug effect is dose-related.The daily dose of alprazolam depends on the individual patient,and averages 1.5to 4mg.Although a major metabolite,a-hydroxyalprazolam,shows some pharmacological activity,no alprazolam metabolites possess any clinically important role,and they do not accumulate to any significant degree.
Pharmacokinetics— Alprazolam is readily absorbed (more than 90%)after oral administration and is distributed widely with a volume of distribution of approximately 1Lper kg in healthy men and women.Peak concentrations in the plasma occur 1to 2hours after administration.With doses ranging from 0.5to 3.0mg given on an empty stomach,plasma peak levels of 8.0to 37ng per mLwere observed.The mean plasma elimination half-life of alprazolam has been reported to be about 11.2hours (range:6.3to 26.9hours)in healthy adults.The drug absorption is slower when alprazolam is taken after a meal than on an empty stomach,but total absorption is unchanged.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Xanax®(Upjohn)1-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10%of the largest lot planned for full production or a minimum of 100,000units,whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— Asingle-dose,randomized,fasting,two-period,two-treatment,two-sequence crossover study comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose,Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose,two-treatment,two-period,two-sequence crossover with a 1-week washout period between Phase Iand Phase IIdosing.An equal number of subjects should be randomly assigned to the two possible dosing sequences.Before the study begins,the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names,titles,and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsors should enroll a number of subjects sufficient to ensure statistical validity of the study.It is recommended that a minimum of 24subjects be used in this study.Subjects should be healthy,preferably nonsmoking,volunteers aged 18to 50years and within 10%of ideal body weight for height and build,although within 15%of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin,1983).Subjects should be selected on the basis of acceptable medical history,physical examination,and clinical laboratory test results.Female subjects must be given a pregnancy test prior to beginning the study.Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study.If smokers are included,they should be identified as such.Written,informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10hours,subjects should be administered a single dose of the test or reference product with 240mLof water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1hour before and after drug administration,when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4hours after administration of the test or reference treatment.All meals should be standardized during the study,and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications,including oral contraceptives,or OTCmedications beginning 2weeks before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0hours)and at 0.25,0.50,0.75,1.0,1.50,2.0,2.50,3.0,4.0,6.0,8.0,12,24,36,48,and 72hours post-dose.Plasma should be separated promptly and immediately frozen until assayed.Following at least a minimum 1-week washout period,subjects should begin the second phase of the study.
Analytical Methods— Alprazolam should be assayed using a suitable method fully validated with respect to adequate sensitivity,specificity,linearity,recovery,and accuracy and precision (both within and between days).Stability of the samples under frozen conditions,at room temperature,and during freeze-thaw cycles,if appropriate,should be determined.Chromatograms of the analysis of the unknown samples,including all associated standard curve and quality control chromatograms,should be available for regulatory authorities.The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data (Plasma)— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Designunder General Guidances.
Clinical Report,Side Effects,and Adverse Reactions— Subject medical histories,physical examinations and laboratory reports,and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application.Each reserve sample should consist of at least 200dosage units and should be retained for a period of not less than 5years following approval of the application.For more information on retention of bioequivalence samples,refer to 21CFR320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12dosage units of the test product and the reference product,employing the biostudy lots.The current official USPdissolution method (see Dissolution á711ñ)should be followed and should be referenced by the sponsor.The following method and tolerances are currently recommended for this product:
Medium: phosphate buffer solution (pH6.0);500mL.
Apparatus 1: 100rpm.
Times: 10,20,30,and 45minutes.
Procedure— Use USPif available,or other validated method (see Validation of Compendial Methods á1225ñ).
Tolerances (Q): not less than 80%in 30minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit.The mean percentage dissolved,the range (highest,lowest)of dissolution,and the coefficient of variation (relative standard deviation)should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 0.25-,0.5-,and 2-mg strengths of the generic product may be granted pursuant to 21CFR320.22(d)(2),provided the following conditions are met:
  1. The 0.25-,0.5-,and 2-mg strengths are proportionally similar in both active and inactive ingredients to another strength (1mg),which has been demonstrated to be bioequivalent to a reference (1mg)product in vivo.
  2. The 0.25-,0.5-,and 2-mg strengths of the generic product meet dissolution requirements.
  3. The drug product follows linear kinetics over its range of available strengths.

Bumetanide Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Bumetanide is a potent diuretic indicated for the treatment of edema associated with congestive heart failure and hepatic and renal disease,including the nephrotic syndrome.Bumetanide is contraindicated in anuria,hepatic coma,states of severe electrolyte depletion,and in patients hypersensitive to the drug.
The major site of action of bumetanide is the ascending limb of the loop of Henle,where it inhibits the sodium-potassium-2chloride absorptive pump.The usual daily dosing range for bumetanide is 0.5to 2.0mg.
Pharmacokinetics— Bumetanide is reported to be readily absorbed from the gastrointestinal (GI)tract with a Tmaxof 0.5to 2hours,and a bioavailability of about 80%to 90%.Several pharmacokinetic studies have shown that bumetanide,administered orally,is eliminated rapidly in humans,with a half-life of between 1and 2hours.Plasma protein-binding is approximately 95%.Oral administration of bumetanide resulted in 36%to 60%recovery of the unchanged drug from urine.The volume of distribution (Vd)is approximately 0.2Lper kg.Following oral administration of bumetanide,the onset of diuresis occurs in 30to 60minutes.Peak activity is reached between 0.5and 3hours.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Bumex®(Roche Laboratories)2.0-mg tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10%of the largest lot planned for full production or a minimum of 100,000units,whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— Asingle-dose,randomized,two-period,two-treatment,two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
Recommended Protocol for Conducting a Single-Dose,Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose,two-treatment,two-period,two-sequence crossover with at least a three-day washout period between Phase Iand Phase IIdosing.An equal number of subjects should be randomly assigned to the two possible dosing sequences.Before the study begins,the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names,titles,and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study.It is recommended that a minimum of 24subjects be used in this study.Subjects should be healthy,preferably nonsmoking,volunteers aged 18to 50years and within 10%of ideal body weight for height and build,although within 15%of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin,1983).Subjects should be selected on the basis of acceptable medical history,physical examination,and clinical laboratory test results.Female subjects must be given a pregnancy test prior to beginning the study.Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study.If smokers are included,they should be identified as such.Written,informed consent must be obtained from all study participants before they are accepted into the studies.
Procedure— Following an overnight fast of at least 10hours,subjects should be administered a single dose (2.0mg)of the test or reference product with 240mLof water.The subjects should drink 240mLof water at the following times:-2,-1,0,1,2,4,6,8,and 10hours relative to dosing.
Restrictions— Study volunteers should observe the following restrictions:
a.Subjects should fast for at least 4hours after administration of the test or reference treatment.All meals should be standardized during the study,and the same meals should be served during both phases of the study.
b.No alcohol or xanthine-containing foods or beverages should be consumed for 48hours prior to dosing and until after the last blood sample is collected.
c.Subjects should take no Rx medications,including oral contraceptives,beginning 2weeks and no OTCmedications beginning 1week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples in a volume sufficient for sample analysis and anticoagulated as appropriate should be collected pre-dose (0hours)and at 0.25,0.50,0.75,1.0,1.25,1.50,1.75,2.0,2.50,3,4,5,6,8,and 12hours post-dose.Plasma should be separated promptly,immediately frozen,and stored at –20until assayed.Following a minimum 1-week washout period,subjects should begin the second phase of the study.
Urine Sampling— Urine may be collected over the following time intervals:-2to 0,0to 1,1to 2,2to 4,4to 6,6to 8,8to 10,and 10to 12hours post-dosing.The urine volume and pHshould be noted.One 50-mLaliquot should be stored refrigerated for possible analysis of sodium and potassium.A15-mLsample should be stored frozen at –20for possible bumetanide assay.
Analytical Methods— High-performance liquid chromatographic methods have been reported for assaying bumetanide in human plasma and urine.
Bumetanide should be assayed using a suitable method fully validated with respect to stability,sensitivity,specificity,linearity,recovery,and accuracy and precision (both within and between days).Stability of the samples under frozen conditions,at room temperature,and during freeze-thaw cycles,if appropriate,should be determined.Chromatograms of the analysis of the unknown samples,including all associated standard curve and quality control chromatograms,should be available for regulatory authorities.The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data (Plasma)— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Designunder General Guidances.
Diuretic Effects and Bumetanide Urinary Excretion Data— Urine samples may be analyzed to determine the following parameters:
  1. The mean rate of excretion of sodium,potassium,water,and bumetanide for each collection period.
  2. The maximum excretion rate (excretion rate calculated for that time interval during which the rate is highest)for sodium,potassium,water,and bumetanide.
  3. Time to maximum excretion rate for sodium,potassium,water,and bumetanide.
  4. Cumulative excretion of sodium,potassium,water,and bumetanide over all sampling time intervals.
Clinical Report,Side Effects,and Adverse Reactions— Subject medical histories,physical examinations and laboratory reports,and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test product and the reference standard used to perform the in vivo bioequivalence studies for approval of the application.Each reserve sample should consist of at least 200dosage units and should be retained for a period of not less than 5years following approval of the application.For more information on retention of bioequivalence samples,refer to 21CFR320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12dosage units of the test product and the reference product,employing the biostudy lots.The current official USPdissolution method (see Dissolution á711ñ)should be followed and should be referenced by the sponsor.The following method and tolerances are currently recommended for this product:
Medium: water;900mL.
Apparatus 2: 50rpm.
Times: 10,20,30,and 45minutes.
Procedure— Use USPif available,or other validated method (see Validation of Compendial Methods á1225ñ).
Tolerances (Q): not less than 85%in 30minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit.The mean percentage dissolved,the range (highest,lowest)of dissolution,and the coefficient of variation (relative standard deviation)should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 0.5-and 1.0-mg strengths of the generic product may be granted pursuant to 21CFR320.22(d)(2),provided the following conditions are met:
  1. The 0.5-and 1-mg tablets are proportionally similar in both active and inactive ingredients to the 2-mg tablet,which has been demonstrated to be bioequivalent to the reference 2-mg product in vivo.
  2. The 0.5-and 1-mg tablets of the generic product meet dissolution test requirements.

Buspirone Hydrochloride Tablets—

In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Pharmacology/Usage— Buspirone hydrochloride is an anti-anxiety agent.Clinically it is effective in the management of anxiety disorders or short-term relief of symptoms of anxiety.Buspirone has no anticonvulsant or muscle relaxant activity and has little sedative effect.It does not substantially affect psychomotor function.There is no evidence that the drug causes either physical or psychological dependence.The mechanism of action of buspirone is not known.Some in vitro preclinical studies indicate that buspirone has a high affinity for serotonin (5-HT1A)receptors and a moderate affinity for brain D2receptors.
For the management of anxiety disorders,the usual initial adult dosage of buspirone is 10to 15mg daily,usually in 2or 3divided doses.Dosage is increased,as necessary,in increments of 5mg daily to achieve an optimal therapeutic response.The maximum daily dose should not exceed 60mg per day.
Pharmacokinetics— Buspirone is rapidly and almost completely absorbed from the GItract.The drug undergoes extensive first-pass metabolism,with about 4%of a dose reaching the systemic circulation unchanged following oral administration.Following oral administration of a single dose of 20mg in healthy volunteers,peak plasma buspirone concentrations of 1to 6ng per mLhave been observed to occur within 40to 90minutes.Plasma concentrations of unchanged buspirone are low and exhibit substantial interindividual variation with oral administration of the drug.Approximately 95%of buspirone is bound to plasma proteins.
Buspirone is rapidly metabolized by oxidation to produce several hydroxylated derivatives and a pharmacologically active metabolite,1-pyrimidinylpiperazine(1-PP).Because of rapid metabolism,less than 1%of the parent drug is excreted unchanged in the urine.The pharmacologically active metabolite has about 20%to 25%of the anxiolytic activity of buspirone.In humans,blood concentrations of 1-PPremain low even after chronic administration of buspirone.The contribution of 1-PPto the pharmacologic and toxic effect thus remains to be elucidated fully.
The average elimination half-life of unchanged buspirone after single doses of 10to 40mg is reported to be 2to 3hours.Buspirone exhibits linear kinetics following administration of doses (10to 40mg)in the therapeutic range.Although food increases the bioavailability of buspirone by decreasing first-pass metabolism,the total amount of drug (changed and unchanged)in plasma is not affected.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Buspar®(Bristol-Meyers Squibb)10-mg tablet.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size at least 10%that of the largest lot planned for full production or a minimum of 100,000units,whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. Asingle-dose,randomized,two-period,two-treatment,two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. Asingle-dose,randomized,three-treatment,three-period,six-sequence crossover,limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.
Recommended Protocol for Conducting a Single-Dose,Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of buspirone and 1-PPfrom a generic formulation with that from a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose,two-treatment,two-period,two-sequence crossover with a 1-week washout period between Phase Iand Phase IIdosing or a single-dose,two-treatment,four-period,four-sequence replicate design study with a 1-week washout period between each phase of dosing.An equal number of subjects should be randomly assigned to each of the two possible dosing sequences.Before the study begins,the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names,titles,and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study.Subjects should be healthy,preferably nonsmoking,volunteers aged 18to 55and within 10%of ideal body weight for height and build,although within 15%of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin,1983).Subjects should be selected on the basis of acceptable medical history,physical examination,and clinical laboratory test results.Female subjects must be given a pregnancy test prior to beginning the study.Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study.If smokers are included,they should be identified as such.Written,informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10hours,subjects should be administered four 10-mg tablets of the test or reference product with 240mLof water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken,except for 1hour before and after drug administration,when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4hours after administration of the test or reference treatment.All meals should be standardized during the study,and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications,including oral contraceptives,beginning 2weeks and no OTCmedications beginning 1week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0hours)and at 0.25,0.50,0.75,1,1.5,2,2.5,3,4,6,7,8,12,and 24hours post-dose.Plasma should be separated promptly and immediately frozen until assayed.Following a minimum 1-week washout period,subjects should begin the second phase of the study.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4hours of the study.Subjects with a heart rate less than 45bpm or greater than 110bpm should have an electrocardiogram (lead II)performed and have their pulse monitored hourly.Subjects should report any unusual symptoms observed during the study.Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Analytical Methods— Buspirone and 1-PPshould be assayed using a suitable method fully validated with respect to adequate sensitivity,specificity,linearity,recovery,accuracy,and precision (both within and between days).Stability of the samples under frozen conditions,at room temperature,and during freeze-thaw cycles,if appropriate,should be determined.Chromatograms of the analysis of the unknown samples,including all associated standard curve and quality control chromatograms,should be available for regulatory authorities.The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Designunder General Guidances.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of buspirone and 1-PPfrom a generic formulation with that from a reference formulation under nonfasting conditions and to compare the rate and extent of absorption of the drug from a generic formulation under fasting and nonfasting conditions when given as equal labeled doses.
Design— The study design is a single-dose,three-treatment,three-period,six-sequence crossover with a 1-week washout period between Phase Iand Phase II,and Phase IIand Phase III.
The limited food effects study (with a minimum of 12subjects)should be performed in the same manner as the single-dose study under fasting conditions,with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6dosing sequences possible in a three-treatment,three-period study design.Each subject will receive the following treatments:
TREATMENT1: Generic product,four 10-mg tablets administered after a standard high-fat breakfast.
TREATMENT2: Reference product,four 10-mg Buspar®tablets administered after a standard high-fat breakfast.
TREATMENT3: Generic product,four 10-mg tablets administered under fasting conditions.
Following a 10-hour fast,the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast.The subjects should have 30minutes to finish the entire breakfast and then should immediately receive Treatment 1or Treatment 2,taken with 240mLof water.Subjects receiving the treatment under fasting conditions should receive Treatment 3,taken with 240mLof water only.The same lots of the test and reference products should be used in the fasted and fed studies.No food should be allowed for at least 4hours post-dose,with water allowed after the first hour.Subjects should be served scheduled standardized meals throughout the study,and the same meals should be served during all phases of the study.
Statistical Analysis— In general,a comparable food effect will be assumed if the AUC0–T,AUC0–¥,and Cmaxmean values for the test product administered under fed conditions are within 20%of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report,Side Effects,and Adverse Reactions— Subject medical histories,physical examinations and laboratory reports,and all incidents of possible adverse reactions and side effects to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application.Each reserve sample should consist of at least 200dosage units or 5times the amount needed for complete release testing,whichever is greater,and should be retained for a minimum of 5years following approval of the application.For more information on retention of bioequivalence samples,refer to 21CFR320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12dosage units of the test and reference products,employing the biostudy lots.The current official USPdissolution method (see Dissolution á711ñ)should be followed and should be referenced by the sponsor.The following times should be used to determine the dissolution profile.
Times: 10,20,30,and 45minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit.The mean percentage dissolved,the range of dissolution (highest,lowest),and the coefficient of variation (relative standard deviation)should be reported.
Content Uniformity Test— Content uniformity testing on the test and reference product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 5-mg strength of the generic product may be granted pursuant to 21CFR320.22(d)(2)provided the following conditions are met:
  1. The 5-mg tablet is proportionally similar in both active and inactive ingredients to the firm's 10-mg tablet,which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 5-mg tablet of the generic product meets dissolution test requirements.

Captopril Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Captopril,an antihypertensive,inhibits the enzyme that converts angiotensin I,a relatively inactive decapeptide,to angiotensin II,a potent endogenous vasoconstrictor substance.This enzyme,angiotensin converting enzyme,or ACE,is a peptidyldipeptide carboxy hydrolase.
The recommended initial dosage for treatment of hypertension is 25mg twice a day (b.i.d.)or three times a day (t.i.d.).This can be increased to 50mg b.i.d.or t.i.d.after 1or 2weeks if the lower dose is ineffective.Doses of captopril higher than 50mg b.i.d.are recommended only with concomitant administration of a thiazide diuretic.
Pharmacokinetics— Following oral administration,approximately 60%to 75%of the dose of captopril is rapidly absorbed from the gastrointestinal tract in fasting healthy adults or hypertensive patients.Peak blood levels of unchanged captopril occur about 1hour after oral administration.Areas under the concentration-time curve and maximum blood concentrations after single oral doses of captopril appear to be dose-related over a range of 10to 100mg.Approximately 25%to 30%of the drug in the systemic circulation is bound to plasma proteins.
Because the presence of food in the GItract is reported to reduce absorption of the drug by 30%to 40%,captopril is labeled to be dosed 1hour before meals.Blood pressure reduction is usually maximal 60to 90minutes post-dose.The elimination half-life of captopril is reported to be about 2hours.
About half the absorbed dose of captopril is rapidly metabolized,mainly to captopril-cysteine disulfide and to the disulfide dimer of captopril.Captopril and its metabolites are excreted in the urine.Renal excretion of unchanged captopril occurs via tubular secretion.In patients with normal renal function,more than 95%of the absorbed dose is excreted in the urine in 24hours.About 40%to 50%of the drug excreted in the urine is unchanged captopril.
IN VIVO BIOEQUIVALENCE STUDY5
Product Information—
  1. FDA Designated Reference Product:Capoten®(Squibb)100-mg tablets.
  2. Batch Size—The test batch or lot must be manufactured under production conditions and must be of a size at least 10%that of the largest lot planned for full production or a minimum of 100,000units,whichever is larger.
  3. Potency—The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Type of Study Required— Asingle-dose,randomized,two-period,two-treatment,two-sequence crossover study under fasting conditions comparing 100-mg doses of the test and reference products is required.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study Under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose,two-treatment,two-period,two-sequence crossover with a 1-week washout period between Phase Iand Phase IIdosing.An equal number of subjects should be randomly assigned to each of the two possible dosing sequences.Before the study begins,the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names,titles,and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study.It is recommended that a minimum of 24subjects be used in this study.Subjects should be healthy volunteers aged 18to 50years and within 10%of ideal body weight for height and build,although within 15%of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin,1983).Subjects should be selected on the basis of acceptable medical history,physical examination,and clinical laboratory test results.Female subjects must be given a pregnancy test prior to beginning the study.Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study.Written,informed consent must be obtained from all study participants before they are accepted into the studies.
Procedure— Following an overnight fast of at least 10hours,subjects should be given a single 100-mg dose of the test or reference product with 240mLof water.Subject blood pressure and pulse rate should be measured immediately before drug administration (pre-dose)and at 0.5,1,1.5,2,3,4,6,and 8hours post-dose.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1hour before and after drug administration,when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4hours after administration of the test or reference treatment.All meals should be standardized during the study,and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications,including oral contraceptives,beginning 2weeks and no OTCmedications beginning 1week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0hours)and at 0.25,0.50,0.75,1,1.25,1.50,1.75,2,2.50,3,3.504,4.50,5,6,7,8,and 10hours post-dose.Whole blood or blood plasma may be used for the assay.In either case,the blood samples should be chemically stabilized immediately after collection (see Analytical Methodsbelow).If plasma is to be assayed,it should be separated promptly from the stabilized whole blood.The whole blood or blood plasma samples should then be immediately frozen until assayed.Following a minimum 1-week washout period,subjects should begin the second phase of the study.
Analytical Methods— Captopril should be measured in an appropriate biological matrix such as whole blood or blood plasma.Several assay methods are available.The method chosen should be fully validated with respect to adequate sensitivity,specificity,linearity,recovery,and accuracy and precision (both within and between days).Because the thiol group of captopril is readily oxidized,a chemical stabilizer must be added to the blood samples immediately after collection.Stability of the samples under frozen storage conditions,at room temperature,and during freeze-thaw cycles,if appropriate,should be determined.Chromatograms of the analysis of the unknown samples,including all associated standard curve and quality control chromatograms,should be available for regulatory authorities.The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Designunder General Guidances.
Clinical Report,Side Effects,and Adverse Reactions— Subject medical histories,physical examinations and laboratory reports,and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application.Each reserve sample should consist of at least 200dosage units and should be retained for a period of not less than 5years following approval of the application.For more information on retention of bioequivalence samples,refer to 21CFR320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12dosage units of the test product versus 12units of the reference product.The biostudy lots should be used for those product strengths tested in vivo.The current official USPdissolution method (see Dissolution á711ñ)should be followed and should be referenced by the sponsor.The following method and tolerances are currently recommended for this product:
Medium: 0.1Nhydrochloric acid;900mL.
Apparatus 1: 50rpm.
Times: 10,20,and 30minutes.
Procedure— Use USPif available,or other validated method (see Validation of Compendial Methods á1225ñ).
Tolerances (Q): not less than 80%in 20minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit.The mean percentage dissolved,the range (highest,lowest)of dissolution,and the coefficient of variation (relative standard deviation)should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 12.5-,25-,and 50-mg strengths of the generic product may be granted pursuant to 21CFR320.22(d)(2),provided the following conditions are met:
1.The lower strengths are proportionally similar in both active and inactive ingredients to the 100-mg strength,which has been demonstrated to be bioequivalent to the reference product in vivo.
2.The 12.5-,25-,and 50-mg strengths of the generic product meet the dissolution test requirements.

Cefaclor Capsules and Suspensions—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Cefaclor is a cephalosporin antibiotic that inhibits bacterial cell-wall synthesis in a manner similar to that of penicillin.Cefaclor is used in the treatment of otitis media caused by susceptible Streptococcus pneumoniae,Haemophilus influenzae(including ampicillin-resistant strains),staphylococci,and group Ab-hemolytic streptococci;of lower respiratory tract infections (including pneumonia)caused by susceptible Streptococcus pneumoniae,Haemophilus influenzae,or group Ab-hemolytic streptococci;of upper respiratory tract infections (including pharyngitis and tonsillitis)caused by susceptible group Ab-hemolytic streptococci;of urinary tract infections (including pyelonephritis and cystitis)caused by susceptible Escherichia coli,Proteus mirabilis,Klebsiella,or staphylococci;or of skin and skin structure infections caused by susceptible Staphylococcus aureusor group Ab-hemolytic streptococci.
The usual adult dosage is 250mg every 8hours.For more severe infections (such as pneumonia),doses may be doubled.
Pharmacokinetics— Cefaclor is well absorbed after oral administration to fasting subjects.Total absorption is similar in the fasted and the fed states.When it is taken with food,the peak concentration achieved is 50%to 75%of that observed in fasting subjects and generally appears about 1hour later.
Following administration of 250-mg,500-mg,and 1-g doses to fasting subjects,average peak serum levels of approximately 7,13,and 23µg per mL,respectively,were obtained within 30to 60minutes.Approximately 60%to 85%of the drug is excreted unchanged in urine within 8hours,the major portion being excreted within the first 2hours.The serum elimination half-life in subjects with normal renal function is 0.6to 0.9hour.In patients with severely reduced renal function,the plasma elimination half-life of the drug is 2.3to 2.8hours.Hemodialysis shortens the half-life by 25%to 30%.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
  1. FDA Designated Reference Product:Ceclor®(Eli Lilly)500-mg capsules;or powder for oral suspension containing 375mg per 5mL(Eli Lilly).
  2. Batch Size—The test batch or lot must be manufactured under production conditions and must be of a size at least 10%that of the largest lot planned for full production or a minimum of 100,000capsules,whichever is larger.
  3. Potency—The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. Asingle-dose,randomized,two-period,two-treatment,two-sequence crossover study under fasting conditions comparing 500-mg doses of the test and reference capsules and 375-mg-per-5-mLdoses of the test and reference suspensions is required.
  2. Asingle-dose,randomized,three-treatment,three-period,six-sequence,crossover,limited food effects study comparing equal doses of the test product under fasting conditions as well as the test and reference products when administered immediately following a standard high-fat breakfast6is required.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose,two-treatment,two-period,two-sequence crossover with a 1-week washout period between Phase Iand Phase IIdosing.An equal number of subjects should be randomly assigned to the two possible dosing sequences.Before the study begins,the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names,titles,and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study.It is recommended that a minimum of 24subjects be used in this study.Subjects should be healthy volunteers aged 18to 50years and within 10%of ideal body weight for height and build,although within 15%of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin,1983).Subjects should be selected on the basis of acceptable medical history,physical examination,and clinical laboratory test results.Female subjects must be given a pregnancy test prior to beginning the study.Subjects with any current or past medical condition,including sensitivity to cephalosporin or penicillin analogues,that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study.Written,informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10hours,subjects should be administered a single dose of the test or reference product with 240mLof water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4hours after administration of the test or reference treatment.All meals should be standardized during the study,and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications,including oral contraceptives,or OTCmedications beginning 2weeks before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0hours)and at 0.25,0.50,0.75,1.0,1.25,1.50,2,3,4,5,6,and 8hours post-dose.Due to the chemical instability of cefaclor,the blood samples should be analyzed as soon as they are drawn,or plasma samples should be promptly frozen at –70until analysis.Following a minimum 1-week washout period,subjects should begin Phase II.For each subject,the sponsor should state the time elapsed between sample collection and its assay.An explanation should be given for any missing samples.
Analytical Methods— For the measurement of cefaclor in plasma samples,a microbiological assay,HPLCmethod,or other suitable method should be selected.The method used should be described in detail,and references,if any,should be cited.The method should include detailed calculation procedures for the assay results.The method chosen should be fully validated with respect to adequate sensitivity,specificity,linearity,recovery,and accuracy and precision (both within and between days).Stability of the analyte in plasma samples under frozen conditions,at room temperature,and during freeze-thaw cycles,if appropriate,should be determined.It should be noted that cefaclor is most stable in the acid pHrange at a temperature of 4.For analytical work,cefaclor reference standard stock solutions should be prepared fresh daily in pH4.5buffer.
Chromatograms of the analysis of the unknown samples,including all associated standard curve and quality control chromatograms,should be available for regulatory authorities.The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Designunder General Guidances.
Limited Food Effects Study— The limited food effects study should be performed in the same manner as the single-dose study under fasting conditions,with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6dosing sequences possible in a three-treatment,three-period study design (total 18subjects).Each subject will receive the following treatments:
TREATMENT1: Generic product,a 500-mg cefaclor capsule or 375-mg-per-5-mLsuspension administered after a standard high-fat breakfast.
TREATMENT2: Reference product,a 500-mg Ceclor®capsule or 375-mg-per-5-mLsuspension (Eli Lilly)administered after a standard high-fat breakfast.
TREATMENT3: Generic product,a 500-mg cefaclor capsule or 375-mg-per-5-mLsuspension administered under fasting conditions.
Following a 10-hour fast,the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast.The subjects should have 30minutes to finish the entire breakfast,and then should immediately receive Treatment 1or Treatment 2,taken with 240mLof water.Subjects receiving the treatment under fasting conditions should be dosed with Treatment 3,taken with 240mLof water only.The same lots of the test and reference products should be used in the fasted and fed studies.No food should be allowed for at least 4hours post-dose with water allowed after the first hour.Subjects should be served scheduled standardized meals throughout the study,and the same meals should be served during both phases of the study.
Statistical Analysis— In general,a comparable food effect will be assumed if the AUC0–T,AUC0–¥,and Cmaxmean values for the test product administered under fed conditions are within 20%of the respective mean values obtained for the reference product administered under fed conditions.
Clinical Report,Side Effects,and Adverse Reactions— Subject medical histories,physical examinations and laboratory reports,and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application.Each reserve sample should consist of at least 200dosage units and should be retained for a minimum of 5years following approval of the application.For more information on retention of bioequivalence samples,refer to 21CFR320.63.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12dosage units of the test product versus 12units of the reference product.The biostudy lots should be used for those product strengths tested in vivo.The current official USPdissolution method (see Dissolution á711ñshould be followed and should be referenced by the sponsor.The following method and tolerances are currently recommended for this product:
Medium: water;900mL.
Apparatus 2: 50rpm.
Times: 10,20,30,and 45minutes.
Procedure— Use USPif available,or other validated method (see Validation of Compendial Methods á1225ñ).
Tolerance (Q) not less than 80%in 30minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit.The mean percentage dissolved,the range (highest,lowest)of dissolution,and the coefficient of variation (relative standard deviation)should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 250-mg strength of the generic capsule may be granted pursuant to 21CFR320.22(d)(2),provided the following conditions are met:
  1. The 250-mg capsule is proportionally similar in both active and inactive ingredients to the firm's 500-mg capsule,which has been demonstrated to be bioequivalent to the reference product in vivo.
  2. The 250-mg capsule of the generic product meets dissolution test requirements.
Waiver of in vivo bioequivalence study requirements for the powder for reconstitution as a suspension for oral administration,125-,187-,and 250-mg-per-5-mLstrengths of the generic product may be granted pursuant to 21CFR320.22(d)(2)provided the following conditions are met:
  1. The 125-,187-,and 250-mg-per-5-mLsuspensions are proportionally similar in their active and inactive ingredients to the firm's 375-mg-per-5-mLsuspension.
  2. An acceptable in vivo bioequivalence study has been conducted for the 375-mg-per-5-mLsuspension.

Cimetidine Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Cimetidine is a histamine H2-receptor antagonist,used for the treatment of endoscopically or radiographically confirmed duodenal ulcer,pathologic GIhypersecretory conditions (e.g.,Zollinger-Ellison syndrome,systemic mastocytosis,and multiple endocrine adenomas),and active,benign,gastric ulcer.
Cimetidine inhibits competitively and selectively the interaction of histamine with H2receptors.It inhibits basal (fasting)and nocturnal acid secretion,acid secretion stimulated by food,sham feeding,fundic distention,and various pharmacological agents.
Effective cimetidine concentrations between 0.5and 1.0µg per mLare required to suppress gastric acid secretion under basal or stimulated conditions.However,no correlation to the therapeutic response from pharmacokinetic data has been established between plasma concentrations of cimetidine or any of the pharmacodynamic parameters and duodenal ulcer healing rate.
For treatment of active duodenal ulcer,the usual adult oral dosage of cimetidine is 800mg daily at bedtime.For maintenance therapy following healing of acute duodenal ulcer,the usual oral dosage of cimetidine is 400mg daily at bedtime.For the treatment of pathologic hypersecretory conditions,the usual adult oral dosage is 300mg,4times daily with meals and at bedtime.For the treatment of active benign gastric ulcer,the usual adult oral dosage is 800mg at bedtime or 300mg,4times daily with meals and at bedtime.
Pharmacokinetics— Following intravenous administration,the plasma concentration profile follows multicompartmental characteristics.The total systemic clearance is high (500to 600mLper minute)and is mainly determined by renal clearance.The volume of distribution is of the order of 1Lper kg.Elimination half-life is approximately 2hours.Following oral administration of cimetidine,2plasma concentration peaks are frequently observed at about 1hour and after about 3hours,probably due to discontinuous absorption in the intestine or individual variation in gastric emptying (but not enterohepatic recycling since the biliary excretion rate in humans is less than 2%).The absolute bioavailability is about 60%in healthy subjects and around 70%in peptic ulcer patients.Absorption and clearance of cimetidine are linear after 200-and 800-mg doses.When given with food,the extent of absorption of the drug remains unchanged,but the time to reach the maximum peak concentration is delayed with only 1peak in the plasma concentration curve observed at about 2hours following dosing.Plasma protein binding of cimetidine is 20%and does not significantly affect the pharmacokinetics of the drug.Cimetidine distributes extensively into the kidney,lung,and muscle tissue but less than 1%into the cerebrospinal fluid.
Following IVadministration,about 50%to 80%of the dose is recovered in urine as unchanged cimetidine.This fraction is less after oral doses.Approximately 2%of cimetidine is excreted in the bile.About 25%to 40%of the cimetidine dose is eliminated as metabolites,formed mainly in the liver.The metabolites are sulfoxide and 5-hydroxymethyl derivatives,and possibly guanylurea,although this latter compound may result from in vitro degradation.Elimination of cimetidine is accelerated in the presence of phenobarbital due to induction of its metabolism.Clearance of the drug is higher in children,who have greater renal elimination rates.With increasing age,the volume of distribution of the drug decreases,total plasma clearance decreases as a function of decreasing renal clearance,and plasma half-life increases.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
  1. FDA Designated Reference Product:Tagamet®(SmithKline Beecham)800-mg tablets.
  2. Batch Size—The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10%of the largest lot planned for full production or a minimum of 100,000units,whichever is larger.
  3. Potency—The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. Asingle-dose,randomized,fasting,two-period,two-treatment,two-sequence crossover study comparing the 800-mg strength of the test and reference products.This protocol may also be used for lower strength tablets if an 800-mg tablet is not being manufactured.In this case,equal doses of the test and reference products should be used.
  2. Asingle-dose,two-way crossover,full food study comparing the 800-mg strength of the test and reference products.Due to the double-peak phenomenon,which is only observed under fasting conditions,the food study may be more reliable for determining the bioequivalency of the test product and therefore should be done with a full complement of subjects.This protocol may also be used for lower strength tablets if an 800-mg tablet is not being manufactured.In this case,equal doses of the test and reference products should be used.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study under Fasting Conditions—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose,two-treatment,two-period,two-sequence crossover with a 1-week washout period between Phase Iand Phase IIdosing.An equal number of subjects should be randomly assigned to the two possible dosing sequences.Before the study begins,the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names,titles,and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study.It is recommended that a minimum of 24subjects be used in this study.Subjects should be healthy volunteers aged 18to 50years and within 10%of ideal body weight for height and build,although within 15%of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin,1983).Subjects should be selected on the basis of acceptable medical history,physical examination,and clinical laboratory test results.Female subjects must be given a pregnancy test prior to beginning the study.Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study.Written,informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10hours,subjects should be administered a single 800-mg dose of the test or reference product with 240mLof water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1hour before and after drug administration,when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4hours after administration of the test or reference treatment.All meals should be standardized during the study,and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medications,including oral contraceptives,or OTCmedications beginning 2weeks before drug administration until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0hours)and at 0.25,0.50,0.75,1.0,1.50,2.0,2.50,3.0,3.50,4.0,5.0,6.0,8.0,10,12,16,20,and 24hours post-dose.Plasma should be separated promptly and immediately frozen until assayed.Following at least a minimum 1-week washout period,subjects should begin the second phase of the study.
Analytical Methods— Cimetidine should be assayed using a suitable method fully validated with respect to adequate sensitivity,specificity,linearity,recovery,accuracy,and precision (both within and between days).Stability of the samples under frozen conditions,at room temperature,and during freeze-thaw cycles,if appropriate,should be determined.Chromatograms of the analysis of the unknown samples,including all associated standard curve and quality control chromatograms,should be available for regulatory authorities.The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Statistical Analysis of Pharmacokinetic Data: See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Designunder General Guidances.
Clinical Report,Side Effects,and Adverse Reactions— Subject medical histories,physical examinations and laboratory reports,and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain an appropriately identified reserve sample of both the test and reference products used to perform the in vivo bioequivalence study for approval of the application.Each reserve sample should consist of at least 200dosage units and should be retained for a period of not less than 5years following approval of the application.For more information on retention of bioequivalence samples,refer to 21CFR320.63.
Recommended Protocol for Conducting a Single-Dose Bioequivalence Study Under Postprandial Conditions— There should be at least 24healthy volunteers.The postprandial study should be conducted in the same manner as described for the fasting study except under fed conditions.The subjects should be given a standard high-fat breakfast6after an overnight fast of approximately 10hours.The dose should be given with 240mLof water approximately 30minutes after beginning breakfast.The plasma cimetidine data should be obtained and analyzed in the same manner as described for the fasting study.Due to the double-peak phenomenon,which is only observed under fasting conditions,the postprandial study may be more reliable for determining the bioequivalency of a test product.
IN VITRO TESTING REQUIREMENTS
Dissolution Testing— Conduct dissolution testing on 12dosage units of the test product versus 12units of the reference product.The biostudy lots should be used for those product strengths tested in vivo.The current official USPdissolution method (see Dissolution á711ñ)should be followed and should be referenced by the sponsor.The following method and tolerances are currently recommended for this product:
Medium: deaerated water;900mL.
Apparatus 1: 100rpm.
Times: 10,15,and 30minutes.
Procedure— Use USPif available,or other validated method (see Validation of Compendial Methods á1225ñ).
Tolerances (Q): not less than 75%in 15minutes.
The percentage of label claim dissolved at each specified testing interval should be reported for each individual dosage unit.The mean percentage dissolved,the range (highest,lowest)of dissolution,and the coefficient of variation (relative standard deviation)should be reported.
Content Uniformity Test— Content uniformity testing on the test product lots should be performed as described in USP.
WAIVER REQUIREMENTS
Waiver of in vivo bioequivalence study requirements for the 200-,300-,and 400-mg strengths of the generic product may be granted pursuant to 21CFR320.22(d)(2),provided the following conditions are met:
  1. The 200-,300-,and 400-mg strengths are proportionally similar in both active and inactive ingredients to another strength (800mg),which has been demonstrated to be bioequivalent to a reference (800mg)product in vivo.
  2. The 200-,300-,and 400-mg strengths of the generic product meet all applicable compendial requirements,including the specified dissolution and content uniformity testing requirements.
  3. The drug product follows linear kinetics over its range of available strengths.

Diclofenac Sodium Delayed-Release Tablets—In Vivo Bioequivalence and In Vitro Dissolution Testing4
Clinical Usage/Pharmacology— Diclofenac sodium is an orally administered nonsteroidal anti-inflammatory drug (NSAID),which also has analgesic and antipyretic properties.Currently approved indications for diclofenac sodium are for the acute or chronic treatment of the signs and symptoms of rheumatoid arthritis (RA),osteoarthritis,and ankylosing spondylitis.Doses above 200mg a day in 3or 4divided doses have not been studied in RApatients.Diclofenac sodium inhibits prostaglandin synthesis,which may be involved in its mechanism of action.Due to possible cross-reactivity,diclofenac sodium is contraindicated in patients in whom aspirin or other NSAIDhas produced asthma,urticaria,or allergic reactions.
Pharmacokinetics— Diclofenac sodium is rapidly absorbed following oral administration with reported time of maximum concentration (Tmax)mean values of 1to 3hours and ranges of 1to 5hours under fasting conditions in normal volunteers.After single oral doses of 25-,50-,and 75-mg delayed-release tablets,reported maximum concentrations (Cmax)in normal fasting subjects were 0.5to 1,0.9to 1.5,and 1.9to 2µg per mL,respectively.Area under the plasma concentration-time curve (AUC)increases linearly over the dose range 25to 150mg;however,Cmaxis less than dose-proportional with values of 1,1.5,and 2µg per mLafter doses of 25,50,and 75mg,respectively.
Diclofenac sodium undergoes first-pass metabolism to give a systemic availability of 50%to 60%.Food may markedly delay the rate of absorption from delayed-release tablets but does not appear to change AUCsignificantly.In two single-dose (50mg)studies,the nonfasting mean Tmaxvalues were 5.4hours (N=12,range 2.5to 12hours)and 9.7hours (N=6,range 8to 10hours).The volume of distribution (Vd)of diclofenac sodium is about 0.12to 0.17Lper kg and it is greater than 99%bound to plasma proteins.After an IVdose,elimination of diclofenac sodium from plasma appears triphasic;after oral dosing,reported terminal b-phase half-life (t½)values are 1to 2hours with a range of 0.5to 4.3hours.The apparent elimination t½of total radiolabeled compounds in patients with normal renal function is 25to 33hours.
The major route of elimination is hepatic clearance with 90%of a dose eliminated in 96hours with about 65%of the dose in urine and 35%of the dose in bile.The dose is excreted as glucuronide and sulfate conjugates of unchanged drug and four metabolites.The principal metabolite (4¢-hydroxy)has about 1/40the activity of the parent drug in animal models of arthritis.It is possible that this metabolite may contribute to the overall activity because it accounts for 30%to 40%of the dose.
IN VIVO BIOEQUIVALENCE STUDIES5
Product Information—
FDA Designated Reference Product: Voltaren®(Geigy)75-,50-,or 25-mg delayed-release oral tablets.
Batch Size— The test batch or lot must be manufactured under production conditions and must be of a size that is at least 10%that of the largest lot planned for full production or a minimum of 100,000units,whichever is larger.
Potency— The assayed potency of the reference product should not differ from that of the test product by more than 5%.
Types of Studies Required—
  1. Asingle-dose,randomized,two-period,two-treatment,two-sequence crossover study under fasting conditions comparing equal doses of the test and reference products.
  2. Asingle-dose,randomized,three-treatment,three-period,six-sequence crossover,limited food effects study comparing equal doses of the test and reference products when administered immediately following a standard high-fat breakfast.6
  3. As stated in 21CFR320.22(d)(2)(iv),no waiver may be granted for the 25-or 50-mg strengths based on the 75-mg strength.Separate fasting studies must be performed for all of the strengths.
Recommended Protocol for Conducting a Single-Dose,Fasted Bioequivalence Study—
Objective— The objective is to compare the rate and extent of absorption of a generic formulation with that of a reference formulation when given as equal labeled doses.
Design— The study design is a single-dose,two-treatment,two-period,two-sequence crossover with a 2-week washout period between phases.An equal number of subjects should be randomly assigned to the two possible dosing sequences.Before the study begins,the proposed protocol should be approved by an institutional review board.
Facilities— The clinical facilities and analytical laboratory used for the study should be identified along with the names,titles,and curriculum vitae of the medical and scientific or analytical directors.
Selection of Subjects— The sponsor should enroll a number of subjects sufficient to ensure statistical validity of the study.It is recommended that a minimum of 36subjects be used in this study.Subjects should be healthy,preferably nonsmoking,volunteers aged 18to 50years and within 10%of ideal body weight for height and build,although within 15%of ideal body weight is acceptable (Metropolitan Life Insurance Company Statistical Bulletin,1983).Subjects should be selected on the basis of acceptable medical history,physical examination,and clinical laboratory test results.Female subjects must be given a pregnancy test prior to beginning the study.Subjects with any current or past medical condition that might significantly affect their pharmacokinetic or pharmacodynamic response to the administered drug should be excluded from the study.If smokers are included,they should be identified as such.Written,informed consent must be obtained from all study participants before they are accepted into the study.
Procedure— Following an overnight fast of at least 10hours,subjects should be administered a single dose of the test or reference product with 240mLof water.
Restrictions— Study volunteers should observe the following restrictions:
  1. Water may be taken except for 1hour before and after drug administration when no liquid is allowed other than that needed for drug dosing.
  2. Subjects should fast for at least 4hours after administration of the test or reference treatment.All meals should be standardized during the study,and the same meals should be served during both phases of the study.
  3. No alcohol or xanthine-containing foods or beverages should be consumed for 48hours prior to dosing and until after the last blood sample is collected.
  4. Subjects should take no Rx medication,including oral contraceptives,or OTCmedication beginning 1week before drug administration and until after the study is completed.
Blood Sampling— Venous blood samples should be collected pre-dose (0hours)and at 0.50,0.75,1,1.25,1.50,1.75,2,2.25,2.50,2.75,3,3.33,3.67,4,5,6,8,10,and 12hours post-dose.Plasma serum should be separated promptly and immediately frozen until assayed.Following a 2-week washout period,subjects should begin the second phase of the study.
Analytical Methods— The active ingredient should be assayed using a suitable method fully validated with respect to adequate sensitivity,specificity,linearity,recovery,accuracy,and precision (both within and between days).Stability of the samples under frozen conditions,at room temperature,and during freeze-thaw cycles,if appropriate,should be determined.Chromatograms of the analysis of the unknown samples,including all associated standard curve and quality control chromatograms,should be available for regulatory authorities.The sponsor should justify the rejection of any analytical data and provide a rationale for selection of the reported values.
Subject Monitoring— Blood pressure and pulse rate should be monitored hourly during the first 4hours of the study.Any subject with a heart rate less than 45bpm or greater than 110bpm should have an electrocardiogram (lead II)performed and have their pulse monitored hourly.Subjects should report any unusual symptoms observed during the study.Subjects should be periodically questioned during each phase of the study for any unusual symptoms experienced after drug administration.
Statistical Analysis of Pharmacokinetic Data— See Statistical Procedures for Bioequivalence Studies Using a Standard Two-Treatment Crossover Designunder General Guidances.In addition,the following parameters should be tabulated:lag time (Tlag),the time of the last zero concentration before the first non-zero concentration;and adjusted Tmax(Tmaxadj),which is the observed Tmaxminus Tlag.
Limited Food Effects Study—
Objective— The objective is to compare the rate and extent of absorption of diclofenac sodium from a generic formulation with that from a reference formulation under nonfasting conditions,and to compare the rate and extent of absorption of the drug form from a generic product under fasting and nonfasting conditions when given in equal labeled doses.
Design— The study design is a single-dose,three-treatment,three-period,six-sequence crossover with a 2-week washout period between Phase Iand Phase II,and Phase IIand Phase IIIof dosing.
In view of the wide range of reported values for Tmaxin the presence of food,it is recommended that the sponsor perform a pilot study to determine appropriate sampling times for the limited food effects study.
The limited food effects study (12to 18subjects)should be performed in the same manner as the single-dose fasting study,with the following exceptions:
Procedure— An equal number of subjects should be assigned to each of the 6dosing sequences possible in a three-treatment,three-period study design.Each subject will receive the following treatments:
TREATMENT1: Generic product,a 75-mg tablet,administered after a standard high-fat breakfast.
TREATMENT2: Reference product,a 75-mg Voltaren®tablet,administered after a standard high-fat breakfast.
TREATMENT3: Generic product,a 75-mg tablet,administered under fasting conditions.
Following a 10-hour fast,the subjects receiving treatments under fed conditions should be served a standard high-fat breakfast.The subjects should have 30minutes to finish the entire breakfast,and then should immediately receive Treatment 1or Treatment 2,with 240mLof water.Subjects receiving the treatment under fasting conditions should receive Treatment 3,with 240mLof water.The same lots of the test and reference products should be used in the fasted and fed studies.No food should be allowed for at least 4hours post-dose,with water allowed after the first hour.Subjects should be served scheduled standardized meals throughout the study,and the same meals should be served during all phases of the study.
Statistical Analysis— In general,a comparable food effect will be assumed if the AUC0-t,AUC0-¥,and Cmaxmean values for the test product administered under fed conditions are within 20%of the respective mean values for the reference product administered under fed conditions.
Clinical Report,Side Effects,and Adverse Reactions— Subject medical histories,physical examinations and laboratory reports,and all incidents of possible adverse reactions to the study formulations should be reported.
Retention of Samples— The laboratory conducting the bioequivalence testing should retain appropriately identified reserve samples of both the test and reference products used to perform the in vivo bioequivalence studies for approval of the application.Each reserve sample should consist of at least 200dosage units,and should be retained for a minimum of 5years following approval of the application.For more information on retention of bioequivalence samples,refer to 21CFR320.63.
IN VITRO TESTING REQUIREMENTS
There is currently no official USPdissolution method for a diclofenac sodium dosage form.Atentative method recommended by FDAis described below.
Drug Release Testing— Conduct dissolution testing on 12dosage units of the test product and the reference product,employing the biostudy lots.The following method and tolerances are currently recommended for this product:
Method Bá724ñ—
Medium: 2hours in 0.1Nhydrochloric acid followed by 45minutes in pH6.8,0.05Mphosphate buffer prepared as follows.Dissolve 76g of tribasic sodium phosphate in water to obtain 1000mLof solution.Mix 250mLof this solution with 750mLof 0.1Nhydrochloric acid and,if necessary,adjust with 2Nhydrochloric acid or 2Nsodium hydroxide to a pHof 6.8±0.05;900mL.
Apparatus 2: 50rpm.
Times: ACID STAGE:30,60,and 120minutes;BUFFER STAGE:5,10,20,30,45,and 60minutes.
Procedure— Use USPif available,or other validated method (see Validation of Comp