development of an in vitro method to help predict...

DEVELOPMENT OF AN IN VITRO METHOD TO HELP PREDICT IN VIVO BEHAVIOR OF CONTROLLED RELEASE PRODUCTS

Jill S. Hall

A Thesis Submitted to the University of North Carolina at Wilmington in Partial Fulfillment

of the Requirements for the Degree of Master of Science

Department of Chemistry and Biochemistry

University of North Carolina Wilmington

2009

Approved by

Advisory Committee

___Robert Kieber__________________ _____Elsie Melsopp_____________

_________Ned Martin__________ Chair

Accepted by

______________________________ Dean, Graduate School

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TABLE OF CONTENTS

ABSTRACT ............................................................................................................................ v

ACKNOWLEDGEMENTS................................................................................................... vii

DEDICATION...................................................................................................................... viii

LIST OF TABLES.................................................................................................................. ix

LIST OF FIGURES ................................................................................................................. x

LIST OF EQUATIONS........................................................................................................ xiii

INTRODUCTION ................................................................................................................... 1

Overview.............................................................................................................................. 1

Bioavailability...................................................................................................................... 1

Food Effects on Drug Bioavailability and the Gastrointestinal Tract ................................. 2

In vitro/In vivo correlations ................................................................................................. 3

Controlled or Sustained Release Solid Dosage Form.......................................................... 5

Problems .............................................................................................................................. 6

Relevant Literature .............................................................................................................. 6

MATERIALS AND METHODS .......................................................................................... 12

List of Materials and Equipment ....................................................................................... 12

Materials ........................................................................................................................ 12

Equipment...................................................................................................................... 12

Test Article – Uniphyl® (theophylline, anhydrous) Tablets, 400 mg............................... 13

Apparatus........................................................................................................................... 16

Apparatus 1 and 2 .......................................................................................................... 17

Apparatus 3.................................................................................................................... 18

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Media Preparation.............................................................................................................. 23

Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) ........................... 23

Phosphate Buffer pH 3.0, 6.6, 6.8 (SIF without enzymes) and 7.8 ............................... 24

0.1 N HCl pH 1.2 (SGF without enzymes).................................................................... 24

Acetate Buffer pH 4.5.................................................................................................... 25

Emulsion (Liposyn®III 10% and Intralipid® 30%)...................................................... 25

Sample Analysis – Ultraviolet Spectrophotometer............................................................ 26

Filter Study .................................................................................................................... 27

Standard Preparation and Standard Curves ................................................................... 27

RESULTS AND DISCUSSION............................................................................................ 32

Mathematical Equations .................................................................................................... 32

Equation 1. % Released of active pharmaceutical ingredient (theophylline) ............... 32

Fit Factor (Moore and Flanner, 1996 and US Department of Health and Human

Services, 1997)...................................................................................................................... 33

In vitro/In vivo Correlation ............................................................................................ 33

Identification of Parameters for Apparatus 3..................................................................... 36

Dips/Minute Determination ........................................................................................... 37

Apparatus 3 Screen Selection ........................................................................................ 40

Simulated Fasted Conditions / Apparatus 1 (Baskets) and 2 (Paddles)............................. 41

Simulated Fasted Conditions / Apparatus 3....................................................................... 44

Simulated GI pH............................................................................................................ 44

Range of pH (1.2 – 7.8) ................................................................................................. 49

Simulated Fed Conditions / Apparatus 2 (Paddles)........................................................... 50

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Simulated Fed Conditions / Apparatus 3 ........................................................................... 52

Oil Soak ......................................................................................................................... 52

Emulsion ........................................................................................................................ 53

Comparison of In Vitro Data with Reported In Vivo Data ............................................... 57

Simulated Fasted Conditions ......................................................................................... 58

Simulated Fed Conditions.............................................................................................. 61

CONCLUSIONS ................................................................................................................... 65

REFERENCES ...................................................................................................................... 68

v

ABSTRACT

Drug discovery and development are very important because new and improved drugs are in

demand to fight diseases. Pharmaceutical companies desire the fastest and safest route to bring

new or generic drugs to the market. An analytical method that would predict in vivo behavior

can save time and money in the development of these drugs.

The primary objective of this research was to evaluate the in vitro release of the active

ingredient (theophylline anhydrous) from a controlled release dosage form (Uniphyl® 400 mg

Tablets) using an emulsion (Liposyn® and Intralipid®) intended for use as total parenteral

nutrition as a medium that would simulate in vivo fed conditions utilizing a reciprocating

cylinder testing apparatus (Apparatus 3).

The in vitro data from the emulsion tests in the Apparatus 3 collected in this research were

compared to bioavailability information (in vivo) obtained from the literature (Karim et al., 1985)

using in vitro/in vivo correlation (IVIVC) concepts. Additionally, the results obtained from

simulated fasted and fed in vitro profiles in the Apparatus 3 were compared to the dissolution

profiles from an Apparatus 2.

In both the 10% (Liposyn®) and 30% (Intralipid®) fat content emulsions studied, good

correlations with reported in vivo data (0.980 – 0.989) were obtained. The higher soybean oil

content in Intralipid® 30% did not have a significant effect on the release profile. The

Intralipid® was slightly more difficult to work with therefore, 10% fatty emulsion is

recommended for future studies.

Another method evaluated to simulate fed conditions was Maturu’s oil soak method (Maturu

et al., 1986 utilized an Apparatus 2 for their studies). The data correlated well with reported in

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vivo data (R2 = 0.984), however the oil soak in vitro profile began to show a significant increase

after 9 hours whereas the emulsion profiles began to show a significant increase after 5 hours.

Simulated fasted conditions were mimicked by using the compendial simulated gastric fluid

(SGF) and simulated intestinal fluid (SIF) without enzymes. The correlation with reported in

vivo data produced good correlations [0.967 (pH 1.2) and 0.934 (pH 6.8)] and were similar to the

correlation results from the Apparatus 2 test (R2 = 0.964).

Also an evaluation of pH dependability (tests included pH 1.2 – 7.8) of Uniphyl® Tablets

was conducted in the Apparatus 3. Results from the tests performed in the Apparatus 3 shows

that the release rate and amount released of Uniphyl® Tablets is not affected by changing the

pH. Fit factors were calculated for comparing the SGF (pH 1.2) and SIF (pH 6.8) curves (f1 = 10

and f2 = 67) demonstrating their similarity and Uniphyl® Tablets are pH independent when

tested in an Apparatus 3. Additionally, a dissolution test was performed which varied the pH

from pH 1.2 – 7.8 over the course of 12 hours to mimic the pH of the GI tract from the stomach

to the colon (Figure 1). No change in the release profile was noted when comparing the profile

to the release profile in pH 1.2 and pH 6.8 over 24 hours.

Results from this research show that emulsions intended for IV administration for parental

nutrition (Liposyn® and Intralipid®) can predict in vivo behavior of controlled release drugs

tested in an Apparatus 3 (reciprocating cylinder) and also in an Apparatus 2 (paddles).

Additionally, this research demonstrated that an Apparatus 3 may be more applicable for

controlled release dosage forms utilizing a cellulose matrix release system than an Apparatus 1

or 2.

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ACKNOWLEDGEMENTS

I would like to thank my committee members, Dr. Melsopp, Dr. Martin, and Dr. Kieber for

their commitment and assistance in seeing this work through to completion. Many thanks go out

to Dr. Tyrell for his role as liaison for the AAI students, assistance in registering for classes,

following up on current goals and accomplishments, and listening to me practice my thesis

defense.

To my formulation colleagues, thanks for allowing me to continue to take up valuable bench

top space in the main lab. To both formulation and analytical colleagues, thank you for your

assistance with questions and road blocks and taking the time to discuss aspects of my research.

I would like to thank my family and friends for the constant encouragement through the

years to “just finish” and for their many prayers during my defense.

And most importantly to AAIPharma for the opportunity to allow me to further pursue my

education in science.

viii

DEDICATION

This work is dedicated to my wonderful children, Bree and Ty.

ix

LIST OF TABLES

Table Page

1. Relevant literature evaluating the effects of food and GI tract physical stresses on controlled release dosage forms........................................................................................... 9 2. Chemical and biological properties of theophylline .......................................................... 14 3. In vivo % absorbed for Uniphyl® Tablets from Karim et al., 1985. ................................. 34 4. In vivo % absorbed for Uniphyl® Tablets from Maturu et al., 1986................................. 35 5. In-vitro results for apparatus 3 dips/minute determination for Uniphyl® Tablets. ........... 37 6. Comparison of FDA accepted data and specifications for Uniphyl® Tablets collected from the SBA and JSH1-009. ...................................................................................................... 44 7. Apparatus 3 tests utilizing media of varying pH to simulate the conditions of the gastrointestinal tract under fasted conditions. .............................................................................. 45 8. In vitro dissolution data of Uniphyl® Tablets tested in an Apparatus 3 evaluating media of varying pH of the GI-tract.................................................................................................. 47 9. In vitro data of Uniphyl® Tablets in simulated fed conditions using Apparatus 3. .......... 55 10. R2 value for correlations with Apparatus 2 and 3 simulated fasted and fed conditions..... 57 11. In vitro data for Uniphyl® Tablets simulated fasted and fed conditions in an Apparatus 2. ............................................................................................................................................ 61

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LIST OF FIGURES Figure Page

1. Varying pH anatomy of the GI tract under fasted conditions (Gibaldi 1984). .................... 3

2. Chemical structure of theophylline.................................................................................... 14

3. USP Apparatus 1 (basket).................................................................................................. 17

4. USP Apparatus 2 (paddle). ................................................................................................ 18

5. USP Apparatus 3 (reciprocating cylinder)......................................................................... 20

6. USP Apparatus 3 – schematic of single vessel/reciprocating cylinder.............................. 21

7. Standard curves for theophylline powder in pH 1.2 medium and equation of the regression line for JSH2-009 (♦) and JSH1-062 (■). .......................................................................... 28





12. Standard curve for theophylline powder in Liposyn® medium and equation of the regression line for JSH2-051 (♦) and JSH2-059 (■).......................................................... 31

13. Apparatus 3 dips/minute determination using Uniphyl® Tablets in phosphate pH 6.6 medium at 5 dips/min – JSH1-032 (■), 7 dips/min – JSH1-047 (×), 10 dips/min – JSH1- 038 (▲), and 15 dips/min – JSH1-021 (♦). ....................................................................... 38

14. Correlation for 5 dips/minute (A), 7 dips/minute (B), and 10 dips/minute (C) in vitro data using % absorbed in vivo (Maturu et al., 1986). ................................................................ 38

15. Apparatus 3 screen selection for Uniphyl® Tablets in phosphate buffer pH 6.6 at 5 dips/minute (A) and 10 dips/minute (B) using stainless steel 20 mesh (♦) and a polypropylene 40 mesh (■) screens. .................................................................................. 41

16. In vitro dissolution of Uniphyl® Tablets using Apparatus 1 in pH 6.6 phosphate buffer, test 1 - JSH2-011 (♦) and test 2 - JSH1-083 (■). ............................................................... 43

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Figure Page

17. In vitro dissolution of Uniphyl® Tablets in pH 6.6 phosphate buffer using an Apparatus 1 (♦) vs. Apparatus 2 (■) with dissolution profile from Karim et al 1985 (▲) included for comparison........................................................................................................................ 43

18. Apparatus 3 in vitro dissolution profiles of Uniphyl® Tablets in pH 1.2 – JSH1-092A (♦), pH 2.9 – JSH1-088 (■), pH 4.5 – JSH1-092B (▲), pH 6.6 – JSH1-047 (×), pH 6.8 – JSH1-100 (◊), and pH 7.8 – JSH1-071 (□) media; pH 1.2 (JSH2-078) and pH 6.8 (JSH2- 072) profiles are similar (f1 = 10 and f2 = 67). ................................................................ 48

19. Apparatus 3 in vitro dissolution profiles of Uniphyl® Tablets in pH 1.2 medium (7 dips/minute) for tests JSH2-078 (♦) and JSH1-092 (■); f1 = 9 and f2 = 81. ..................... 48

20. Apparatus 3 in vitro dissolution profiles of Uniphyl® Tablets in pH 6.8 medium (7 dips/minute) for tests JSH1-100 (■), and JSH2-072 (▲); f1 = 4 and f2 = 91. ................... 49

21. Apparatus 3 in vitro dissolution profile in media ranging from pH 1.2 – pH 7.8 - JSH2- 015 (♦) plotted with profiles in pH 6.8 - JSH2-072 (■), and pH 1.2 - JSH2-078 (▲) for comparison........................................................................................................................ 50

22. Uniphyl® Tablet in vitro dissolution profile tested in an Apparatus 2 in Liposyn® medium (simulated fed) – JSH2-049 (♦) and plotted with in vitro profile in pH 6.6 medium (simulated fasted) – JSH1-013 (■),..................................................................... 51

23. Uniphyl® Tablet in vitro dissolution profiles under simulated fed conditions using an oil soak method in an Apparatus 3 - JSH2-062A pH 6.8 (♦) and JSH2-062B pH1.2 and 6.8 (■) [(f1 = 3 and f2 = 86)]. In vitro dissolution profiles under simulated fasted conditions [(JSH2-072, pH 6.8 (▲)] are also plotted for comparison [(f1 = 28 and f2 = 47)]............ 54

24. Uniphyl® Tablet in vitro dissolution profiles under simulated fed conditions in an Apparatus 3. ...................................................................................................................... 56

25. Uniphyl® Tablet in vitro dissolution profiles under simulated fed conditions – Liposyn® (n=12) (■) and Intralipid® (n=12) (▲) [10% oil vs. 30% oil; f1 = 1 and f2 = 98] versus simulated fasted conditions [JSH2-072, pH 6.8 (♦)] are also plotted for comparison [(Liposyn vs. pH 6.8; f1 = 25 and f2 = 50)(Intralipid® vs pH 6.8; f1 = 17 and f2 = 65)] in an Apparatus 3. ................................................................................................................. 56

26. Correlation for Uniphyl® Tablets in vitro data (JSH1-013 - ♦) using Apparatus 2 in pH 6.6 medium vs. reported fasted in vivo data (Karim et al., 1985). .................................... 58

27. Correlation for Uniphyl® Tablets in vitro data (JSH1-009 - ♦) using Apparatus 1 in pH 6.6 medium vs. reported fasted in vivo data (Karim et al., 1985)...................................... 59

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Figure Page



30. Correlation for Uniphyl® Tablets in vitro data (JSH2-049 - ♦) using Apparatus 2 in Liposyn® medium vs. reported fed in vivo data (Karim et al., 1985). ............................. 62

31. Correlation for Uniphyl® Tablets in vitro data in Liposyn® medium [JSH2-099 (A) and JSH3-009 (B)] using Apparatus 3 vs. reported fed in vivo data (Karim et al., 1985)....... 63

32. Correlation for Uniphyl® Tablets in vitro data in Intralipid® medium [JSH3-019 (A) and JSH3-029 (B)] using Apparatus 3 vs. reported fed in vivo data (Karim et al., 1985)....... 64

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LIST OF EQUATIONS

Equation Page

1. % Released of active pharmaceutical ingredient (theophylline)..................................... 32 2. Fit Factor equations f1 and f2. ......................................................................................... 33 3. Equation for in vivo % absorbed (Aiache et al., 1989, Karim et al, 1985, Weinberger et al., 1978). ........................................................................................................................ 35 4. Correlation linear equation. (Sunkara and Chilukuri, 2003).......................................... 36

1

INTRODUCTION

Overview

Pharmaceutical companies endeavor to discover new drugs and develop them into the

dosage form that provides the best therapeutic result. They also work to further optimize

existing drugs to give an even better therapeutic response. Many are successful and continue

their drug development efforts because new and improved drugs are in demand. When a new

drug is discovered or a generic drug is being developed, one of the first things that is studied or

considered is its bioavailability. A drug substance’s bioavailability is used to determine the dose

that will give the best therapeutic result. Bioavailability information is collected during in vivo

studies or clinical trials during the development of a drug.

Bioavailability

In the early stages of development of a new drug or a generic drug one vital characteristic

that is studied is the bioavailability of the active ingredient. “Bioavailability is a

pharmacokinetic term that describes the rate and extent to which the active drug ingredient is

absorbed from a drug product and becomes available at the site of drug action” (Makoid, 1996-

2000). Bioavailability of an active ingredient can be determined by measuring hthe drug

concentration in the blood or urine. This determination is based on the presumption “that the

drug at the site of action is in equilibrium with the drug in the blood” (or urine) (Makoid, 1996-

2000). The greater the bioavailability of an active ingredient the smaller the therapeutic dose can

be which in turn means a smaller tablet, which is easier to swallow, increasing patient

compliance. There are a number of factors that can affect the bioavailability of an active

ingredient. One that has been studied for many years is the effect of food.

2

Food Effects on Drug Bioavailability and the Gastrointestinal Tract

In order to be able to determine bioavailability the first thing that needs to happen is the

active ingredient needs to be absorbed. The absorption of an active ingredient in a solid dosage

form is comprised of three steps:

1. “disintegration of the drug product,

2. dissolution of the drug in the fluids at the absorption site and

3. the transfer of drug molecule across the membrane lining the gastrointestinal tract”

(Makoid, 1996-2000).

The solubility of the active ingredient also plays an important role in absorption. Anything

that inhibits any of the steps mentioned above will in turn affect the bioavailability of the active

ingredient. There are a number of factors that inhibit bioavailability and can be patient related or

dosage form related (Makoid, 1996-2000). One of these factors is the presence of food in the

gastrointestinal tract. This is an important factor to study because many people take medication

with food (Makoid, 1996-2000).

The presence of food alters the motility and ionic strength, slows gastric emptying, increases

pH, and secretes bile, pancreatic fluids, digestive enzymes and gastric hormones in the

gastrointestinal (GI) tract (Wearley et al., 1985). Figure 1 shows the range in pH along the GI

tract in the fasted stomach. After meal intake the pH of the stomach increases to a pH of 3-7 and

then returns to normal (pH under 3) after 2-3 hours (Dressman et al., 1998). The pH of the small

intestine increases slightly in the fed state. Different pH conditions, ranging from pH 1.2 – 7.8

were studied during this research.

3

Figure 1. Varying pH anatomy of the GI tract under fasted conditions (Gibaldi 1984).

Food taken with drugs can affect the drug, before and during gastrointestinal absorption,

during distribution, during metabolism and during elimination (Singh, 1999). The effects

include decreased, delayed, increased, accelerated absorption or no effect on the bioavailability

of a drug (Singh, 1999 and Welling, 1996). B.N. Singh has tabulated research done on several

different drugs, and grouped them in their therapeutic class and the type of food effect reported

(Singh, 1999).

In vitro/In vivo correlations

In vivo testing is one way to determine the bioavailability of an active ingredient. In vivo

studies take place within a living organism and often involve the measurement of drug

concentration in the blood or urine. In vitro testing is performed outside of a living organism, in

a laboratory and measures the drug release from the dosage form. Both of these kinds of tests

4

are essential in developing an optimal formulation for a new drug product. An in vitro test is

also commonly used to monitor the drug product quality of a commercial product. In vivo tests

can be expensive and time consuming which can be detrimental for pharmaceutical companies

who are rushing to be the first company to develop a generic product for an already marketed

product or who are bringing a new drug to the market. In vitro tests are much less expensive and

do not take as much time. To perform an in vitro dissolution test on a dosage form an apparatus

is needed to facilitate the dissolution and an instrument is needed to analyze the samples. The

data is then used to calculate the % dissolved (or released) of the active ingredient over time.

A hopeful solution to this set back is the development of an in vitro/in vivo correlation

(IVIVC). The Food and Drug Administration (FDA) defines an IVIVC as “a predictive

mathematical model for the relationship between the entire in vitro dissolution/release time

course and the entire in vivo response time course, e.g., the time course of plasma drug

concentration or amount of drug absorbed.” (Sirisuth and Eddington; U.S. Department of Health

and Human Services, 1997). The United States Pharmacopoeia (USP) describes IVIVC as “the

establishment of a rational relationship between a biological property, or a parameter derived

from a biological property produced by a dosage form, and a physicochemical property or

characteristic of the same dosage form.” Cmax (maximum concentration) or AUC (area under the

plasma time curve) are the most commonly used biological properties and the percent of drug

released over time is the physicochemical property most commonly used. The relationship

between the two properties, biological and physicochemical, is then expressed quantitatively

(The United States Pharmacopoeia, 2008 <1088>). IVIVCs are beneficial because typical

compendial media does not take into account for food effects, range in pH, etc. (Nicolaides et al.,

1999). Scientists and pharmacists have tried for years to develop a correlation that could replace

5

in vivo studies with in vitro dissolution tests. Such a correlation is nearly impossible. But many

people have been successful in the development of in vitro methods that are able to predict

certain aspects of in vivo behavior. One of the objectives of this research was to evaluate media

that would simulate fed conditions and potentially help predict in vivo behavior of a drug

utilizing an Apparatus 3. “Efficient product-medium interaction appears to provide an improved

and bio-relevant dissolution testing environment”(Qureshi, 2007). Hence the purpose of using

an emulsion as media containing fats and oils to simulate fed conditions. The medium,

temperature, sink conditions, stirring or agitation are all in vitro parameters that are manipulated

to mimic human gastric physiology.

Although it is doubtful that in vitro dissolution tests could ever fully replace in vivo studies,

they can help develop an ideal formulation, help prevent in vivo failures, and help as a quality

control test during manufacturing (Kahn, 1996; Sirisuth and Eddington). Therefore it is

beneficial to move a drug product through development with an analytical method that would

help predict food effects on drug absorption.

Controlled or Sustained Release Solid Dosage Form

Controlled release (CR) or sustained release (SR) tablets are a solid dosage form that

initially releases a sufficient amount of active ingredient to produce the desired therapeutic effect

and then the remaining active is released over an extended period of time (Lieberman et al.,

1989). One common way to achieve this type of release of active is to create a “matrix” which

forms a gel layer around the tablet and releases a certain amount of active at a time. “The three

major types of materials used in the preparation of matrix devices are insoluble plastics,

hydrophilic polymers and fatty compounds” (Gennaro, 1990). In vitro dissolution testing is a

crucial tool for the development of a CR or SR formulation.

6

Problems

All of the physiological changes that occur in the GI tract due to food make simulating the

environment very difficult during in vitro testing (Makoid, 1996-2000; Khan 1996). In turn this

is a prime example of why IVIVCs are very difficult to accomplish. The popular dissolution

USP compliant apparatuses (paddles and baskets) that are typically used do not allow an

automated flow of media with varying pH that would simulate the GI tract. In order to simulate

the range of physiological pH the media has to be changed manually and is very time consuming

and it often can give erroneous analytical results. These in vitro dissolution systems are also not

very fitting for low solubility drugs (Kahn, 1996). There have been many methods developed

over the past several years that have been able to overcome these problems. Maybe in vitro

testing cannot fully replace in vivo studies but it can be used as a qualitative tool to predict some

in vivo behavior, therefore it plays an essential part in the development of a formulation and can

help avoid in vivo failures (Nicolaides et al., 1999; Sirisuth and Eddington). Furthermore, a valid

IVIVC will allow post approval flexibility in formulation and scale-up changes without having to

perform in vivo bioequivalence studies (Sirisuth and Eddington and U.S. Department of Health

and Human Services, 1995)

The goal of this project will be to develop an in vitro dissolution method simulating fed

conditions that could predict the in vivo behavior of a CR dosage form.

Relevant Literature

Several in vitro dissolution test methods that simulate the GI tract fed conditions have been

investigated to predict the in vivo bioavailability. Many of these methods involve the use of a

model drug(s) and compare its in vitro data with its in vivo data. Some of these studies explore

several different dosage forms of a model drug to determine if one dosage form is effected by the

7

presence of food more than another form. Maturu et al., 1986 observed different dosage forms

of theophylline and fasting vs. fed conditions. The “fed” condition was simulated by soaking the

dosage form in peanut oil for a specified amount of time and then performing a dissolution

profile. El-Arini et al., 1989 also utilized soaking the product in oil prior to performing a

dissolution on four different CR product forms of propanolol. Several other researchers have

utilized an oil soak to simulate fed conditions of the GI tract (Aiache et al., 1989, Wearley et al.,

1985, Esbelin et al., 1991, and Mu et al., 2003). Abrahamsson et al., 1994 studied the effect of

different solubilizers added to the dissolution media. Aoki et al., 1992 compared the very

common paddle method vs. a paddle method with polystyrene beads in the dissolution vessel.

The polystyrene beads were used to mimic the mechanical destruction or frictional forces present

in the GI tract. Katori et al., 1996 also utilized polystyrene beads to simulate the mechanical

stresses caused by the intake of food, but used the rotating dialysis cell method as opposed to the

Apparatus 2. Garbacz et al., 2008 also studied the physical stresses of the GI tract on a

controlled release diclofenac product using a novel apparatus.

El-Arini et al., 1990 developed a dialysis cell method which involved many changes to the

media to best simulate the GI tract before and after the presence of food and compared four

different product forms of theophylline to their in vivo data using an in vitro oil soak method.

Macheras et al., 1989 compared two different forms of theophylline by testing each in milk with

varying fat content. Another study conducted by Diakidou et al., 2009 utilized homongenized

long-life milk as medium and gradually added a 1.83M HCl solution containing 1.1 mg/mL of

pepsin. Additionally, studies were conduct with and without the addition of lipase to simulate

intragastric lipolysis (Diakidou et al., 2009). Some have prepared emulsions to simulate fed

conditions (Al-Behaisi et al., 2002 and Kostwicz et al., 2002).

8

The majority of information found in the literature utilized the Apparatus 1 (baskets) and 2

(paddles) with less research reported using an Apparatus 3 (Reciprocating Cylinder) or 4 (Flow-

Through Cell). Table 1 summarizes prior work done using various in vitro apparatus on

evaluation of food effects on controlled release drugs. In a literature review prepared by M.

Zahirul I. Khan, 1996, summaries of previous studies on the effects of food were provided in

tabular format. Table 1 below includes the summaries from Khan’s (1996) review and adds

summaries of research conducted since 1996 or omitted from his summary table.

Rohrs et al., 1995 studied top mesh, bottom mesh and dips/minute parameters in an

Apparatus 3 by testing six hydrophilic matrix formulations and one coated-bead formulation.

Khamanga and Walker, 2007 evaluated the effects of buffer molarity, dips/minute and mesh size

on verapamil controlled release tablets using an Apparatus 3.

Yu et al., 2002 studied immediate release (IR) products in an Apparatus 3 and compared the

data with data produced from testing in an Apparatus 2. Other relevant literature reviewed

involving IR products include Gaila et al., 1998, Dressman et al., 1998, Nicolaides et al., 1999,

Löbenberg et al., 2000, Nicolaides et al., 2001 and Al-Behaisi et al., 2002. These publications

and the FDA guidelines indicated that this research topic is novel and relevant to both the

pharmaceutical industry and academia.

9

Table 1. Relevant literature evaluating the effects of food and GI tract physical stresses on controlled release dosage forms.

In vitro Apparatus Factor(s) Studied

Method Modifications Model Drug/Dosage Form

Results Reference

Apparatus 2 and modified with polystyrene beads in the medium

GI motility/mucin plugs

Paddle-bead method – Paddle method with polystyrene beads inserted into the dissolution medium

Phenyl-propanolamine HCl/matrix tablet

The modified method resulted in a better in vitro-in vivo correlation than the paddle method when compared with the release profile obtained in fasted beagle dogs

Aoki et al., 1992

Novel Rotating Dialysis Cell

pH of the GI tract/food induced changes

A dialysis cell containing the dosage form in a small volume of fluid is immersed in the dissolution medium in a dissolution vessel. The physiological conditions are simulated by adjusting the fluid of the dialysis cell

Theophylline/ beads, either embedded in matrix tablet or filled in capsule

The method allowed testing of the extended release dosage forms under various food induced conditions. Novel in vitro method was successful in predicting in vivo behavior under fed conditions (with exception of one product)

El-Arini et al., 1990

Rotating bottle apparatus method according to National Formulary XIII

High fat food Pretreatment of the dosage form (or content) in peanut oil for 2 h prior to standard dissolution testing

Theophylline/matrix tablet, and beads filled in capsule

The in vitro dissolution data correlated well with in vivo percent dissolved in humans after high fat breakfast

Maturu et al., 1986

Apparatus 1 High fat food Pretreatment of the dosage form content in peanut oil for 1 h at 37°C prior to standard dissolution testing

Propanolol HCl/capsule

A significant decrease in dissolution rate of the drug was observed as a result of pretreatment of the dosage form with peanut oil when compared with untreated dosage form

El-Arini et al., 1989

Apparatus 2 Fatty Food Milk with various levels of fat content (0.1%, 2.0%, 5.0% and 7.5%) was used as dissolution medium

Theophylline/matrix tablet, and capsule

A direct relationship was established between fat contents of milk and dissolution data with good correlation between data obtained in humans after a high fat meal

Macheras et al., 1989

Apparatus 2 modified with a stationary basket

Poor aqueous solubility of the drug

Addition of solubilizer in the dissolution medium, and a stationary basket to hold the dosage form above the paddle to achieve reproducible hydrodynamic conditions

Felodipine/matrix tablet

The method produced dissolution data with good in-vitro-in vivo correlation, and was capable of discriminating formulations with different in vivo performance

Wingstrand et al., 1990

Apparatus 2 modified with a stationary basket

Poor aqueous solubility of the drug

Addition of solubilizer in the dissolution medium

Felodipine/matrix tablet

The method provided data with a good in vitro-in vivo correlation, and the choice of solubilizer affected the results

Abrahamsson et al., 1994

1 Table modeled after Table 1 in Khan, 1996. Information in italics taken from Khan, 1996.

10

Table 1, continued.



Results Reference

Rotating bottle apparatus method according to National Formulary XIII

High fat food Uilized Maturu et al. in vitro method (oil soak) to test 3 marketed theophylline controlled release products from France

Theophylline Hydrophilic matrix tablets;coated micropellets

Linear correlations were obtained for two of the products tested. One product obtained a curvilinear relationship. Concluded that the in vitro oil soak method was again successful in simulating the complexities of the GI tract.

Aiache et al., 1989

Apparatus 1 Baskets

Food effects - pH 6.6 buffer (reference system) - 0.04M sodium deoxycholate (bile salt) - 1-3hr soak in oleic acid followed by dissolution in pH 6.6 buffer (fatty acid) - 1-3hr soak in oleic acid followed by dissolution in bile salt

Four controlled release theophylline products

The combination of fatty acids and bile salts (1-3hr oleic acid soak/bile salt) resulted in changes in release rates for all four products which correlated to in vivo AUC changes after a high fat meal. Other media alone did not correlate to in vivo behavior.

Wearley et al., 1985

Apparatus 3 Rotating bottle

Food effects Comparison of Apparatus 3 with rotating bottle

Simulated fasted (SGF, pH 1.2 and SIF, pH 6.8 and buffer solutions of intermediate pH) Simulated fed (Maturu et al., 1986 oil soak)

Theophylline controlled release Theostat Dilatrane Armophylline

The in vitro data correlated well with the in vivo data, therefore the Apparatus 3 can be used as an alternative to the rotating bottle apparatus.

Esbelin et al., 1991

Apparatus 1 Apparatus 2 Apparatus 4 Rotating dialysis cell method (El-Arini et al., 1990)

Effects of mechnical destruction forces

No modifications were made to the official apparatuses 1, 2 and 4 Modifications to the rotating dialysis cell method included the addition of 20 polystyrene beads

Two formulations of controlled release spherical granules containing acetaminophen

Food effects were noted for the formulation with the greater hardness of the granules. In vitro release of both prototypes in all official methods (1,2 and 4) showed similar release rates. However, the formulation with a lower hardness released much faster with use of the polystyrene beads in the rotating dialysis cell apparatus, similar to in vivo results.

Katori et al., 1996

Apparatus 3

Food effects Plasticizer type, concentration& coating level in ethylcellulose coated beads

Modified Maturu et al. in vitro method (oil soak) in an Apparatus 3

Chlorpheniramine maleate ehtylcellulose coated beads

Dissolution rates of beads coated with ethylcellulose films plasticized with TEC or DBS may be influenced when administered concomitantly with a high-fat meal. The DBS plasticizer was more effected by the oil soak that the TEC-plasticized films.

Williams et al., 1997

Apparatus 3

Food effects Presoak in peanut oil (Maturu et al. and Esbelin et al.) Continuous oil contact

Propanolol hydrochloride controlled release formulations Inderal® LA Capsules

Both methods studied, pre-soak and continuous contact, demonstrated ability to predict the effect of food in vivo.

Mu et al., 2003

1 Table modeled after Table 1 in Khan, 1996.

11

Table 1, continued.



Results Reference

Apparatus 3 Effect of pH and ionic strength

Range of pH media was studied in buffer solutions having pH values of 1.2, 2.5, 4.5, 6.8, and 7.5, mimicking the pH environment under fed and fasted conditions of the GI tract. Range of ionic strength (0 – 0.4) to mimic the physiological conditions of the GI tract

Heterodisperse polysaccharide-based controlled release system (HPCRS) (using xanthan and locust bean gum) formulations of propanolol HCl

HPCRS was pH dependent releasing slightly faster in acidic medium. Possibly would predict a food effect. Changing the ionic strength had a more limited effect. Mu et al., 2003

Novel apparatus Apparatus 2

Physical stress conditions of the GI tract

Novel apparatus could apply two physical stresses; 1. rotational movement where the chamber passes during each rotation through the air-water interface, 2. simulated peristaltic ‘squeeze’ (pressure waves) No modifications were made to the apparatus 2

Diclofenac extended release matrix tablets

The multiple peaks observed in the in vivo plasma profiles could be attributed to the physical stress events of the GI-tract based on the in vitro data obtained by using the novel stress test apparatus. The dissolution profile obtained using the Apparatus 2 showed almost continuous release, not predictive of the in vivo behavior observed.

Garbacz et al., 2008

Apparatus 2 Modified by adhering the tablets to the paddle on a steel wire

Food effects Simulation of gastric lipolysis

Intragastric lipolysis was simulated in vitro by the gradual addition of acidic solutions (1.83M HCl) containing pepsin with and without the addition of lipase, 9.375 and 3.125g, initially and after 90 minutes, respectively.

Felodipine extended release tablets

Results show the addition of the lipase to the media helped predict in vivo behavior of the fed stomach (intragastric lipolysis) for the release of felodipine from extended release formulation.

Diakidou et al., 2009

Apparatus 1 Apparatus 2 Apparatus 3 Apparatus 4

Food effects Apparatus comparison

Simulated fed and fasted conditions were performed in vitro by using biorelevant media developed previously (Vertzoni et al., 2005; Fotaki et al., 2005, Dressman et al., 2007, Janatrid et al., 2008) No modifications were made to the compendial Apparatuses 1 and 2

Modified release formulation of diclofenac sodium

Good correlations were obtained using the biorelevant in vitro dissolution tests (simulating pre-and postprandial states). The compendial Apparatuses 1 and 2 did not predict the food effect (slower absorption, release) when compared to the in vivo data.

Jantratid et al., 2009 Article in press

Apparatus 2 Apparatus 3 Apparatus 4

In vitro simulated Fasted conditions Apparatus comparison

No modifications to the USP methods. Media utilized during testing each apparatus was simulated gastric fluid (SGF) with pH 1.8; fasted state simulated intestinal fluid (FaSSIF); and simulated colonic fluid (SCoF)

New chemical entity, BRL-49653 extended release tablet formulation Volmax® salbutamol sulphate osmotic pump formulation

In vitro results from each apparatus studied, 2, 3 and 4, show that each can be equally useful in predicting the actual in vivo profiles on an average basis for both of the formulations studied. However, some differences in the in vitro data were observed but attributed to the effects of the hydrodynamics of each apparatus.

Fotaki et al., 2009 Article in press

1 Table modeled after Table 1 in Khan, 1996.

12

MATERIALS AND METHODS

This section provides a list of materials and equipment, a description of the items needed

(apparatus and instrument to analyze samples) and some of the method development techniques

utilized during this research. Tests performed were each designated a lot number which

corresponds to a notebook and page number. Nomenclature for lot numbers was dictated by my

initials (JSH), notebook number (1-3) followed by a dash and the corresponding page number (1-

100), e.g. JSH1-009.

List of Materials and Equipment

Materials

Theophylline anhydrous powder was provided as a gift from BASF. Uniphyl® Tablets, 400

mg were purchased from Purdue Pharmaceutical Products. Acetic Acid, Glacial was purchased

from Fisher Laboratories. The following chemicals were purchased from Mallinckrodt:

hydrochloric acid, potassium phosphate dibasic, potassium phosphate monobasic, phosphoric

acid, and sodium hydroxide. Potassium chloride was purchased from Spectrum. Potassium

hydroxide (KOH) was purchased from Ricca Chemical Company. Sodium acetate (NaC2H3O2

3H2O) was purchased from JT Baker and isopropyl alcohol was purchased from Burdick and

Jackson. Milli-Q water was obtained from the main lab of the formulations development

laboratory at AAIPharma.

Equipment

To perform an in vitro dissolution test on a dosage form an apparatus is needed equipped

with an agitation device to facilitate dissolution and an instrument is needed to analyze the

13

samples. The data obtained upon analysis of the sample is then used to calculate the % dissolved

(or released) of the active ingredient over time. The medium, temperature, sink conditions,

stirring or agitation are all factors that are manipulated to mimic human gastric physiology.

Various dissolution baths equipped with either baskets (Apparatus 1) or paddles (Apparatus

2) and reciprocating cylinders (Apparatus 3) were utilized during these studies. Secondary

equipment utilized were thermometer, pH meter, balance and filters (0.45 µm Gelmen nylon

filter and 1 µm glass filters). A UV spectrophotometer was utilized to analyze the samples.

Test Article – Uniphyl® (theophylline, anhydrous) Tablets, 400 mg

Uniphyl® Tablets, 400 mg were utilized as the model drug for this research. Theophylline

is the active pharmaceutical ingredient in Uniphyl® Tablets. According to reported in vivo data,

(Karim et al., 1985) the rate and extent of absorption of Uniphyl® Tablets are significantly

increased under the presence of food. Labeling for drugs containing theophylline instructs to

dose consistently with or without food. Theophylline is frequently used as a model drug in

dissolution studies (El-Arini et al., 1990; Karim et al., 1985; Maturu et al., 1986; Wearley et al.,

1985). One attribute of this drug that causes interest is that several different formulations and

dosage forms of theophylline are available on the market and are readily available to purchase

for research. Additionally, this model drug was chosen based on the availability of reported in

vivo data for comparison with the in vitro data collected during this research.

Theophylline is a methylxanthine or xanthine derivative and is structurally similar to

caffeine, only differing by a methyl group. The major metabolite of theophylline is 1,3-

dimethyluric acid (Delgado and Remers, 1998). The structure of theophylline is provided in

Figure 2. Chemical and biologic properties are also listed in Table 2.

14

Figure 2. Chemical structure of theophylline.

Table 2. Chemical and biological properties of theophylline

Chemical formula C7H8N4O2 Name(s) 3,7-Dihydro-1,3-dimethyl-1H-purine-2,6-dione

(1,3-dimethylxanthine) Methylxanthine or a xanthine derivative

Description White powder Solubility Soluble in hot water, in alkali hydroxides,

ammonia, dil HCl or HNO3, sparingly soluble in ether

pKa 8.77 Elimination half life (t1/2)

8 hours

Indication For treatment of symptoms with chronic asthma and other lung diseases

Mechanism of action Unknown with certainty Drug class Bronchodilator

Merk Index, 12th edition 1996.

Several researchers have determined that Uniphyl® Tablets have pH-independent

dissolution rates (Maturu et al., 1986, Karim et al., 1985 and Wearly et al., 1985), however pH-

dependence was studied during this research as well in an Apparatus 3 (Table 6, Figures 18-20).

Uniphyl® Tablets were approved by the Food and Drug Administration (FDA) on

September 1, 1982, ANDA 87-571 and are currently marketed by Purdue Fredrick. The method

originally filed in the marketing application by the sponsor was a USP basket method (Apparatus

N

N N

HN

CH3

H3C

O

O

15

1) at 100 rpm in 900 mL of simulated gastric fluid without enzymes and thereafter 900 mL of

simulated intestinal fluid without enzymes. Sample times were 1, 2, 4, 5, 6, 9 and 12 hours.

Data for n=12 tablets from a batch utilized in a clinical trial is outlined below. This information

was collected from the Summary Basis of Approval (SBA) for Uniphyl® Tablets (ANDA 87-

571) which was prepared by the FDA reviewers upon review and approval of the marketing

application.

Time (hr) %Dissolved S.D. 1 4.6 0.31 2 10.4 0.41 3 14.5 0.51 4 17.8 0.66 6 23.6 0.96 9 30.3 1.20 12 36.5 1.49

In the SBA, there was data for one lot tested out to 24 hours but the average release of 12

tablets was still only 52.8%. Even though the dissolution methodology was not acceptable

(typically at least 80% released is desired) to the FDA reviewing chemist at the time of launch to

market the following specifications were utilized until an acceptable method was developed.

Time (hr) Specification (% released) 1 6-11 2 9-17 4 14-24 8 20-34 12 28-42 18 38-52 24 45-61 The comments from the FDA were that the amount released should be at least 80% for a

meaningful test. Furthermore, they commented that because of the poor dissolution of the

formulation the method filed was not a predictor of in vivo behavior and could not be used to

compare other strengths of Uniphyl® Tablets to the 400 mg tablet. An additional clinical trial

16

would have to be conducted to obtain approval for other strengths. The formulation may have

changed since approval in 1982 as well as the method utilized currently to verify the quality of

the commercial tablet, however that information is proprietary and therefore was not found

publicly available.

Apparatus

The typical assembly utilized for a dissolution test consists of a vessel which holds a known

quantity of medium that may be covered to prevent evaporation and is submerged in a

temperature controlled water bath. The vessel is made of glass or other inert, transparent

material, a motor, a metallic drive shaft and a stirring apparatus (The United States

Pharmacopoeia, 2008 <711>). There are several accepted stirring apparatuses. The most

commonly used for solid oral dosage forms are the cylindrical basket (Apparatus 1) and paddle

(Apparatus 2). The focus of this research utilizes a reciprocating cylinder (Apparatus 3) which

reciprocates the samples vertically in the media. For each apparatus mentioned, the vessel is

partially immersed in a water bath that is kept at 37 ± 0.5°C (The United States Pharmacopoeia,

2008 <711>). Samples are taken at specified time intervals, filtered and analyzed using a UV

spectrophotometer or HPLC. For this research a UV spectrophotometer was utilized to analyze

the samples. The percent of active dissolved (released) at each pull point is calculated using the

absorbance of the sample plotted against time in order to display the release of the drug over

time (dissolution profile).

The media used in these studies were intended to simulate the physiological changes in the

gastrointestinal tract due to fatty foods and also under fasted conditions. The media and method

were developed utilizing parameters from existing methods, some of which are described below.

17

Apparatus 1 and 2

Apparatus 1 is most commonly known as the basket apparatus and Apparatus 2 the paddle

apparatus. A picture of an Apparatus 1 and 2 dissolution instrument is shown in Figures 3 and 4,

respectively. Parameters to develop consist of media, rotations of the basket or paddle per min,

use of sinkers (more common with capsules), pull volume, sample times and filter size and

shape. Typically, filter studies are conducted evaluating different size and types of filters. In

analyzing samples the parameters to evaluate include standard concentration, wavelength and

cell length.

Figure 3. USP Apparatus 1 (basket).

18

Figure 4. USP Apparatus 2 (paddle).

Apparatus 3

The Apparatus 3 (Figure 5) was included in the USP general chapters <711> as an

acceptable apparatus for dissolution testing in 1991. It was developed due the to growing

popularity of in vitro/in vivo correlations and the recognition of potential issues (% release

affected by shaft wobble, location of dosage form, centering and coning) with the popular

Apparatus 1 and 2 (Yu, L.X et al., 2002 and Borst et al., 1997). The USP Apparatus 3 is most

commonly used on controlled release dosage forms but research has been done to demonstrate

that immediate release dosage forms can also be tested in an Apparatus 3 (Yu, L.X et al., 2002

and Al-Behaisi et al., 2002).

19

Figure 5 shows an entire Apparatus 3 dissolution set-up and Figure 6 is a schematic close-up

of a vessel, the reciprocating glass cylinder and the evaporation cap in which the reciprocating

cylinder is held. USP Apparatus 3 consists of 6 rows of cylindrical flat-bottomed glass vessels;

each row may contain up to 7 vessels. The seventh vessel is used for standard solutions when

automated analysis is utilized. Manual collection of samples from the vessels were conducted

therefore the seventh vessel was not utilized during this research. A reciprocating glass cylinder

which houses the tablet via an end cap equipped with a mesh screen (stainless steel type 316 or

other suitable material) on the bottom and top, moves the tablet into the dissolution media in an

up and down motion. The bottom end cap/screen is where the product (tablet or capsule) rests

during the upward motion. On the downward motion the product moves freely within the

medium. Collectively the upward and downward motion carries the product being tested through

a moving medium (Borst et al., 1997). The evaporation cap contains air holes to allow for air to

be released as the cylinder moves down into the medium, which prevents splashing and also

helps in preventing evaporation as it also provides a cover over each vessel. Additionally the

entire apparatus contains a vinyl cover that moves along with the shaft containing the

reciprocating cylinders to reduce evaporation. One of the advantages of the 6 rows of glass

vessels is the ability to use up to six different types of dissolution media without the rigors of

having to change media during the dissolution test.

Parameters to develop for this apparatus consist of dissolution medium, dips/minute, dip

time intervals, hold time, drain time, and mesh size. Each is described in more detail below.

20

Figure 5. USP Apparatus 3 (reciprocating cylinder).

21

Figure 6. USP Apparatus 3 – schematic of single vessel/reciprocating cylinder.

General Chapters <711> Dissolution

Reprinted with permission. The United States Pharmacopeial Convention. Copyright 2009. All rights reserved.

22

Dissolution Medium

Several different dissolution medium are used to measure the release of a drug. The most

common dissolution media utilized are water, pH 6.8 potassium phosphate and pH 1.2

hydrochloric acid medium. Sometimes the intent is to mimic the gastro-intestinal environment

as closely as possible in vitro and other times the method is developed for routing testing to

assure the quality of a marketed pharmaceutical.

Dips/Minute

The dips/minute parameter controls the amount of agitation (up and down motion of the

glass reciprocating cylinder) in the dissolution medium. Parameters used during this research

ranged from 5 dips/minute to 20 dips/minute with 7 and 10 dips/minute being used during the

majority of the dissolution tests.

Dip Time Intervals

The dip time interval parameter is the amount of time that the glass reciprocating cylinder

(Figure 5) dips in a specified row, therefore this parameter is set for each of the 6 rows. This

parameter is additive, for example, if the first and second pull points desired were 1 and 2 hours

and in rows 1 and 2, respectively, the dip time intervals for rows 1 and 2 would be 1 hour and 1

hour. In other words if the dip time intervals for the 6 rows were added together this includes the

total time that the tablets were in the dissolution medium.

Hold Time

If desired, the hold time parameter can be used to keep the tablets immersed in the

dissolution medium at the end of the dip time interval parameter. This parameter is set for each

23

row. During this research the only occasion that this parameter was used was during the

emulsion media studies and only in the “rinse rows” containing isopropyl alcohol.

Drain Time

The drain time is also set for each of the 6 rows and occurs at the end of the dip time interval

parameter (will occur after the hold time parameter if used) when the reciprocating cylinder

(Figures 4 and 5) rises above the dissolution medium and allows for the medium to “drain” from

the reciprocating glass cylinder.

Mesh Size

The reciprocating glass cylinder is enclosed by two plastic end caps equipped with either a

polypropylene or stainless steel mesh. During some of the studies no mesh was utilized in the

top enclosure because the Uniphyl® Tablets simply eroded and did not break up into multiple

smaller pieces, therefore there was no concern for pieces of the tablets floating out of the

reciprocating cylinder.

Media Preparation

Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF)

Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) are widely used media in

dissolution tests in the pharmaceutical industry. More commonly SGF and SIF media are

prepared and utilized in dissolution tests without enzymes. Enzymes were not utilized in this

research; they are typically used to test capsule dosage forms to help with cross-linking issues

from the gelatin in the capsule shell.

24

SGF is pH 1.2 HCl solution with purified pepsin (800-2500 units of activity) (Test

Solutions, The United States Pharmacopoeia, 2008). Simulated intestinal fluid (SIF) is pH 6.8

monobasic potassium phosphate solution with pancreatin (Test Solutions, The United States

Pharmacopoeia, 2008).

Phosphate Buffer pH 3.0, 6.6, 6.8 (SIF without enzymes) and 7.8

The phosphate buffers with pH from 3.0-7.8 were prepared with potassium phosphate

monobasic and milli-Q water. The initial studies (Apparatus 1 and 2 and early Apparatus 3 pH

range studies, fasted conditions) utilized potassium phosphate monobasic solutions and were

prepared in accordance with procedures defined in the current USP. However, final studies

utilized phosphate buffer prepared according to the following procedure. For 20 liters of buffer,

136 g of potassium phosphate monobasic was weighed and charged to a carboy. The carboy was

filled to 20 L with Milli-Q water and the initial pH was recorded. While stirring, potassium

hydroxide was added to adjust the pH of the solution to 6.6 (or 6.8; 7.8) and the final pH was

recorded. The solution was degassed with helium gas for at least 15 minutes. This step is

performed to minimize bubbles, air pockets which can adhere to the tablet and affect the surface

area that is exposed to the media.

The procedure to prepare the pH 3.0 is identical to the one outlined above with the exception

that the pH of the solution is adjusted with phosphoric acid (H3PO4).

0.1 N HCl pH 1.2 (SGF without enzymes)

The 0.1 N HCl solutions were prepared by combining a 0.2 M potassium chloride solution

with a 0.2 M hydrochloric acid solution. The 0.2 M potassium chloride solution was prepared by

charging 74.55 g of potassium chloride to a carboy and filling to 5 L with Milli-Q water. The 0.2

25

M hydrochloric acid solution was prepared by diluting 165.3 mL of HCl with milli-Q water to 10

L. To prepare the final solution (0.1N HCl), 5 L of potassium chloride solution was charged to a

carboy followed by 8.5 L of 0.2 M hydrochloric acid solution. Milli-Q water was then added to

20 L. The final pH was recorded and then the solution was degassed with helium for at least 15

minutes.

Acetate Buffer pH 4.5

To prepare the 10 liters of the acetate buffer pH 4.5, 29.9 g of sodium acetate (NaC2H3O2

3H2O) was charged to a carboy followed by 140 mL of 2 N acetic acid solution (140 mL of

acetic acid diluted to 1 L with Milli-Q water). The carboy was then charged to 10 L with milli-Q

water. The pH of the solution was recorded while stirring. The final solution was degassed with

helium for at least 15 minutes.

Emulsion (Liposyn®III 10% and Intralipid® 30%)

Liposyn® III 10% is a commercially available parenteral product intended for IV

administration and indicated for use as a source of calories for patients requiring parenteral

nutrition. (Liposyn® III package insert, Hospira 2005) Liposyn® was purchased and used as

dissolution medium to mimic fed conditions. Liposyn® consists of 10% soybean oil, up to 1.2%

egg phosphatides added as an emulsifier and 2.5% glycerin in water for injection. Sodium

hydroxide is used to adjust the pH to 8.3. The total caloric value of Liposyn III 10% is 1.1

kcal/mL. The FDA recommended test meal for a food-effect bioavailability and fed

bioequivalence study is to be a high fat meal (approximately 50 percent of total caloric content of

the meal and high-calorie (approximately 800 – 1000 calories) (Guidance for Industry Food

26

Effect Bioavailability and Fed Bioequivalence Studies, December 2002). It is similar in caloric

content to a high fat breakfast given to subjects in a clinical trial evaluating food effects.

A higher fat content (30%) emulsion was also utilized as a medium to simulate fed

conditions. Intralipid® 30% is also a commercially available product and intended as a source of

calories and essential fatty acids for use in a pharmacy admixture program. Intralipid® consists

of 30% soybean oil, 1.2% egg yolk phospholipids, 1.7% glycerin and water for injection. The

total caloric value is 3.0 kcal/mL. The pH of the final product is 8 (range is 6-8.9)(Intralipid®

package insert, Baxter Healthcare Corporation, April 2000).

Sample Analysis – Ultraviolet Spectrophotometer

The samples are analyzed using an ultraviolet spectrophotometer (UV). The samples need

to be clear and free of particles to allow the light to pass through the sample and record the

absorbance. When the emulsions were used as medium the samples had to be diluted 10 fold

with IPA in order to break up the emulsion and produce a clear solution. With each different

medium used a pure sample of the medium (sample) was used as a blank.

The USP monograph for theophylline controlled release capsules and test procedures

developed at AAIPharma (TP #s 21913B and 23599) were utilized as a guideline for

determination of the wavelength (271 nm) and the cell length (0.05 cm). A 10 mL sample was

pulled from the dissolution vessel and filtered through a 0.45 µm nylon filter and read at 271 nm

with a 0.05 cm cell. A blank was prepared consisting of the medium minus the contents of the

tablet. Each time point was analyzed between standard solution readings.

27

Filter Study

Several filters were evaluated by comparing the absorbances of a standard solution and a

sample solution. The sample solution was prepared by dissolving one 400 mg Uniphyl® Tablet

in 1000 mL of pH 6.6 phosphate buffer in a dissolution bath equipped with paddles rotating at

250 rpms. The standard solution was prepared by weighing 800 mg of theophylline anhydrous

powder into a 2 L volumetric flask and stirring until dissolved. The following filters were

evaluated: Gelman acrodisc 0.45 µm nylon, Gelman acrodisc 0.45 µm PTFE, Millex-HV PVDF

hydrophilic 0.45 µm, and Millex-HN Nylon 0.45 µm

A 10 mL sample was filtered and analyzed using a UV spectrophotometer at 271 nm. The

absorbances were very similar for each filter. Both the standard solution and the sample solution

were very difficult to press through the PTFE filter. Based on feasibility and cost the Gelman

acrodic 0.45 µm nylon filter was chosen and used to filter samples for all dissolution tests

performed with exception of the tests in Intalipid® media. The Intralipid® would not filter

through the nylon filter, therefore glass filters were used to filter samples for these dissolution

profiles.

Standard Preparation and Standard Curves

Calculating the percent dissolved of an active ingredient from a dosage form, i.e., tablet or

capsule is done against a standard solution prepared with the active ingredient at a known purity.

The standards are prepared in the same medium used in the dissolution test and therefore

identical to the sample.

Standard curves for each media studied were generated to aid in the choice of an appropriate

standard for sample analysis and have an assurance of the accuracy of the absorbance recorded

among the samples tested. The standard curves were generated to bracket the expected

28

concentration range of the samples for analysis. (Fundamentals of UV-visible Spectroscopy,

1996).

Standard Curves

Standard Curves were generated for each media utilized. The plots are shown in Figures 7-

12.

Figure 7. Standard curves for theophylline powder in pH 1.2 medium and equation of the

regression line for JSH2-009 (♦) and JSH1-062 (■).

(♦) y = 2.69x + 0.0194R2 = 0.999

(■) y = 2.56x + 0.0579R2 = 0.999

0

0.4

0.8

1.2

1.6

2

0 0.2 0.4 0.6 0.8

Theophylline (mg/mL)

Abs

orba

nce

29



(♦) y = 2.79x + 0.0256R2 = 0.999

(■) y = 2.97x - 0.0549R2 = 0.985

0

0.4

0.8

1.2

1.6

2

0 0.2 0.4 0.6


Abs

orba

nce



(♦) y = 2.78x + 0.0312R2 = 0.999

(■) y = 2.74x + 0.0186R2 = 1.00

0

0.4

0.8

1.2

1.6

2

0 0.2 0.4 0.6


Abs

orba

nce

30



(♦) y = 2.78x + 0.0175R2 = 1.00

(■) y = 2.72x + 0.0279R2 = 1.00

0

0.4

0.8

1.2

1.6

2

0 0.2 0.4 0.6 0.8


Abso

rban

ce



(♦) y = 2.8x + 0.0094R2 = 1.0

(■) y = 2.8x - 0.0033R2 = 1.0

0

0.4

0.8

1.2

1.6

2

0 0.2 0.4 0.6 0.8


Abs

orba

nce

31

Figure 12. Standard curve for theophylline powder in Liposyn® medium and equation of the


(♦) y = 2.8x + 0.0056R2 = 1.0

(■) y = 2.7x - 0.0021R2 = 1.0

0

0.4

0.8

1.2

1.6

2

0 0.2 0.4 0.6


Abs

orba

nce

32

RESULTS AND DISCUSSION

Mathematical Equations

Equation 1. % Released of active pharmaceutical ingredient (theophylline)

% theophylline 1 hour = 100××××

×LC

VsamDsamDstd

VstdPWstd

AstdAsam

% theophylline 2 hours =

VsamPVhourneTheophylli

LCPVVsam

DsamDstd

VstdPxWstd

AstdAsam ])1(%[100)( ×

+×−

×××

% theophylline 3.5 hours =

VsamPVhoursneTheophylli

LCPVVsam

DsamDstd

VstdPxWstd

AstdAsam ])2(%[100)( ×

+×−

×××

Asam = Absorbance of the sample Astd = Absorbance of the standard Wstd = Weight of the standard in mg P = Purity of the standard in decimal form Vstd = Volume of the standard in mL Dsam = Dilution of the sample Dstd = Dilution of the standard Vsam = Volume of the sample PV = Pull Volume LC = Label Claim in mg 100 = Conversion to percent

33

Fit Factor (Moore and Flanner, 1996 and US Department of Health and Human Services,

1997)

Equation 2. Fit Factor equations f1 and f2.

∑∑ ==•−=

111 100]}[/]{[t t

nt tt

n RTRf

}100])()/1(1{[log50 5.0212 •−+•= −=∑ ttt

n TRnf

f1 = Difference factor f2 = Similarity factor Rt = Dissolution value of the reference at time t Tt = Dissolution value of the test at time t n = Number of time points According to the FDA Dissolution Guidance (1997), the difference factor (f1) calculates the

percent (%) difference between the two curves at each time point and is a measurement of the

relative error between the two curves. The similarity factor (f2) is a logarithmic reciprocal square

root transformation of the sum of squared error and is a measurement of the similarity in the

percent (%) dissolution between two curves. For two curves to be considered similar the f1 value

should be between 0-15 and f2 value should be greater than 50 (50-100) (U.S. Department of

Health and Human Services, 1997). When comparing fasted versus fed in vitro profiles, the

fasted was chosen as the reference lot. When comparing two fasted profiles, either lot could be

designated as the reference lot.

In vitro/In vivo Correlation

In vitro data collected during this research was compared to reported in vivo data. The in

vivo data (% absorbed theophylline from Uniphyl® Tablets) was calculated from data provided

in Karim et al., 1985 (Table 3 below) and Maturu et al., 1986 (Table 4 below). In Maturu et al.,

34

1986, the fasted data was provided in tabular format however the fed data was only provided

plotted against the in vitro data, therefore it was extrapolated from the plot. Figured 22 and 23

shows that the simulated fasted and simulated fed curves do not differ significantly until after ~8

and 5 hours, respectively. This is verified by calculating fit factors (f1 and f2) for JSH2-072

(fasted, as reference) and JSH3-009 (fed, as test). When evaluating only up to the 5 hour time

point the f1 = 24 and the f2 = 65 but when evaluating up to 24 hours the f1 = 28 and the f2 = 48.

Therefore the in vivo data calculated from the Karim et al., 1985 paper was utilized because the

in vivo data provided in Maturu et al., 1986 only included up to 7 hour time point and one could

argue that the fasted vs. fed curves are similar up to 5 hour time point. The Maturu et al., 1986

in vivo data was utilized in early determination of Apparatus 3 parameters (screen and

dips/minute selections).

Table 3. In vivo % absorbed for Uniphyl® Tablets from Karim et al., 1985.

Time (hr)

% Absorbed Fed Conditions1

% Absorbed Fasted Conditions1

2 9 11

4 22 20

8 41 29

12 59 34

16 66 37

24 77 44 1 Fraction absorbed calculated by Karim et al., 1985 and multiplied by 100.

35

Table 4. In vivo % absorbed for Uniphyl® Tablets from Maturu et al., 1986.

Time (hr)

% Absorbed Fed Conditions1

% Absorbed Fasted Conditions1

1 2.4 7.1

2 12.5 13.1

3.5 28.5 18.4

5 37 26.9

7 49 33.1 1 Data was extrapolated from IVIVC plot.

According to Maturu et al., 1986 the % absorbed was also calculated from data taken from

Karim et al., 1985. Karim et al., 1985 calculated the rate and extent of theophylline absorption

(fraction absorbed) using a modification of the Wagner-Nelson method (Wagner and Nelson,

1963) which was developed by Weinberger et al., 1978 (see Equation 4). To obtain % absorbed

the result was simply multiplied by 100 (Aiache et al., 1989).

Equation 3. Equation for in vivo % absorbed (Aiache et al., 1989, Karim et al, 1985, Weinberger

et al., 1978).

100/

%0

0 xAUC

AUCkCabsorbed tet

∞−

−+=

Ct = Concentration at time t ke = Elimination rate constant AUC0-t = Serum area under the curve (AUC) from time zero to the last measurable

data point AUC0-∞ = Serum AUC from time zero to infinity

36

To correlate, the in vitro data was plotted against the in vivo data and expressed as a simple

linear equation.

Equation 4. Correlation linear equation. (Sunkara and Chilukuri, 2003)

Y (in vivo absorbed) = mX (in vitro drug dissolved) + C (intercept)

This type of correlation is known as a Level A correlation and is recognized by the FDA as

the most informative and is recommended for use where possible (U.S. Department of Health

and Human Services, 1997). Level A correlations are generally linear and represents a point-to-

point relationship between in vitro drug release and in vivo behavior (Sirisuth and Eddington,

U.S. Department of Health and Human Services, 1997).

An IVIVC should predict in vivo performance. These correlations are typically established

during development and utilizing three or more formulations with different release rates.

Correlation concepts (comparing in vitro data to in vivo data) are utilized here to be able to

evaluate the in vivo predictability from the in vitro data collected utilizing emulsion media in an

Apparatus 3.

Identification of Parameters for Apparatus 3

Initial studies in the Apparatus 3 included evaluation of the top and bottom screens of the

cylinder and determination of dips/minute parameter that most correlated with the reported in

vivo data.

37

Dips/Minute Determination

The dips/minute studies were conducted using Uniphyl® Tablets in pH 6.6 phosphate buffer

(Table 5). A test was performed initially with 20 dips/minute but the test was not completed due

to equipment malfunction. However, based on the tablet appearance (significantly more eroded

compared to tablets tested in Apparatus 1 and 2) after the one hour pull point it was determined

that the next test should be performed at 15 dips/minute. The dissolution profile at 15

dips/minute was much faster than the data collected from the basket apparatus (Apparatus 1); as

listed in Table 5. Other evaluations included 5, 7 and 10 dips/minute. For the latter three

evaluations the in vitro % released data was plotted against the % absorbed data reported in

Maturu et al., 1986 (Figure 14). For subsequent studies 7 and 10 dips per minute were used

based on linear comparisons in Figure 14. The in vitro data is listed in Table 5 for each

evaluation and profiles are plotted in Figure 13. This data (Uniphyl® Tablets) is in agreement

with studies on hydrophilic matrix controlled release formulations performed in an Apparatus 3,

as dips/minute parameter is increased the drug release rate is also increased (Rhors et al., 1995

and Khamanga et al., 2007).

Table 5. In-vitro results for apparatus 3 dips/minute determination for Uniphyl® Tablets.

Lot # JSH1-032 5 dips/min

JSH1-047 7 dips/min

JSH1-038 10 dips/min

JSH1-021 15 dips/min b

Time Point (hr) % Dissolved a (SD) 1 10 (0.6) 10.3 (0.5) 12.7 (0.7) 13.9 2 14.7 (0.6) 16.3 (1.1) 19.3 (1.3) 23.3 4 23.0 (1.3) 27.4 (1.0) 30.8 (3.2) 38.2 5 26.7 (1.6) 32.5 (0.7) 35.9 (4.1) 59.0 8 36.7 (2.8) 44.5 (1.1) 49.3 (5.3) 75.8

12 47.8 (4.8) 58.1 (1.6) 64.3 (5.0) 93.1 24 71.5 (7.4) 87.1 (1.4) 93.7 (2.6) 120.4

SD = Standard deviation a n=6 tablets b Note: Original data wasn’t available to calculate the standard deviation for error bars on JSH1-021 – 15 dips/min.

38

Figure 13. Apparatus 3 dips/minute determination using Uniphyl® Tablets in phosphate pH 6.6

medium at 5 dips/min – JSH1-032 (■), 7 dips/min – JSH1-047 (×), 10 dips/min – JSH1-038 (▲),

and 15 dips/min – JSH1-021 (♦).

0

20

40

60

80

100

120

0 5 10 15 20 25Time (hr)

% D

isso

lved

Figure 14. Correlation for 5 dips/minute (A), 7 dips/minute (B), and 10 dips/minute (C) in vitro

data using % absorbed in vivo (Maturu et al., 1986).

(A)

y = 1.206x - 5.265R2 = 0.9913

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35

% Dissolved - in vitro

% A

bsor

bed

- in

vivo

39

(B)

y = 0.8526x - 1.578R2 = 0.9926

0

5

10

15

20

25

30

35

0 10 20 30 40 50

% Dissolved - in vitro

% A

bsor

bed

- in

vivo

(C)

y = 0.8192x - 3.316R2 = 0.9933

0

5

10

15

20

25

30

35

0 10 20 30 40 50

% Dissolved in vitro

% A

bsor

bed

- in

vivo

40

Apparatus 3 Screen Selection

The cylinders which house the unit dose (tablet or capsule) during a dissolution test using an

Apparatus 3 require a mesh screen on the bottom on which the unit dose rests while the cylinder

reciprocates up and down in the media (as depicted in Figure 6). A mesh screen can be placed

on the top of the glass cylinder as well. Two different mesh screens were evaluated, a #20 mesh

stainless steel and a #40 mesh polypropylene. Both screens were evaluated at 5 dips/minute and

10 dips/minute in pH 6.6 phosphate buffer (Figure 15).

At 5 dips/minute, the dissolution profiles are practically identical for both the 20 mesh and

the 40 mesh screens. At 10 dips/minute the profiles are similar with a very slight increase in %

dissolved for the 20 mesh at the 2-12 hour time points (Figure 15). The 20 mesh stainless steel

screens were chosen for the remaining Apparatus 3 evaluations based on its durability and ease

in assembly with reciprocating glass cylinder. Rohrs et al., 1995 also reported no effect on

release rate when evaluating different bottom screen mesh sizes when evaluating the drug release

rate on six different hydrophilic matrix formulations and one coated-bead formulation. In their

study they also evaluated top mesh screen size and dips/minute and also reported no significant

difference in the amount release when evaluating the bottom screen. However, the top mesh

screen had an effect on the formulations containing an erosion controlled drug release

mechanism (Rhors et al., 1995).

41

Figure 15. Apparatus 3 screen selection for Uniphyl® Tablets in phosphate buffer pH 6.6 at 5

dips/minute (A) and 10 dips/minute (B) using stainless steel 20 mesh (♦) and a polypropylene 40

mesh (■) screens.

(A)

0

20

40

60

80

0 5 10 15 20 25 30

Time (hr)

% D

isso

lved

(B)

0

20

40

60

80

100

0 5 10 15 20 25 30

Time (hr)

% D

isso

lved

Simulated Fasted Conditions / Apparatus 1 (Baskets) and 2 (Paddles)

To simulate physiologic fasted conditions in the stomach and upper small intestine, simple

aqueous solutions (without enzymes) in 1-liter vessels were used as described in the USP.

Preliminary in vitro evaluations of the Uniphyl® Tablets consisted of performing dissolution

42

studies in an Apparatus 1 and 2. The USP monograph test 6 for theophylline extended release

capsules was used a general guideline as well as AAIPharma in-house test procedures (TPs

21913B and 23599). Six tablets were tested in pH 6.6 potassium phosphate monobasic buffer

with spindle rotation speed at 100 rpms. Sample times included 1, 2, 4, 5, 6, 8, 10, 12, 14, and

24 hour pull points. No replenishment of medium was performed during the test. A 0.45 µm

nylon syringe filter was used to filter the samples prior to analyzing with a UV

spectrophotometer.

Dissolution tests were performed twice using an Apparatus 2 (paddles) and once using the

Apparatus 1 (baskets) using n= 6 tablets in pH 6.6 potassium phosphate medium. Complete

dissolution (80-100% release of theophylline) was not achieved using either of these apparatuses

even with an infinity time point (sample collected after 15 minutes of rotations at 250 rpm) after

the 24 hour time point. These tests were conducted as initial studies with the Uniphyl® Tablets

and to aid in development of Apparatus 3 methods and to later compare with results collected

from an Apparatus 3. The results were plotted and are shown in Figures 16 and 17. Karim et al.,

1985 also performed an in vitro dissolution of Uniphyl® Tablets in an Apparatus 1 and data from

this reference are plotted in Figure 17 for comparison.

43

Figure 16. In vitro dissolution of Uniphyl® Tablets using Apparatus 1 in pH 6.6 phosphate

buffer, test 1 - JSH2-011 (♦) and test 2 - JSH1-083 (■).

0

20

40

60

80

0 5 10 15 20 25 30

Time (hr)

% D

isso

lved

Figure 17. In vitro dissolution of Uniphyl® Tablets in pH 6.6 phosphate buffer using an

Apparatus 1 (♦) vs. Apparatus 2 (■) with dissolution profile from Karim et al 1985 (▲) included

for comparison.

0

20

40

60

80

0 5 10 15 20 25 30

Time (hour)

% D

isso

lved

44

The data compares well with data included in the Summary Basis of Approval (SBA)

(discussed in Test Article section) and meets the acceptance criteria in the SBA with exception

of the 4 hour time point (Table 6).

Table 6. Comparison of FDA accepted data and specifications for Uniphyl® Tablets collected

from the SBA and JSH1-009.

Time (hr) % Dissolved Data

(n=12 from Uniphyl® SBA)

Specification (% Dissolved) for Uniphyl® Tablets Upon

Approval to Market

% Dissolved Data for Test JSH1-009

(n=6) 1 4.6 6-11 10.3 2 10.4 9-17 15.4 3 14.5 -- -- 4 17.8 14-24 25.4 6 23.6 -- 28.0 8 -- 20-34 32.7 9 30.3 -- -- 12 36.5 28-42 40.6 18 -- 38-52 -- 24 -- 45-61 59.2 SBA = Summary basis of Approval -- = no data collected at these time points

Simulated Fasted Conditions / Apparatus 3

Potassium phosphate monobasic pH 6.8 media (SIF without enzymes) and hydrochloric acid

solution pH 1.2 media (SGF without enzymes) was utilized to simulate fasted conditions of the

GI tract. Additionally, the in vitro dissolution of Uniphyl® Tablets was evaluated in several

different media ranging in pH. The results of these tests are discussed below.

Simulated GI pH

Several dissolution tests were performed using the Apparatus 3 in different media with

varying pH. Three to six tablets were tested during each dissolution test. Table 7 lists the

different media used along with lot number and pH. An in vitro dissolution test was performed

45

on Uniphyl Tablets in each of the following media, pH 1.2 HCl, pH 3.0 potassium phosphate

monobasic, pH 4.5 acetate, pH 6.6, pH 6.8 and pH 7.8 potassium phosphate monobasic. Other

parameters for these tests included 7 dips/minute, hold time was 0 seconds, drip time was 10

seconds and the dip time intervals were at various time points for a maximum of 24 hours. The

media temperature was kept at 37°C ± 0.5°C as defined in the general chapter <711> on

Dissolution in the USP (The United States Pharmacopoeia, 2008 <711>).

Table 7. Apparatus 3 tests utilizing media of varying pH to simulate the conditions of the gastro-

intestinal tract under fasted conditions.

Lot number(s) [data collected]

Media used pH Figure(s)

JSH1-065A [4 hrs] JSH1-092A [8 hrs] JSH2-078 [24 hrs]

0.1N HCl 1.2 18, 19

JSH1-088 [6 hrs] 50 mmol potassium phosphate monobasic 3.0 18

JSH1-065B [4 hrs] JSH1-092B [8 hrs]

sodium acetate 4.5 18

JSH1-021 [24 hrs] JSH1-032 [24 hrs] JSH1-038 [24 hrs] JSH1-047 [24 hrs]

Potassium phosphate monobasic 6.6 18

JSH1-071A [8 hrs] JSH1-100 [8 hrs] JSH2-072 [24 hrs]

Potassium phosphate monobasic 6.8 18, 20

JSH1-071B [8 hrs] Potassium phosphate monobasic 7.8 18

Table 8 and Figure 18 shows the results up to 8 hours for each of these media evaluations.

The dissolution profile up to 24 hours in pH 1.2 and pH 6.8 are plotted in Figures 19 and 20,

respectively, along with earlier profiles in the same media which were initially collected up to 8

hours. The 8 hour and 24 hour profiles are considered similar when comparing using fit factors

46

(Figure 19, pH 1.2, f1 = 9 and f2 = 81) (Figure 20, pH 6.8, f1 = 4 and f2 = 91). According to the

FDA Guidance for Industry Dissolution Testing of Immediate Release Solid Oral Dosage Forms

August 1997, two dissolution curves are considered similar if f1 is closer to 0 (0-15) and f2 is

closer to 100 (50-100). Based on the similarity of the 8 and 24 hour profiles [pH 1.2 (Figure 19)

and pH 6.8 (Figure 20)], dissolution profiles were not repeated to collect 24 hour profiles in pH

3.0, 4.5, and 7.8.

According to the data provided in Table 8 and as can be noted from Figure 18, Uniphyl®

Tablets appear to dissolve very slightly faster in pH 1.2 media than in pH 6.8 media. However,

the curves are similar according to a fit factor test (Moore and Flanner, 1996), f1 = 10 and f2 =

67.

47

Table 8. In vitro dissolution data of Uniphyl® Tablets tested in an Apparatus 3 evaluating media of varying pH of the GI-tract. Lot #

JSH1-065A1

pH 1.2

JSH1-092A1

pH 1.2

JSH2-0782

pH 1.2

JSH1-088

pH 3.0

JSH1-065B

pH 4.5

JSH1-092B

pH 4.5

JSH1-0212,3

pH 6.6

JSH1-0322

pH 6.6

JSH1-0382

pH 6.6

JSH1-0472

pH 6.6

JSH1-071A1

pH 6.8

JSH1-100

pH 6.8

JSH2-0722,3

pH 6.8

JSH1-071B1

pH 7.8

JSH2-015 pH

range

Time Point (hr)

% Dissolved3 (SD)

1 12.4 (0.3)

12.2 (0.1)

11.9 (0.1)

11.8 (0.2)

11.7 (0.5)

11.3 (0.1) 13.9 10.0

(0.6) 12.7 (0.7)

10.3 (0.5)

10.2 (0.4)

10.1 (0.2) 9.9 11.4

(0.2) --

2 20.6 (0.3)

20.1 (0.1)

18.8 (0.2)

19.1 (0.2)

19.3 (0.6)

18.1 (0.3) 23.3 14.7

(0.6) 19.3 (1.3)

16.3 (1.1)

16.6 (1.1)

16.2 (0.2) 15.9 18.4

(0.3) 19.8 (0.4)

3 27 (0.6)

26.5 (0.2) -- 25

(0.4) 25.4 (0.9)

25 (0.1) -- -- -- -- 21.2

(1.6) 21.7 (0.4) -- 23.5

(0.8) 25.8 (0.7)

3.5 -- -- 27.4 (0.5) -- -- -- -- -- -- -- -- -- 23.3 -- --

4 33 (0.8)

32.3 (0.2) -- 29.9

(0.4) 30.6 (1.4)

31.3 (0.3) 38.2 23.1

(1.3) 30.8 (3.2)

27.5 (1.0)

25.4 (2.3)

26.6 (0.6) -- 28.1

(0.9) 31.2 (0.6)

5 -- 37.7 (0.2)

34.3 (0.6) -- -- 37.4

(0.3) 59.0 26.8 (1.6)

35.9 (4.1)

32.6 (0.7)

29.2 (3.0)

31 (1.2) 29.5 32.3

(1.5) --

6 -- 42.1 (0.2) -- 37.6

(0.8) -- 42.5 (0.5) -- -- -- -- 33.2

(3.1) -- -- 36.3 (1.8)

39.5 (0.8)

7 -- -- 42.8 (0.9) -- -- -- -- -- -- -- -- -- 36.6 -- --

8 -- 50.3 (0.8) -- -- -- 51.3

(0.9) 75.8 36.8 (2.8)

49.3 (5.3)

44.6 (1.1)

39.6 (3.0)

42.1 (1.7) -- 42.7

(2.4) 47

(1.2)

9 -- -- 50.1 (1.0) -- -- -- -- -- -- -- -- -- 42.8 -- --

12 -- -- 60.5 (1.0) --- -- -- 93.1 47.9

(4.8) 64.3 (5.0)

58.2 (1.6) -- -- 52.4 -- 60

(1.9)

16 -- -- 72.1 (1.1) -- -- -- -- -- -- -- -- -- 64.6 -- --

24 -- -- 90.1 (1.6) -- -- -- 120.4 71.5

(7.4) 93.8 (2.6)

87.2 (1.4) -- -- 86.1 -- --

SD = Standard deviation -- = No data collected at these time points 1 n=3 tablets 2 n=6 tablets 3 Standard deviations not calculated. Did not have original data (individual data points).

48

Figure 18. Apparatus 3 in vitro dissolution profiles of Uniphyl® Tablets in pH 1.2 – JSH1-092A

(♦), pH 2.9 – JSH1-088 (■), pH 4.5 – JSH1-092B (▲), pH 6.6 – JSH1-047 (×), pH 6.8 – JSH1-

100 (◊), and pH 7.8 – JSH1-071 (□) media; pH 1.2 (JSH2-078) and pH 6.8 (JSH2-072) profiles

are similar (f1 = 10 and f2 = 67).

0

20

40

60

0 2 4 6 8 10

Time (hour)

% D

isso

lved

pH 1.2 - JSH1-092A pH 2.9 - JSH1-088 pH 4.5 -JSH1-092BpH 6.6 - JSH1-047 pH 6.8 -JSH1-100 pH 7.8 - JSH1-071

Figure 19. Apparatus 3 in vitro dissolution profiles of Uniphyl® Tablets in pH 1.2 medium (7

dips/minute) for tests JSH2-078 (♦) and JSH1-092 (■); f1 = 9 and f2 = 81.

0

20

40

60

80

100

0 5 10 15 20 25 30

Time (hour)

% D

isso

lved

49

Figure 20. Apparatus 3 in vitro dissolution profiles of Uniphyl® Tablets in pH 6.8 medium (7

dips/minute) for tests JSH1-100 (■), and JSH2-072 (▲); f1 = 4 and f2 = 91.

0

20

40

60

80

100

0 5 10 15 20 25 30

Time (hr)

% D

isso

lved

Range of pH (1.2 – 7.8)

A test (JSH2-015) was performed utilizing each of these media ranging the pH from 1.2-7.8

utilized in rows 1-6 similar to the fasted conditions of the GI tract (Figure 1) to determine the

drug’s dissolution dependence on pH. The data is included in Table 8 and plotted in Figure 21

along with pH 1.2 (JSH2-078) and pH 6.8 (JSH2-072), which were held constant over 24 hours.

Changing the pH over 12 hours did not have an effect on the release of theophylline in Uniphyl®

Tablets. This was also shown by Maturu et al., 1986, Karim et al., 1985 and Wearley et al., 1985

in either the NF XIII rotating bottle apparatus, Apparatus 1 or 2.

50

The media utilized for this profile in the Apparatus 3 consisted of:

Row 1: HCl pH 1.2 Row 2: Potassium phosphate monobasic pH 3.0 Row 3: Sodium acetate pH 4.5 Row 4: Potassium phosphate monobasic pH 6.6 Row 5: Potassium phosphate monobasic pH 6.6 Row 6: Potassium phosphate monobasic pH 7.8

Other parameters for this test included, 7 dips/minute, hold time was 0 seconds, drip time

was 10 seconds and pull points were 2, 3, 4, 6, 8 and 12 hours

Figure 21. Apparatus 3 in vitro dissolution profile in media ranging from pH 1.2 – pH 7.8 -

JSH2-015 (♦) plotted with profiles in pH 6.8 - JSH2-072 (■), and pH 1.2 - JSH2-078 (▲) for

comparison.

0

20

40

60

80

100

0 5 10 15 20 25 30

Time (hr)

% D

isso

lved

Simulated Fed Conditions / Apparatus 2 (Paddles)

In order to simulate fed physiologic conditions in the intestine a marketed emulsion product,

Liposyn® III 10%, intended for use as parenteral nutrition, consisting of 10% soybean oil was

utilized. One test (JSH2-049) was conducted in an Apparatus 2 for comparison with the in-vitro

51

results from the Apparatus 3 under simulated fed conditions. Three tablets were tested in

volumes of 400-600 mL of medium at 37°C ± 5°C. The spindle rotation was 100 rpm. Sample

times were 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24 hours and infinity (additional 15 minutes at 250

rpm). No replenishment was performed. The samples were filtered through a 0.45 µm nylon

syringe filter and analyzed using a UV spectrophotometer. JSH2-049 (Liposyn, Apparatus 2)

was performed on n=3 tablets. However, only n=1 tablet (vessel 3) is plotted here because 600

mL of media was used and 24 hours of data obtained. For the other two tablets (vessels 1 and 2)

400 mL of media was used and not all of the time points were obtained due to sampling error

whereas, the autosamplers were unable to reach enough sample.

Figure 22 shows the simulated fasted and simulated fed profiles in the Apparatus 2 are

similar up to the 8 hour time point (f1 = 3 and f2 = 94) but the simulated fed profile begins to

release faster after the 8 hour time point (f1 = 19 and f2 = 57).

Figure 22. Uniphyl® Tablet in vitro dissolution profile tested in an Apparatus 2 in Liposyn®

medium (simulated fed) – JSH2-049 (♦) and plotted with in vitro profile in pH 6.6 medium

(simulated fasted) – JSH1-013 (■),

0

20

40

60

80

100

0 5 10 15 20 25 30

Time (hr)

% D

isso

lved

52

Simulated Fed Conditions / Apparatus 3

The main objective of this research was to test fatty emulsions as a dissolution medium to

simulate fed conditions and evaluating the dissolution profiles of a controlled release product in

an Apparatus 3. Therefore, dissolution profiles were performed using Liposyn® 10% fatty

soybean oil and Intralipid® 30% soybean oil to emulate a bio-relevant environment in-vitro.

Additionally, an oil soak method similar to Maturu et al. 1986 was conducted in an Apparatus 3.

The data from these simulated “fed” dissolution profiles are collected in tabular format in Table

9. The simulated “fed” profiles are plotted in Figure 23.

Oil Soak

Some researchers have been able to mimic in vivo behavior by soaking the dosage form in

oil prior to performing the dissolution test. Maturu et al., 1986 evaluated the influence of a high

fat breakfast on the bioavailability of different controlled-release theophylline commercial

products. Uniphyl® was one of the formulations evaluated in this paper. The in vivo data was

compared to in vitro evaluation and they were able to obtain IVIVCs for each of the four

products. The in vitro method to simulate a high fat breakfast consisted of shaking the tablet

with peanut oil (10 mL) for 2 hours. The tablets were removed from the oil and the dissolution

profile was determined using digestive enzymes and the NF XIII in vitro dissolution test

procedure for timed-release tablets and capsules. El Arini et al., 1989 also modified the method

that Maturu et al. had developed by placing the capsule contents into a 40-mesh basket and

immersing it in a small beaker with 10 mL of peanut oil. The beaker was in a water bath which

was heated to 37°C and gently agitated horizontally for one hour.

For the dissolution test performed for this research, the Uniphyl® tablets were placed into a

test tube containing 10 mL of peanut oil and shaken for 1 hour (on a rotating device). Then the

53

oil was gently blotted from the tablets and placed into the bottom mesh screen of the cylinder

and the dissolution was performed in SIF without enzymes (pH 6.8 potassium phosphate

monobasic media), n=3 tablets at 7 dips/minute. Sample times were 1, 2, 5.5, 9.5, 12.5, 21.5 and

24.5 hour. Hold times were 0 seconds and drain time was 10 seconds for all rows. Figure 23

shows the dissolution profile is different from the simulated fasted profile in pH 6.8 (f1 = 28 and

f2 = 47).

Additionally, dissolution was completed (n=3 tablets) using SGF without enzymes (pH 1.2

HCl) in rows 1 and 2 for the first 5.5 hours (sample times 1, 2 and 5.5 hours), then SIF without

enzymes (pH 6.8) in rows 3, 4 and 5 for the remainder of the test (sample times 9.5, 12.5, 21.5

and 24.5 hours. The other parameters were the same as mentioned above. There was no

significant difference (f1 = 3 and f2 = 86) between the two curves, further supporting that

Uniphyl® Tablets are pH independent (Figure 23).

Emulsion

The in vitro dissolution studies using Liposyn® as the medium utilized the following

parameters on the Apparatus 3: row 1 medium: Liposyn III 10%, rows 3 – 6 medium:

Potassium phosphate monobasic pH 6.8, 7 or 10 dips/minute, hold time of 0 seconds, drip time

of 10 seconds, pull points varied to a maximum of 24 hours. Row 2 varied in the hold time and

drip time parameters as this row was utilized as a “rinse” process to minimize the number of

samples required for dilution when analyzing by UV spectrophotometer. Row 3 was also

utilized as a “rinse” row in some tests and consisted of pH 6.8 potassium phosphate monobasic

media. Sample preparation included a dilution by almost 10 fold with isopropyl alcohol (IPA) in

order to break up the emulsion and make a clear solution in order for the UV analysis.

54

Figure 23. Uniphyl® Tablet in vitro dissolution profiles under simulated fed conditions using an

oil soak method in an Apparatus 3 - JSH2-062A pH 6.8 (♦) and JSH2-062B pH1.2 and 6.8 (■)

[(f1 = 3 and f2 = 86)]. In vitro dissolution profiles under simulated fasted conditions [(JSH2-072,

pH 6.8 (▲)] are also plotted for comparison [(f1 = 28 and f2 = 47)].

0

20

40

60

80

100

120

0 5 10 15 20 25 30

Time (hr)

% D

isso

lved

The same parameters were utilized for Intralipid® as was used for Liposyn® dissolutions,

with exception of the preparation of the samples and standards. The higher percentage of

fats/lipids required more dilution with IPA.

Results show that the higher soybean oil content in Intralipid® 30% did not have a

significant effect on the dissolution profile when compared to the dissolution profiles obtained in

Liposyn® which has 10% soybean oil content (Table 9, Figures 24 and 25) [(f1 = 1 and f2 = 98)].

Figure 25 combines lots JSH2-099 and JSH3-009 for the Liposyn® profile and also combines

lots JSH3-019 and JSH3-029 for the Intralipid® profile to provide n=12. The Intralipid®

required more dilution of the samples in order to be analyzed by UV.

55

Both the oil soak and emulsion methods exhibited an increase in release of theophylline

drug from Uniphyl® Tablets in comparison to the dissolution profile in pH 6.8 media (SIF). The

oil soak profile (JSH2-062) began to show a significant increase after 9 hours whereas the

emulsion profiles began to show a significant increase after 5 hours.

Table 9. In vitro data of Uniphyl® Tablets in simulated fed conditions using Apparatus 3.

Lot # JSH2-

062A1,5 JSH2-062B2,5

JSH2-0913,6

JSH2-0993,6

JSH3-0093,6,7

JSH3-0194,6

JSH3-0294,6

Media Time Point

(hr) % Dissolved3

Emulsion 1 12.7 15.2 11.9 14.1 12.2 7.9 5.4 Emulsion 2 19.6 22.6 19.1 22.2 19.5 15.8 15.9 Emulsion 3.5 -- -- 27.4 27 28.9 23.2 23.7 Emulsion 5 36.1 41.0 35.5 34.9 37.5 30.8 31.5

pH 6.8 7 -- -- 45.3 49.4 52.1 48.7 49.7 pH 6.8 9 50.7 54.9 53.4 57.6 60.2 57.1 58.2 pH 6.8 12 74.9 78.8 65.8 68.0 71.0 67.9 68.8 pH 6.8 16 -- -- - 80.1 83.9 80.6 81.7 pH 6.8 24 102.6 104.5 - 98.9 101.5 102.1 101.0

-- = No data collected at these time points. 1 Oil Soak/pH 6.8 2 Oil Soak/pH 1.2 and 6.8 3 Emulsion was Liposyn III 10% 4 Emulsion was Intralipid 30% 5 n=3 tablets 6 n=6 tablets 7 Did not record weights of standards. Used 100% of target weight.

56

Figure 24. Uniphyl® Tablet in vitro dissolution profiles under simulated fed conditions in an

Apparatus 3.

0

20

40

60

80

100

120

0 5 10 15 20 25 30

TIME(HR)

% D

ISSO

LVED

JSH2-099 - Liposyn/pH 6.8 JSH2-091 - Liposyn/pH 6.8JSH3-009 - Liposyn/pH 6.8 JSH3-019 - Intralipid/pH 6.8JSH3-029 - Intralipid/pH 6.8 JSH2-062A - Oil Soak/pH6.8JSH2-062B - Oil Soak/pH1.2 and pH6.8

Figure 25. Uniphyl® Tablet in vitro dissolution profiles under simulated fed conditions –

Liposyn® (n=12) (■) and Intralipid® (n=12) (▲) [10% oil vs. 30% oil; f1 = 1 and f2 = 98] versus

simulated fasted conditions [JSH2-072, pH 6.8 (♦)] are also plotted for comparison [(Liposyn vs.

pH 6.8; f1 = 25 and f2 = 50)(Intralipid® vs pH 6.8; f1 = 17 and f2 = 65)] in an Apparatus 3.

0

20

40

60

80

100

120

0 5 10 15 20 25 30

TIME(HR)

% D

ISSO

LVED

57

Comparison of In Vitro Data with Reported In Vivo Data

Reported in vivo data (Table 3, % absorbed calculated from Karim et al., 1985) was plotted

against the % released in vitro data to evaluate the usefulness of emulsion as a biorelevent

medium in predicting in vivo behavior using an Apparatus 3. Good correlations (Level A) were

obtained with the both the fasted (SIF and SGF without enzymes) and fed (emulsion) in vitro

dissolution data collected during this research.

Additionally the in vitro data from the initial studies performed in Apparatus 2 were plotted

against the reported in vivo data as well and used to compare with the data obtained using the

Apparatus 3. Table 10 provides the R2 value for each correlation reported.

Table 10. R2 value for correlations with Apparatus 2 and 3 simulated fasted and fed conditions.

Apparatus 2 R2

Apparatus 3 R2

Lot #/Media Simulated Fasted Simulated Fed Simulated

Fasted Simulated Fed

JSH1-013 pH 6.6 0.964 -- -- --

JSH2-049 Liposyn® -- 0.959 -- --

JSH2-072 pH 6.8 -- -- 0.934 --

JSH2-078 pH 1.2 -- -- 0.967 --

JSH2-099 Liposyn® -- -- -- 0.978

JSH3-009 Liposyn® -- -- -- 0.989

JSH3-019 Intralipid® -- -- -- 0.981

JSH3-029 Intralipid® -- -- -- 0.989

-- = Not applicable

58

Simulated Fasted Conditions

Apparatus 2

Figure 26 shows that even though the Apparatus 2 method in pH 6.6 media results in

approximately 60% recovery after 24 hours in SIF without enzymes (Figure 17), there is still a

good Level A correlation (R2 > 0.9) according to the R2 value (0.964). Figure 27 includes the

plot for the % dissolved of Uniphyl® Tablets in an Apparatus 1 with reported % absorbed (Table

3). The correlation is slightly more linear with an R2 value of 0.971.

Figure 26. Correlation for Uniphyl® Tablets in vitro data (JSH1-013 - ♦) using Apparatus 2 in

pH 6.6 medium vs. reported fasted in vivo data (Karim et al., 1985).

y = 0.671x + 2.80R2 = 0.964

0

20

40

60

80

100

0 10 20 30 40 50 60 70


% A

bsor

bed

in v

ivo

59



y = 0.7528x + 1.489R2 = 0.9706

0

20

40

60

80

100

0 20 40 60 80


% A

bsor

bed

in v

ivo

Apparatus 3

Figures 28 and 29 demonstrate that the dissolution profiles tested in an Apparatus 3 in pH

1.2 and pH 6.8, respectively, also have good Level A correlations, R2 values of 0.967 and 0.939,

respectively. In pH 6.8 (6.6 for Apparatus 2) the Apparatus 2 provides a better correlation in

simulated fasted condition of the small intestine based on the R2 value. The R2 value for pH 1.2

media in an Apparatus 3 is better than the pH 6.8 (R2 = 0.967 and 0.934, respectively).

60



y = 0.4424x + 5.763R2 = 0.9665

0

20

40

60

80

100

0 20 40 60 80 100

% Dissolved in-vitro

% A

bsor

bed

in-v

ivo



y = 0.4467x + 8.112R2 = 0.9385

0

20

40

60

80

100

0 20 40 60 80 100


% A

bsor

bed

in v

ivo

61

Simulated Fed Conditions

Apparatus 2

Although the in vitro data collected in an Apparatus 2 did predict the increase in overall

absorption of Uniphyl® Tablets under the presence of food (JSH2-049) the correlation (R2 =

0.959) with the reported in vivo data (Table 3) was not as good as the correlation in an Apparatus

3 (R2 = 0.989). The in vitro raw data is provided in Table 11 and the correlation is provided in

Figure 30.

Table 11. In vitro data for Uniphyl® Tablets simulated fasted and fed conditions in an

Apparatus 2.

Time point (hr)

JSH1-013 Simulated Fasted

JSH2-049 Simulated Fed

1 10.3 12.2 2 16.9 13.3 3 -- 17.7 4 24.4 23.2 5 27.5 -- 6 30.3 30.1 8 35.2 36.7 10 39.6 -- 12 43.7 50.9 14 47.5 -- 16 -- 64.8 20 -- 77.3 24 64.7 83.2

-- = No data collected at these time points

62


Liposyn® medium vs. reported fed in vivo data (Karim et al., 1985).

y = 0.989x + 0.834R2 = 0.959

0

20

40

60

80

100

0 20 40 60 80 100


% A

bsor

bed

in v

ivo

Apparatus 3

Figure 31 includes the IVIVC correlation plots for the analysis of Uniphyl® Tablets using

Apparatus 3 in simulated fed conditions with the Liposyn® emulsion. Lot JSH2-091 was not

plotted because only up to 12 hr in vitro data was available for analysis. Figure 32 includes the

IVIVC correlation plots for the analysis of Uniphyl® Tablets using Intralipid® emulsion as

simulated fed conditions. Good linear correlations for all tests performed in both emulsions

show that this medium can be used to predict in vivo behavior. The R2 values are nearly identical

for the 10% (Liposyn®) and 30% (Intralipid®) fatty acid emulsion, therefore increasing the fat

content in the emulsion did not improve the predictability of in vivo behavior for Uniphyl®

Tablets tested in an Apparatus 3.

63

Figure 31. Correlation for Uniphyl® Tablets in vitro data in Liposyn® medium [JSH2-099 (A)

and JSH3-009 (B)] using Apparatus 3 vs. reported fed in vivo data (Karim et al., 1985).

(A)

y = 0.8855x - 6.370R2 = 0.9799

0

20

40

60

80

100

0 20 40 60 80 100 120


% A

bsor

bed

in v

ivo

(B)

y = 0.8457x - 5.740R2 = 0.9894

0

20

40

60

80

100

0 20 40 60 80 100 120


% A

bsor

bed

in v

ivo

64

Figure 32. Correlation for Uniphyl® Tablets in vitro data in Intralipid® medium [JSH3-019 (A)

and JSH3-029 (B)] using Apparatus 3 vs. reported fed in vivo data (Karim et al., 1985).

(A)

y = 0.7964x - 0.2081R2 = 0.9814

0

20

40

60

80

100

0 20 40 60 80 100 120


% A

bsor

bed

in v

ivo

(B)

y = 0.804x - 0.971R2 = 0.986

0

20

40

60

80

100

0 20 40 60 80 100 120


% A

bsor

bed

in v

ivo

65

CONCLUSIONS

It is difficult to predict the in vivo behavior of a particular drug based on its chemical

structure because the dosage form (tablet, capsule, semi-solid, etc) and formulation (immediate

and controlled release) can play a crucial role in the amount of drug release through the gastro-

intestinal tract. Additionally, the solubility of the dosage form is important in determining

bioavailability. A good place to start testing different prototype formulations in vitro to predict

in vivo behavior is to determine pH dependence and possibly ionic effects by using solutions and

buffers mimicking the pH of the GI-tract.

Different aqueous media with varying pH (without enzymes) were utilized to mimic fasted

conditions of the GI tract. Results from the tests performed in the Apparatus 3 shows that the

release rate and amount released of Uniphyl® Tablets is not affected by changing the pH.

Additionally, a dissolution test was performed which varied the pH from pH 1.2 – 7.8 over the

course of 12 hours to mimic the pH of the GI tract from the stomach to the colon (Figure 1). No

change in the release profile was noted when comparing the profile to the release profile in pH

1.2 and pH 6.8 over 24 hours. The correlation with reported in vivo data produced a good

correlation [0.967 (pH 1.2, SGF) and 0.934 (pH 6.8, SIF)]. The correlation of the reported in

vivo data with the release profile in an Apparatus 2 was slightly better in SIF (R2 = 0.964).

According to reported in vivo data, (Karim et al., 1985) the rate and extent of absorption of

Uniphyl® Tablets are significantly increased under the presence of food. The results of this

research demonstrate that using emulsion intended for IV parenteral nutrition as a medium for in

vitro testing can help predict food effects in vivo for Uniphyl® Tablets using in Apparatus 3 (and

also in Apparatuses 1 and 2, although not as well). In both the 10% (Liposyn®) and 30%

(Intralipid®) fat content emulsions studied, good correlations 0.98 – 0.99) were obtained. The

66

Intralipid® was slightly more difficult to work with so 10% fatty emulsion is recommended for

future studies.

The results of this research show that an Apparatus 3 is more suitable than the more popular

Apparatus 1 or 2 for Uniphyl® Tablets simply based on the amount of drug released after 12 and

24 hours in aqueous buffer media (Table 8). Ordinarily, the amount of drug released should be

at least 80% for a routine quality dissolution test. The hydrodynamics (up and down movement

of media on the dosage form) of the Apparatus 3 allow for complete release of the active

ingredient for Uniphyl® Tablets as opposed to the hydrodynamics (swirling of media around the

tablet) of an Apparatus 1 or 2 which showed significantly lower amount of release even after 24

hours and an additional 15 minutes at 250 rpm. Furthermore, the in vitro results from tests in an

Apparatus 2 did not show a significant difference until after 8 hours (Figure 23) but the App 3

showed a difference sooner (Figure 24) it is possible that for this type of controlled release

formulation technology (hydrophilic matrix) of theophylline the Apparatus 3 may be a better

predictor of in vivo behavior. This is also supported by the correlation of the reported in vivo fed

data with the in vitro simulated fed conditions utilizing an Apparatus 2 (R2 = 0.959) and an

Apparatus 3 (R2 values ranged from 0.978 – 0.989).

One advantage of the Apparatus 3 over the basket (Apparatus 1) and the paddle (Apparatus

2) is the allowance of up to 6 different medias (6 rows) without having to change the media from

any vessel during the test. Additionally, USP Apparatus 3 offers the advantages of avoiding

cone formation position and wobbling of the drive shaft and mimicking the changes in

physiochemical conditions and mechanical forces experienced by products in the gastrointestinal

tract and it requires much less water and considerably fewer chemicals. An Apparatus 3 would

67

be preferred for pH dependent drugs for ease of performing dissolution profiles in a range of pH

simulating the GI tract without the cumbersome task of changing out media during the test.

For future studies, analysis by high performance liquid chromatography (HPLC) is

recommended. This would avoid the need for a clear solution (i.e. dilution of the emulsion with

IPA). According to the labeling for the marketed fatty emulsions (Liposyn® and Intralipid®) the

pH of is basic (approximately 8) which is not representative of the pH of the stomach even under

fed conditions. Given that Uniphyl® was shown to be pH-independent, dissolution profiles were

not generated with the pH adjusted to 3-7 with 1N HCl, but this testing may be useful if the drug

product is influenced by a change in pH. The addition of enzymes and bile salts to emulsion

media may also be beneficial information. Lastly, it may also be beneficial to test Uniphyl®

Tablets in other biorelevent media (emulsions with and without bile salts).

In vitro dissolution testing using emulsion media in an Apparatus 3 has proven to be a tool

that formulation scientists can use to discriminate different formulations of controlled release

dosage forms (slow, moderate and fast release profiles) and help predict in vivo behavior.

68

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development of an in vitro method to help predict...

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