Development of a Bioreactor to Simulate
Drug Adsorption from the Small
Intestines into the Blood stream
BSE-4126 Comprehensive Design Project
Drafted May 4, 2009
Purpose: The purpose of this report, at its fundamental level, is to explain the problem and our
approach to its solution. This will be accomplished by clearly defining: the problem, the scope
of work, the necessary background information, the design and its alternatives, the work plan,
and safety measures. The criteria and constraints of the project will be described as well as
evaluated in this document.
Team Name:
Hungry Hippos Engineering
Group Members:
David Morgan
Kevin Richter
*Neil Templeton*
Advisors:
Dr. Mike Zhang
Dr. Robert Grisso
EXECUTIVE SUMMARY:
Development of a Bioreactor to Simulate Drug Adsorption from
the Small Intestines into the Blood stream
When a new drug is released on the market, in general, it is 10 to 15 years in the making. Occasionally,
it takes all of 10 years to learn that the drug being developed, does not meet regulation. This represents
a monumental risk that all pharmaceutical companies take on, and underscores high prescription drug
costs for Americans.
It was determined is estimated that 30-50% of the total developmental cost for a given drug is paid in
clinical trials. All Drug testing on humans represents a significant undertaking on the company’s part,
and a great deal of its budget is tied up in insurance. To quantify this in monetary value, 50 to 300
million US dollars are spent on clinical trials, for any given drug.
Drug kinetics modeling is not a new concept, and on a basic level this task has been performed by the
pharmaceutical industry for many years. What has not been performed is the advancement of this
modeling, especially in a non invasive in vitro environment. A two compartment model (standard of
industry) to explain drug adsorption with a central excreting and a peripheral compartment tells very
little about the bioavailability of a drug. Bioavailability is analogous to Gibbs free energy, except in this
case, the question is what portion of the drug is available to actually do work.
When a drug is taken orally into the body, a great deal of the drug is never adsorbed past the small
intestine’s folds. Of the drug that makes it to the blood stream, only a portion of the drug will ever
make it be transported to the intentional site. This explains the concept of bioavailability, which in a
sense is the fundamental concern of any type of drug introduced into the body. It tells you when the
drug will be available to produce the desired effect. The multi-stage bioreactor developed by Hungry
Hippos Engineering will give pharmaceutical companies this information at lower costs and in less time.
Hungry Hippos has addressed the pharmaceutical industry’s critical challenge in research and
development: cost. Enzyme inhibitor drugs make up nearly half (47%) of drugs currently marketed
today, making it the most common type of drug. Therefore, it seemed appropriate to develop a design
that could model this type of drug. This design can effectively explain the motion of oral drugs through
the stomach, small intestines, and into the blood stream. It can show the impact of food inhibiting the
absorption of the drug through the small intestines, as well as how the stomach’s pH can alter a drug
and its associated encapsulation. Not only can the design predict the mass balance of a given drug, but
the overall rates of reaction in various stages.
Modeling after a drug well studied and still widely used, Captopril, Hungry Hippos can reproduce the invivo performance of the drug in our specific reactor design. With this solution, predictions can be made
on many types of upcoming enzyme inhibiting drugs in the pharmaceutical industry. Tests can be
performed on our bioreactor design in a controlled environment, with no risk to human patients. This
will accomplish the overall goal of this project, an increase in patient safety. This design can decrease
the risk for allergic reaction or overdosing, because scientists will better understand drug absorption at
various stages as well as the reaction kinetics representative of the human body. This technology truly is
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advantageous to pharmaceutical companies, as less clinical trials correspond with decreasing insurance
costs. Conversely, this gain can be returned to the consumer and prescription drug costs will decrease.
Details of design, cost and issues
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PROBLEM STATEMENT .......................................................................................................................7
BACKGROUND SITUATION ..................................................................................................................7
CONNECTION TO CONTEMPORARY ISSUES .........................................................................................7
SCOPE OF WORK ................................................................................................................................8
JUSTIFICATION FOR RESEARCH ...................................................................................................................8
OBJECTIVES ..........................................................................................................................................8
DELIVERABLES .......................................................................................................................................9
DESIGN CRITERIA AND CONSTRAINTS .................................................................................................9
LITERATURE REVIEW ........................................................................................................................ 10
GENERAL INFORMATION ........................................................................................................................ 10
DRUG ADSORPTION .............................................................................................................................. 11
PHARMACOKINETICS ............................................................................................................................. 12
CAPTOPRIL KINETICS ............................................................................................................................. 14
ORAL DRUG INFORMATION .................................................................................................................... 16
CAPTOPRIL INFORMATION ...................................................................................................................... 17
STOMACH AND SMALL INTESTINES ANATOMY ............................................................................................. 18
GENERAL........................................................................................................................................................ 18
STOMACH ...................................................................................................................................................... 19
SMALL INTESTINE............................................................................................................................................. 20
MICROORGANISMS .......................................................................................................................................... 20
OTHER CONSIDERATIONS .................................................................................................................................. 21
SAFETY AND REGULATIONS ..................................................................................................................... 21
FEDERAL REGULATIONS .................................................................................................................................... 21
ANIMAL TESTING ............................................................................................................................................. 22
GLOBAL ENVIRONMENT ................................................................................................................... 22
SAFETY ASSESSMENT ....................................................................................................................... 22
ENVIRONMENTAL CONSIDERATIONS ................................................................................................ 23
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ALTERNATE DESIGNS ........................................................................................................................ 24
STOMACH/ENZYMATIC SI MODELS .......................................................................................................... 25
OTHER CONSIDERATIONS ....................................................................................................................... 26
PROJECT DESIGN .............................................................................................................................. 26
DESIGN 1. .......................................................................................................................................... 26
DESIGN 2 ........................................................................................................................................... 27
DESIGN 3 ........................................................................................................................................... 29
MASS BALANCES.................................................................................................................................. 30
PROJECT EVALUATION ..................................................................................................................... 32
DESIGN SCHEDULE ........................................................................................................................... 33
TIMELINE ........................................................................................................................................... 33
START AND FINISH DATES....................................................................................................................... 36
NON-ROUTINE AND ROUTINE TASKS ......................................................................................................... 36
RESOURCE CONSTRAINTS ....................................................................................................................... 37
ACCOMPLISHMENTS AND FUTURE CONSIDERATIONS ..................................................................................... 37
CONCLUSION ................................................................................................................................... 39
REFLECTIONS ................................................................................................................................... 40
APPENDIX A ..................................................................................................................................... 42
LIST OF FIGURES................................................................................................................................... 42
FIGURE 1. DIFFUSION SIMULATION. ................................................................................................................... 42
FIGURE 2. DRUG KINETICS MODEL. .................................................................................................................... 43
FIGURE 3. TWO COMPARTMENT MODEL OF DRUG KINETICS. .................................................................................. 43
FIGURE 4. GASTROINTESTINAL TRACT (MARIANA RUIZ VILLARREAL, 2006). ............................................................. 44
FIGURE 5.ANATOMY OF THE STOMACH. .............................................................................................................. 45
FIGURE 6. DUODENUM OF THE SMALL INTESTINES. ............................................................................................... 46
FIGURE 7. HIGH PRESSUREFILTRATION. ............................................................................................................... 46
FIGURE 8. HOLLOW FIBER MEMBRANE. .............................................................................................................. 47
FIGURE 9. DOUBLE ENZYME REACTOR. ............................................................................................................... 47
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FIGURE 10. SINGLE ENZYME REACTOR ................................................................................................................ 47
FIGURE 11. SIMPLIFIED BIOREACTOR DESIGN. ...................................................................................................... 48
FIGURE 12. FALL SEMESTER GANTT CHART. ......................................................................................................... 49
FIGURE 13. SPRING SEMESTER GANTT CHART. ..................................................................................................... 50
FIGURE 14. DECISION FLOWCHART..................................................................................................................... 51
FIGURE 15. BREAKDOWN OF CURRENTLY MARKETED DRUGS IN THE UNITED STATES TODAY (COPELAND, 2005)............. 52
FIGURE 16. DESIGN 1. BLOOD STREAM INHIBITION REACTOR. ............................................................................... 53
FIGURE 17. DESIGN 2. PRO-DRUG NON IMMOBILIZED REACTOR............................................................................. 54
FIGURE 19. COMPETITIVE ENZYME INHIBITION. .................................................................................................... 55
FIGURE 20. ENZYME KINETICS OF CAPTOPRIL. ...................................................................................................... 56
FIGURE 21. MASS BALANCE. ............................................................................................................................ 56
LIST OF TABLES .................................................................................................................................... 57
TABLE 1. DESIGN OF ADSORPTION MODELS. ........................................................................................................ 57
TABLE 2.DECISION MATRIX ON ALTERNATE DESIGNS. ............................................................................................. 57
TABLE 3. DESIGN OF ENZYMATIC SYSTEMS FOR STOMACH AND SMALL INTESTINE. ...................................................... 58
TABLE 4. ECONOMIC ANALYSIS. ......................................................................................................................... 59
QUALIFICATIONS .................................................................................................................................. 60
SAMPLE CALCULATIONS ......................................................................................................................... 61
ACID-BASE CALCULATIONS USING HENDERSON-HASSELBALCH EQUATION................................................................. 61
WORKS CITED ..................................................................................................................................... 63
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TITLE: DEVELOPMENT OF A BIOREACTOR TO SIMULATE DRUG ADSORPTION FROM
THE SMALL INTESTINES INTO THE BLOOD STREAM
PROBLEM STATEMENT {NOT SURE YOU HAVE DEFINED THE PROBLEM}
To improve patient safety in oral pharmaceuticals, the design of a multi-stage bioreactor will be
developed. {HOW?} The design will have a simulated stomach, small intestines, and blood
stream component where drug adsorption will take place. This system will improve
understanding of drug delivery considering aspects such as adsorption, enzyme inhibition, and
reaction kinetics.
BACKGROUND SITUATION
Medicine has provided a source of healing and comfort to people throughout time. With each
new drug released, there is rigorous clinical testing to determine the drug’s clinical uses as well
as side effects. In any clinical trials there are potential dangerous side effects and risks are
posed to the volunteer patient or the animal subjects. To limit the risks to patients, a
bioreactor simulating the changes in the stomach and the absorption/adsorption in the
intestines should be developed. Beyond just the prevention of these dangerous practices, the
pharmaceutical industry could benefit by rapidly increasing experimentation of new drug
possibilities, or document the effects of different drug combinations and observe the
absorption of the intestines in a non-harmful or intrusive way. This design could serve as a
predictor for all new drugs coming up the pipeline without any human testing. The successful
design would represent a significant cost savings for the company through a reduction in
insurance cost. The bioreactor representation of the stomach, small intestines, and absorption
into the blood stream would make this possible.
CONNECTION TO CONTEMPORARY ISSUES
Currently in our society, our bioreactor design will deal with issues such as: human and animal
testing, PETA, drug manufacturing and experimentation, and medical advances. Currently the
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cost of prescription drugs is extremely high and sometimes unaffordable for the middle class, so
a successful bioreactor design could potentially reduce the cost to consumer. With the baby
boomer generation reaching retirement age, the prescription drug cost problem is only going to
affect more and more Americans. Not to mention the supply and demand problem that will
occur when more Americans retire than any other prior generation.
This design could potentially improve the public representation of a drug company. When
money is invested in this, it could be portrayed as a commitment to the safety of the consumer.
When drug companies have to deal with the problem of removing drugs off the market (when
determined unsafe when reevaluated by the FDA) our design could provide a welcome change
of pace. Perception of a company’s commitment to safety could lead to great market share and
greater profit margins.
SCOPE OF WORK
Justification for Research
Currently, clinical trials represent between 30 and 50% of the total development costs of any
particular drug. Clinical trials are the stage where human testing actually takes place (as well as
rat testing), and is towards the end of the drug development. This represents a monetary value
of 50 to 300 million dollars, widely varying according to the type of drug.
For this reason, our bioreactor design could potentially significantly reduce this initial and
significant cost to the drug manufacturer and move the product into the market more quickly.
The design would do so by offering more information to the pharmaceutical company,
regarding drug delivery, before clinical trials ever began. Essentially the same information
acquired through blood samples (from a volunteer patient) could potentially be given by the
various stages of our bioreactor design. Less human testing leads to less insurance cost paid by
the drug manufacturer, needed to protect them in the event of a catastrophe of in human
testing (Hughes and Turner, 2002).
Objectives
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1. Increase patient-based drug testing safety This is an indirect objective
2. Better understand drug delivery (Measureable?)
3. Design and model intestine/bloodstream bioreactor
Deliverables
1. Specific Bioreactor Design
2. Mass Balance
3. Economic Plan
The deliverables were chosen based on what information a company may need to invest in the
artificial stomach and small intestine. An investor would need to know that the design is
feasible, that it has correct assumptions during the design, the measures of success, and most
importantly how much the product will cost.
DESIGN CRITERIA AND CONSTRAINTS
The determination of whether or not the chosen design (all designs mentioned in alternative
designs section) is successful is dependent on a number of criteria. With the cost of clinical
trials making up 30-50% of the overall cost for a drug’s development, it won’t be particularly
challenging to offer a cheaper alternative. Currently drug companies pay from 50-300 million
dollars in their clinical trials, and though it’s a high cost, it is a necessary cost to ensure safety
and meet the regulations from the FDA. This project is also unique in that it aims to increase
safety in drug testing, specifically for the patients volunteering. The question can reasonably be
raised in what price you are willing to pay for safety? This point is raised not to start an ethical
discussion, but to draw attention to the point that we aren’t developing a product to compete
in the marketplace. Currently the idea of a bioreactor, of any shape or form, is at best in the
research and development stage in the United States. To this team’s knowledge, no
bioreactors are in use today by pharmaceutical companies for the purpose of modeling the GI
tract. Competition would cloud the overall justifiable cost argument, but it is relatively unclear
just who the competition is at this point.
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Physically {what happen to size?}, the specific design of the bioreactor will be constrained to
match to human body as closely as possible. Therefore it will need to be 37 degrees Celsius,
which can be controlled by insulation and heat application. It will need to be able to withstand
the strong acidic environment of the stomach. It will also need to be partitioned to be the
same size as the stomach and small intestine. The reactions that occur in the stomach will need
to be emulated in the stomach section and the enzymatic changes will need to be accounted
for in the small intestine section. The adsorption will also need to occur at similar rates as the
human small intestine.
LITERATURE REVIEW
General Information
In research and development, companies of any discipline are always working to improve their
technology. Technology developed to increase safety is unique; one of the few cases where
improving efficiency is second hand (to overall safety). In this design, safety and efficiency go
together. Effectively simulating the pharmacokinetics without having to risk a patient’s life, is a
huge advance in efficiency. Insurance and legal costs are significantly decreased now that a
machine is taking the risk that a human once volunteered to do. To improve safety of drugs, as
well as decrease patient based testing, our design aims to provide a simulation that has yet to
be well established in the industry.
This design is unique in that most of the research being done in order to simulate drug delivery
actually uses living tissues or organs. For example, Fortn et al. (2001) has successfully replicate
artificial perforations by means of a bioreactor to create gastric mucosa and fibrous tissue. This
design uses a bioreactor to do the simulation. The bioreactor developed will simulate the
stomach and small intestines, as well as the blood stream. One possible way to simulate the
diffusion of a molecule into the small intestine (Figure 1), is to use a filter design (Stefanyk,
2008). Even though the small intestines and stomach represent a large portion of the digestive
system, important components are being left out. Digestion begins in the mouth. Weisbrod
insists that the mouth should be included for simulation of the digestive tract. There are even
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ways to have drugs pass through the stomach unscathed (Weisbrod, 2008), which raises the
consideration of replacing the stomach with the mouth.
Drug Adsorption
In terms of drug safety, frequently lab tests are done on a small scale to simulate different parts
of pharmacokinetics (literally the movement of drugs). Passive diffusion is predominant for
most forms of drugs. In addition, all forms of drugs must pass through several forms of
biological membranes which are composed of mainly lipids and proteins. Traditionally, the
ability of the drug to permeate across various biological membranes is evaluated by measuring
the partitioning of the drug in octanol and water systems. Octanol is a good representation of
lipid materials. Partitioning is simply the ability of a compound to distribute in two immiscible
systems. Partitioning is often a function of pH, among other things such as temperature. This
current work of partitioning using octanol as the medium will be heavily considered in
development in the bioreactor (Ghosh et al., 2005).
Typically, poor oral drug absorption has been attributed to having poor solubility or poor
membrane permeability (at a given pH). Also, physiology as well as chemistry are the two
predominant factors looked at for drug absorption. However, a new approach to
understanding drug absorption deals with the actual drug’s metabolism in the intestine. The
consideration of a counter-transport process via enzyme is also being considered. Both of
these drug interactions are believed to have a significant impact on the drug’s absorption. The
availability of a large number of drugs is believed to be impacted by these two processes (Benet
et al., 1996).
Passive diffusion occurs when there is a concentration gradient in the system. It is the natural
tendency towards equilibrium. Depending upon the concentration gradient, this passive
diffusion might occur in a quick or sluggish fashion at any given time. In the human body, often
the concentration gradient is quite minimal, so this passive diffusion might not occur fast
enough to actually make a positive impact on the body. Since the human body is incredibly
efficient, often the use of attractive or repulsive forces (often hydrophobic or hydrophilic) are
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used to speed the diffusion of a molecule through a biological membrane. These are all forms
of passive transport.
When passive diffusion cannot provide the necessary rate for molecular movement, there are
several forms of active transport that the body is known to use. In one specific form of active
transport, mentioned by Benet et al. (1996), deals with the counter-transport processes using
enzymes. In all cases, it is worthwhile mentioning that the enzyme doesn’t directly cause any
reaction to take place; it merely plays a role as a catalyst. The human body would not be able
to function without enzymes as catalysts, and when an enzyme is used, an “active” process is
occurring.
Pharmacokinetics
Drug kinetics, meaning literally the movement of drugs throughout the body, is one of the key
points that this design project will address. Tracing the path of the drug can be difficult, and in
many instances the entire system will be simplified as the stomach, small intestine, and
bloodstream (Figure 2) in order to develop mathematical models for it. Two of the most used
models are the one and two compartment system. In the one compartment model, the
assumption is made that the drug reaches rapid equilibrium throughout the body. A good
example of a rapid equilibrium type of drug is Viagra (Stewart et al., 2009). Peak drug
concentrations of Viagra in the bloodstream can be found in as little as 30 minutes. The only
factors that are considered are the drug dose going into the body, the volume of the body for
the drug to rapidly disperse to, and the elimination rate of the drug. This model works best to
simulate an injection of a drug, as the circulatory system can rapidly distribute the drug
throughout the body.
Typically the assumption is also made that the removal of the drug is directly related to current
concentration. This also means that the half-life of the drug is always going to be the same, no
matter what the concentration. Half-life is defined as the amount of time necessary for the
drug to reach half its original concentration in the body. It is an important parameter of drug
models, as it is assumed to be constant throughout the time the drug is taken to complete
removal of the drug (through excretion). The major factor that controls the accuracy of this
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model is how quickly the drug reaches equilibrium throughout the body. If equilibrium is nearly
instantaneous, then this serves as an accurate model for the drug’s metabolism. The other
model that it is actively used is the two compartment model (Gibson, 1994).
The two compartment model (Figure 3) takes into account the fact that not every part of the
body has the potential to eliminate a drug. Drug elimination is often studied in drug models
because adsorption is often more difficult to measure. If the drug is not being eliminated, then
it is most likely interacting with some components of the body. If not part of the GI tract, then
the drug has obviously been absorbed through a biological membrane. Most of adsorption
takes place in the small intestines, and minimal adsorption takes place in the stomach and
mouth. Modeling adsorption in this design project will strictly deal with the small intestines. In
conclusion, when testing humans measuring the removal of the drug is simpler; there is no
invasive testing needed and no guesswork of where the drug might be concentrated in the
body. Invasive testing is done, but it is much more time and effort intensive. A bioreactor
design is being developed to reduce the amount of invasive testing.
The major forms of drug removal are no different than any food, excretion via feces or urine.
For this reason, a compartment is taken into account for all areas of the body that cannot
eliminate the drug. This model is much more appropriate for simulations with the small
intestines and stomach, as neither can directly eliminate a drug out of the body. In this model,
more factors can be taken into account. The factors considered are: drug dose, rate of drug
delivery to non-excreting compartment, rate of drug delivery to the excreting compartment,
and the rate of drug removal. Areas of the body that are highly permeable, such as your lungs,
heart, or glands are represented by the excreting compartment. Areas of the body that are not
very permeable, such as your muscles or skin, are generally represented by the non-excreting
compartment. In the two compartment model, in addition to modeling the change of
concentration over time (like the one compartment model), the area under the concentration
time curve must be taken into account. This area describes the overall load of the drug in the
body. This “bioavailability” is an indirect representation of the therapeutic value of the drug
(Gibson, 1994).
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Kinetic order of a reaction must also be considered. When the assumption is made that the
rate of drug removal is relevant to the concentration, a first order rate of reaction is assumed.
The two compartment model, widely used in the pharmaceutical industry to explain drug
kinetics, uses a first order rate of reaction. However, when the drug concentration is high
enough, the body will remove of it at a maximum rate until a point, at which it will remove of
the drug in accordance to concentration. When it is removing the drug at maximal rate, the
relationship is zero order (Gibson 1994). Zero order reactions typically turn into a first order
reaction as the concentration gradient decreases over time.
The two largest groups of enzyme interactive drugs are pro-drugs and enzyme-inhibition drugs.
Nearly half of all currently marketed drugs are enzyme inhibitors (Copeland 2005). A pro-drug
bonds to an enzyme that speeds the reaction necessary for the substrate to change to a usable
drug molecule (it changes form). An enzyme inhibitor doesn’t typically change in substrate
form; it simply slows down a reaction that was already going to occur in the human body.
Currently there is more research on enzyme inhibiting drugs as opposed to pro-drugs, as well as
more drugs on the market.
Enzyme inhibiting drugs will be used in this design because of greater available information and
because of they will be more readily measureable in the outflow from the bioreactor. The
inhibitor is a known substrate, and since it doesn’t change (in detectable form) using
chromatography techniques, we will know what to measure for in the outlet of the reactor.
Given enough time all of the inhibitors will elute out of the small intestine design. But with the
immobilized enzymes in the column, they should elute out in a measureable quantity and a
respective disassociation constant should be able to be calculated. To determine the contents
of the outflow, samples will be taken at specific time intervals to determine how much enzyme
interaction is taking place in the small intestines. The time intervals will have to be chosen at a
later date. Depending on the drug chosen, a specific naturally produced target enzyme will be
immobilized in the column.
Captopril Kinetics
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Peak reduction of blood pressure occurs roughly 60 to 90 minutes after initial dosage of drug
(Stewart et al, 2009). It has been determined that to reduce the reaction velocity by half, a
concentration of 4.98 x 10-10 M is necessary (Copeland, 2005) in the bloodstream. Specifically,
the reaction that is being slowed by Captopril is the combination of angiotensin I with ACE to
produce angiotensin II. This can be seen in Figure 18. angiotensin I is the substrate and ACE is
the enzyme. The inhibitor, is our drug, Captopril. The half life of the drug is 30 minutes upon
entering the blood stream, so the body is quite capable of readily removing it (Stewart et al,
2009). Due to the fact that analytical chromatography is a slow process in general, this design
will not seek to remove of 50% of the drug in 30 minutes of time.
The rate constant for the forward reaction of the enzyme combining with the substrate is 1.2 x
106 M-1s-1. This is commonly referred to as k1. The rate constant for the forward reaction of the
enzyme combining with the substrate is 2.55 x 106 M-1s-1. This explains to you that at initial
conditions, if the concentration of the substrate is the same as the concentration of the
inhibitor, the enzyme will not only have a greater affinity to the inhibitor but additionally the
reaction will proceed at less than half the possible velocity. Additionally, it can be observed
that the overall rate of the reaction is certainly forward by looking at the rate coefficients.
Whether the inhibitor is added or not the reaction, it will proceed. The rate that the reaction
precedes is what is impacted by the inhibitor, and drug, Captopril. The forward rate coefficient
of the enzyme combining with the substrate is, as previously mentioned, 1.2 x 10 6 M-1s-1. With
the reverse of that being only 4 x 10-4 s-1, the forward reaction is 3 x 109 more powerful at initial
conditions (Copeland, 2005)!
The body cannot have this reaction proceeding so quickly at all given times (reaction would be
out of control) so the body uses allosteric effects regulate or moderate the reaction (Campbell
et al, 2006). The substrate and product of our design’s reaction, as mentioned previously, are
Angiotensin I and Angiotensin II. However, there are several other intermediates that
additionally are made to regulate the blood pressure. The eventual product that impacts blood
pressure is aldosterone. However, angiotensin I and angiotensin II impact blood pressure as
well, as they effect the production of aldosterone. Captopril doesn’t inhibit the production of
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aldosterone, but instead angiotensin II. For this reason, or design simplifies the system and
only deals with the angiotensin I to angiotensin II conversion. The significance of all this is that
the catalytic rate coefficient, kcat, is dynamic. Kcat describes the rate that the enzyme substrate
complex converts to the product and enzyme. The enzyme responds to the product
concentration (aldosterone) and the product works as a negative effector. A diagram of the
enzyme kinetics can be seen in figure 19.
Oral Drug Information
Orally taken drugs are the direct focus of this bioreactor simulation. Protein based drugs are
not significant in this simulation as they have not yet been developed to be able to tolerate oral
dosages. This is because they are particularly susceptible to degradation, especially if they
were to be exposed to the pH of the stomach. Much of the drug technologies on the market
today currently rely on a controlled release mechanism. There are a few different levels of
controlled drug release, and in the spirit of the current jargon, there are six different
generations of it. Generations one through three will be most heavily considered for
development products to be potentially used in the bioreactor. In many drugs on the market
today, the flatter the plasma drug concentration over a period of time, the better. Unlike
modern day medicines that might be site specific, such as protein based drugs, none of these
drugs are at that level of sophistication. Once the drug has entered the body orally, it will
follow natural biological paths into the rest of body through the bloodstream as shown in
Figure 2. Likewise, once drugs have entered the bioreactor that is under development, there
will be no control over where the highest concentration of drug will specifically go. This is
doubly frustrating as it is very difficult to measure the concentrations of any drug in different
parts of the body after it has been ingested (Ghosh, 2005).
Drug tablets can be made of surprisingly numerous materials, which could involve: polymers,
sugars, and gelatins. Depending on the purpose of the drug, one of the many options can be
chosen. Though the tablet coating process can be overall difficult to master (for a given drug),
the benefits of solid tablets are substantial. They can be accurately measured for specific
dosages, they are easily transported in bulk, and they are generally more stable than their
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liquid cousins. Understanding the materials that coat a given drug will allow for the prediction
of where the drug will become active, and how it will reach its given destination (Ghosh, 2005).
Another form of oral drug dosage is a liquid. Solutions, suspensions, and emulsions are the
main forms of orally taken liquid drugs. The predominant solvents that are added to the drugs
are water and alcohol. There are many advantages and disadvantages of oral liquids. Some of
the advantages are: the active agent is homogeneously distributed in the product and the agent
doesn’t need to be dissolved and therefore can have faster therapeutic response. Some of the
disadvantages are: the active agents are often susceptible to chemical degradation and the
solution is often susceptible to microorganisms (Ghosh, 2005).
Captopril Information
Captopril was one of the first blood pressure regulator drugs to ever hit the market, and it was
approved by the FDA in 1981. Early research actually extracted ideas for such a drug by
studying the venom of snakes. As it turns out, snake venom often contains components that
inhibit the conversion of Angiotensin I to II (Case et al, 1980). It is still widely used today for
treatment, and as almost 74 million Americans have hypertension, will likely remain popular for
years to come. It is an enzyme inhibitor, making it among the most common class of drugs
being marketed today (Figure 14). It was chosen as the drug for this design to model because
of the relative simplicity in comparison to other drugs. Pro-drugs, another common type of
drug used for high blood pressure treatment, will change forms at various stages in the body
until its eventual use. This makes pro-drugs an unattractive option for the design, because
what is introduced in the system cannot directly be measured for. Since Captopril remains the
same throughout introduction and excretion out of the body, it was chosen because it would be
easier to measure for.
Captopril works by inhibiting one specific enzyme, ACE, and nothing else. It inhibits one step of
the multiple steps that take place in our body to regulate blood pressure. Some enzyme
inhibitors show affinity to multiple enzymes, further complicating the system, making Captopril
again a logical choice. In short, choosing Captopril allowed for a more feasible design that could
produce more measureable and repeatable results. It was also chosen because of the
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popularity of enzyme inhibiting drugs on the market today. The goal of this design project is to
effectively simulate Captopril, but if this can be accomplished, the design can predict the
kinetics of a drug before ever entering a volunteering test subject.
The reason that Captopril works is because of the impact that Angiotensin I and II have on the
body. Angiotensin II is a vasoconstrictor that is three times more powerful then Angiotensin I
(Copeland, 2005). As will be explained later in the mass balance, the majority of the Captopril
never accomplishes this goal of inhibiting the production of Angiotensin II, but as it turns out
the drug doesn’t have to. The goal of Captopril is to reduce blood pressure, not cease it. If
Captopril was 100% effective, this would be of no use to medicine as it would result in patient
death. Captopril moderates the body’s natural regulatory mechanism called the reninangiotensin system. Rate coefficients of Captopril can be found in figure 19.
Stomach and Small Intestines Anatomy
GENERAL
The digestive tract in the human body is composed of many different major parts including the
mouth, stomach, small intestine, large intestine, pancreas, and liver (Figure 4). Even this is not
the entire system, and for the purposes of this design there will be even more simplification.
The stomach’s main purpose is the mechanical and chemical digestion of food. It does use
pepsinogen to form pepsin to begin the chemical digestion of proteins, but the majority of the
enzymatic digestion occurs in the small intestine. Besides the pepsin in the stomach and the
amylase in the mouth, the section of the small intestine called the duodenum uses enzymes
secreted locally and those transferred from the pancreas and liver to break down lipids, starch,
sugars, and proteins. The enzyme secretion is controlled by a variety of hormones that can
start and stop the secretion process. Bile salts from the liver are also used in to emulsify fats
for easier decomposition. Beyond the duodenum the rest of the small intestine absorbs the
nutrients like amino acids, maltose, and fatty acids. The large intestine is mainly used to
reabsorb water and salts (Hopson, 1992). For the purpose of this design the stomach and the
small intestine are the only major locations that will be focused on.
18
The movement through the tract called peristalsis is mainly controlled by muscle contraction.
Two different sphincters exist on the stomach, one at the entrance and one at the exit. The
cardiac sphincter at the opening is controlled by the swallowing motion from the esophagus as
the food is moved into the stomach. There the food becomes chime and exits through the
pyloric sphincter at an average rate of about 5 ml per sec in a squirting motion. This flow rate
will be used in the reactor to be developed. This is controlled by muscle contraction, but unlike
the cardiac sphincter is not done so consciously (Mader, 1994).
In creating stomach/small intestine bioreactor, the mouth and large intestine may be ignored.
While the secretions from the pancreas and liver can be simulated, the majority of the focus
will be on the stomach and small intestine. With the way food flows through the stomach and
small intestine, the best kind of reactor will be a batch reactor to better simulate the stomach.
The path from the small intestine functions more like a continuous reactor, the batch process
from the stomach will adequately simulate the process (Purves, 2004).
STOMACH
The lining of the stomach produces approximately 2 liters of gastric fluid daily, composed
mainly of: pooled hydrochloric acid, mucus in the layers, and pepsinogen secreted into the
system (Figure 5). The acidic nature of the stomach can change break down lipids by
peroxidation (Kanner, 2001). Proteins are one of the products largely broken down in the
stomach by pepsin created from the reaction of pepsinogen and HCl, though not absorbed.
This is largely the reason why protein based drugs have yet to advance to the level of being
administered orally. Enzyme sources, their location that they are active, and the products that
they break down are well known and studied (Starr, 2004). For example Lactase has been
widely studied in its relation to lactose intolerant people. Many people exhibit problems with
milk’s sugar lactose. Lactase, produced in the cells of the small intestine, has been studied and
shown to break down lactose to aid in digestion. While to people mentioned in National
Digestive Diseases Information Clearinghouse have problems with lactase other people who
have lactase working in their system have no trouble with milk.
19
SMALL INTESTINE
The small intestine is made up of three specific parts, the duodenum, the jejune, and the ileum.
Most of the reactions that occur in the small intestine occur in the duodenum. The pancreatic
duct (Figure 6) leads into this section with enzymes from the pancreas and liver. Most of the
adsorption that occurs in the small intestine occurs in the jejune and ileum. Some adsorption
does occur in the duodenum though, and in total the surface area where adsorption can occur
is about 550 square meters (Purves, 2004).
Due to the fact that recreating 550 square meters in a design to fit in a lab space is nearly
impossible, this design will not incorporate such a large surface area. The human body creates
such a large surface area by having a great deal of overlapping folds. Having overlapping folds
and a consistent experimental runs is highly unlikely and the technology simply does not exist.
For this design, an area of 1 m2 square meter will represent the small intestines.
Transmembrane pressure will be used to overcome the fact that the area for diffusion to take
place is going to be considerably smaller. The surface area will be considered appropriate if
expected diffusion rates are produced.
MICROORGANISMS
There are several microorganisms in the digestive system, yet only a few will be considered in
this simulation. This is because some of the microorganisms in our body are responsible for
producing enzymes that are needed for digestion. The only natural flora of the stomach is the
microorganism Heliobacter Pylori that can set up the housekeeping in the stomach because of
its strong flagella and urease enzyme which can split urea into ammonia and CO 2. The
ammonia dampens the acidic environment making it feasible to live in the stomach. Also food
can pass through the stomach due to a food bolus in certain cases (Daniel, 2008). It is nearly
impossible for any other microorganism to survive in such harsh conditions of the stomach with
the pH around 2.
The small intestines have slightly more microorganisms in them. The pH changes to nearly 4 or
5. This allows the bacteria that are Enterococci and Lactobacilli to survive in the environment.
As the small intestines continue more bacteria can survive (Brock, 2006).
20
OTHER CONSIDERATIONS
A food’s effect upon an impact of a drug can be significant. However, they are difficult to
model. Biological membrane pore size can be altered with red meats and it can allow for more
toxins to be absorbed into the system. Gorelik et al. (2008) discourages us from following food
digestion with the system. This could be a future change we could alter our model at a later
time.
Safety and Regulations
Safety in pharmaceuticals fights impossible odds to get a medicine with no chance of any side
effect. “Every effective medicine has a balance of benefit to risk and anybody taking or
prescribing a medicine, to obtain the anticipated benefit, must recognize and accept the
concomitant risk of an adverse effect” (Inman, 1986).
FEDERAL REGULATIONS
There are two organizations in the US who influence drug safety. The first organization is the
food and drug administration (FDA) and the second organization is the drug regulatory
authorities (DRAs). Ways these companies improve drug safety are by modifying the
formulation of the drugs to decrease gut irritation, aid absorption, or alter the bioavailability in
order to produce more consistent and more appropriate blood levels (Inman, 1986). The words
‘reformulations of drugs’ are strongly discouraged in drug safety improvement. The DRA would
prefer a specific area to be mentioned for drug safety. Reformulation could cause a complete
new set of side effects. The largest area that the DRA pays attention to is the Post Marketing
Surveillance (PMS). This implies that the industries must follow any ideas that relate to safety
as long as they are practical and realistic.
Prescriptions drugs have strict regulations listed due to the potential to do harm if used
incorrectly. The number one problem with prescription drugs is the prescription drugs are
being diverted for illegal uses. The Drug Abuse Warning Network (DAWN) is in charge of
making sure prescription drugs go to their proper use. It is nearly impossible to get addicts off
of prescription drugs. Therefore there are regulations on dosage, labeling, and warnings. Any
positive or negative effect known about the drug must be listed on the bottle.
21
ANIMAL TESTING
Federal regulations have strict rules on animal testing. Animal testing costs amount to
approximately $136 billion per year (Stare and George, 2008). In animal testing are there are
25-50 billion animals killed every year by drug testing. Most of these animals are rats and
hamsters. Half of those tests are done by cosmetic companies. Even the tests that are
successful have harmful effects for 61% of the people who are tested on. This is due to
enzymatic differential in animals versus humans. For instance, cats do not have the enzyme to
digest ibuprofen (Paws, 2008).
GLOBAL ENVIRONMENT
Regulations of medicines in Europe started shortly after the thalidomide affair. The
thalidomide affair was a toxicological disaster caused by several ‘competent authorities’ which
allowed the drug to make the market. The European Union was established to have a common
market for drugs on the market. This organization is decades behind the FDA which was
established in the US in the early 1900s but the EUs branch for drug safety still has very similar
regulations (Mulder, 2006).
SAFETY ASSESSMENT
Good Laboratory Practice (GLP) was developed in the 1970s to ensure quality. All US and
European countries should meet GLP standards. The only portion of pharmaceuticals that do
not follow GLP standards would be the developmental pharmacological section. (Mulder, 2006)
When assessing if the products would be safe for humans there are several different qualities
that each product must pass. For instance if it was a Vaccine, it should have minimal side
effects. Whole-cell pertussis vaccine was taken off of the market due to side effects including
convulsions, persistent, and screaming. If the product was a sleeping pill it would have a strong
effect on blood pressure. It could be pulled off the market if there was a high risk of patients
developing a dependence on the drug by taking a sleeping pill or other drug every night
(Mulder, 2006).
If there was a harmful drug identified, patients must know what medicines they were taking.
The best way to identify these drugs would be leaving them in their original packaging with
22
warning labels shown. If the drugs were not in the original packaging they would be considered
dangerous because the consumer would not be informed of active ingredients. (Inman, 1986)
The ICH, the International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use, and The ICH M3 guidelines specify that before
the first use in humans the following data should be present.

Safety pharmacology with respect to vital functions, cardiovascular, central nervous
system, and respiratory system.

Information on absorption, distribution, metabolism, and animal metabolic pathways.

Single-dose (acute) toxicity in two mammalian species. A dose-escalation study is
considered an acceptable alternative.

Repeated-dose toxicity. Tests should be done on rodents and in non-rodents.

Local tolerance should be studied, preferably in connection with other toxicity studies.

Genotoxicity data from in vitro tests: evaluations of mutations (bacterial mutagenicity,
Ames test) and of chromosomal damage (e.g. mouse lymphoma) are generally needed.
The FDA has developed the “ScreeningIND” (IND = investigational new drug) to take a volunteer
to test the pharmacokinetics in the same person before placing the new drug into their system.
Some tests cannot be altered when testing. For instance, testing maximum allowable dosage
will be tested on dogs rather than humans. (Mulder ,2006)
ENVIRONMENTAL CONSIDERATIONS
The removal of chemical wastes from Virginia Tech is a streamlined process. Similar regulations
could take place in other locations or facilities. The containers for the waste are provided from
the university, as well as the labels. Waste removal is prompt, and only requires a quick phone
call. Of all the wastes in our design, only one item is biological in nature. The enzymes that will
be used are obviously biological products. However, enzymes do not fall in the category of
biological hazards (Egan, 2008). The only significant environmental hazards in this project are
the acids, and the current infrastructure setup at Virginia Tech (VT EHSS, 2008) is experienced
and capable of handling this task.
23
The Hazardous Waste Disposal Program (a division of Environmental, Health, and Safety
Services) explains in detail how to handle the disposal of hazardous chemicals. This particular
design calls for the use of hydrochloric acid, a major component of gastric fluid, to be used.
Instructions for disposal of such acids are as follows:

Wastes should be in sealed containers without any sign of leakage

The container should be full but not overflowing

The maximum container weight is 22.7 kg (50 pounds) and volume is (18.9 liters) 5
gallons

Stored waste should be in appropriate storage cabinet, according to the chemical
compatibility

All containers must be labeled with hazardous waste label and the requested
information all completed

Waste label information of: Department, Building, Room, Name, Phone, Size, Proper
chemical name, Components of waste and their percentages, pH, Written and signed
name

Schedule a waste pickup

Send label waste copy to Environmental, Health, and Safety Services
ALTERNATE DESIGNS
Adsorption Models
The adsorption models shown below are all representing the small intestine. With the small
molecule drugs as the focus of the project, most adsorption takes place in the small intestine.
In Table 2 shown below you can see some of the advantages and drawbacks of the models.
A high pressure filter is our first and most simple design. As shown in Figure 7, this model
places the contents of the stomach on the left side of the filter and places the contents of the
bloodstream on the right side of the filter. There is a mechanical agitator on the left side of the
filter as well to increase the speed of filtration.
24
A hollow fiber membrane is a strong candidate for representing the small intestine. In Figure 8,
the hollow fiber membrane is shown in a cylindrical nature to accurately represent the physical
nature of the small intestine. One of the drawbacks of the hollow fiber membrane is the
membrane will not have uniform filtration when modeling. However, this can be accomplished
by increasing the pressure in the filter and adding a recycle element to the reactor. There are
physical and chemical constraints to the membrane, but those constraints are high heat and
corrosive substances which do not play a factor in our model.
Stomach/Enzymatic SI Models
For modeling the stomach and small intestine several things should be taken into account. For
instance, the stomach is a pretreatment phase. Due to the low pH and very acidic environment
of the stomach, the small molecule drugs are designed to pass through the stomach and get
adsorbed in the small intestine. Most of the enzymatic reactions happen at the beginning of
the small intestine in the duodenum. The list of advantages and drawbacks are shown below in
Table 3.
The double enzyme model (DEM) will be a realistic model for our system. This model will
accurately represent the stomach and small intestine from an enzymatic level. This figure is
shown by Figure 9. This is simply a packed bed of immobilized enzymes that will react with the
small molecule drug. If the drug needs a longer reacting time there could be a recycle portion
of the reactor added to the end of the packed bed and returning it to the beginning of the
column. Unfortunately, one drawback is that not all of the enzymes will play a factor in the
model.
The single enzyme model (SEM) will be less expensive than the double enzyme model. The
single enzyme model only accounts for the enzymes in the small intestine. This is a fair
assumption due to the fact that the enzymes in the stomach do not play a role in the
breakdown of the small molecule drugs. The drugs are typically designed to pass through the
stomach so it can make it to the adsorption stages of the small intestine. In Figure 10 the
model is very similar to the double enzyme model except there are no enzymes in the stomach.
This will reduce the cost of the system.
25
The majority of design analysis of this project will be performed by chromatography analysis.
Specific times that samples are taken from the bioreactor will have to be recorded. Samples
would have to be taken at the exit of the model as well as within the model. These specific
locations (where samples will be taken) will be explicitly defined next semester. Different
components and their concentration could be determined by anion exchange chromatography,
with the knowledge that different compounds of the drug measured for will elude at different
times. A spectrophotometer (commonly embedded within the chromatography model) would
also be necessary. This would provide the information necessary to construct the two
compartment model
Other Considerations
A Pig Stomach could be a very positive way to represent the model due to the stomach being a
living system. This system would include all of the natural flora, enzymes, and proper filter size.
However, there are several drawbacks that include a onetime use for each stomach, variability
in each stomach, and possible ethical issues in taking the stomach from the pig. It would also
be difficult to maintain a living system.
PROJECT DESIGN
The best design choice will be combining the single enzyme model with the perforated column
membrane. This model is shown below in Figure 11 and accurately represents the GI tract for
small molecule drug adsorption. In the GI tract the drug must pass through the stomach, then
pass through the duodenum where the drugs will be broken down, then finally be adsorbed
from the small intestine into the bloodstream. Due to the mechanical nature of this design, it
follows only the theoretical path of the GI tract. The full model shows any enzymatic or
chemical breakdown of drugs along with the adsorption stages of the GI tract.
Design 1.
Since this design follows the path through the GI tract it will start with a stomach simulation.
The stomach tank would store a working volume of 2L of HCl, salts, and the capsulated drug.
26
The tank will be held at a heat of 37 ˚C to properly simulate the heat of the body, and the pH
should be kept around 1-2. Instrumentation such as pH and temperature regulators would be
implanted here to ensure proper conditions throughout the process. After a mixing tank, a
peristaltic pump will pull out about 5 mL/s of the slurry and send it to the perforated column
membrane. The retentate from the membrane would be the waste of the drug. This would be
excreted from the body through the GI tract without affecting the body. The permeate from
the membrane would be the adsorbed drugs and enzymes that will have an effect on the body
in the blood stream. The separation is shown in Figure 1. The assumption in this model is the
enzymes are inhibited in the blood stream. Since this project would use Captopril, an orally fed
high blood pressure medicine that affects the blood cells, the assumption would be valid. To
show the enzymes in the bloodstream there would be an immobilized enzyme column to
simulate the interaction. Once the drug passed through the enzyme column, affinity
chromatography would be used to measure the amount of drug that would interact with those
enzymes. This design is shown in Figure 16.
Assumption: Enzymes are absorbed into and interaction takes place in the blood stream.
Difficulties: Though enzyme immobilization would save on enzyme costs (enzyme would be
reused), it would be difficult to modify the concentration of the enzyme. Enzyme
immobilization would have to be done by the company that the affinity column would be
purchased from, and this would take time and significant cost. (How much more?)
Recommendation: Due to the lower cost overall (enzyme cost would be the most significant
cost in this design) and simulation of food inhibition as well as enzyme drug binding in the
column, this design does receive our top recommendation. Pharmacokinetics could most
effectively be studied with this design; however, the other two designs could simulate portions
of what this design could with less possible investment. Depending upon what the client’s
exact interests would be then design 1, 2, or 3 could all be valid options.
Design 2
27
This design will work well with a pro-drug or enzyme inhibitor {Need to differentiate – Pro-drug
is mostly a protein drug}. A pro-drug requires a specific enzyme to change form and produce a
product. This product functions as a drug in the body. The fact that it can work with either
type of drug is an advantage to this design.
There will be a 2 liter tank containing a few salts such as KCl and NaCl, and a strong
concentration of HCl. The pH will be kept between the range of 1-2, and this tank will represent
the stomach. The temperature of the tank will be 37°C, regulated by a heat exchanger. The
encapsulated drug will be introduced into the stomach and then be dissolved. The stomach will
be removing the gastric fluid at a rate of 5 mL/s.
The second compartment will represent the small intestines. This tank will have to have a pH of
4-5 and be maintained at body temperature. The gastric fluid containing the drug will be mixed
with a buffer solution as well as a sugar solution containing enzymes. These will be allowed to
mix in the second compartment, and will allow sufficient time for enzyme drug interaction, and
flow to the next compartment.
In the third compartment, there will be a perforated cylinder membrane. The enzymes are
quite large in comparison to the small molecular weight pro-drug, and the products of the prodrug; therefore separation will occur. The pro-drug and its products will permeate through the
filter, and the enzymes will be forced to pass through as retentate.
The permeate will be collected and anion exchange chromatography will be run. Due to the
different charges of the product and the pro-drug, these two molecules will elute at different
rates. The pro-drug and its product often have different charges in the human body because to
cross a membrane, the product might have the proper properties to diffuse, but the pro-drug
does not have the right charge and will be forced to exit into the large intestines. This design
can be seen in figure 17.
Assumptions: The pro-drug will be assumed to have sufficient time to react with the enzymes
when in compartment two. Not all of the pro-drug will be converted into its products, just as in
28
the body, but a comparable level of conversion will occur. There are no outside interactions in
the process that have not already been mentioned.
Difficulties: This design would be exceptionally expensive to implement. Enzyme cost would be
extraordinary, and where other designs would be able to reuse the enzymes (as they are
immobilized, this design would require a separation process to recycle the enzymes. This would
be necessarily easy, as the enzymes would have to very well purified, otherwise they would
contain contents of the pro-drug and its product and would likely influence the results of
product produced that we measure in our chromatography step.
The second challenge of this design is achieving a comparable reaction rate in the second
compartment with the enzymes and the pro-drug. This would be nearly impossible, and finding
conversion rates of any type of drug with an enzyme is not readily available information. The
body cannot possibly achieve 100% conversion of the pro-drug into its product, and neither can
this design. However, matching the conversion rate of the body with our design would
challenging and expensive.
Design 3
The stomach is simulated by a tank of hydrochloric acid with a working volume of 2 liters. It is
kept at a temperature of 37oC by a heat coil, and a pH sensor is linked to acid and base
reservoirs to keep the pH between 1 and 2. A slurry of food and drug is added to the tank to
simulate ingestion. The base reservoir will be used as a buffer solution to mix with the slurry
upon entrance into this section to better simulate small intestine conditions. This slurry, after
being well mixed, is pumped out at a constant rate of 5 mL/s into the duodenum section of the
design. The majority of adsorption in the small intestines takes place in the duodenum. This
section has immobilized enzymes placed into the packed column to interact with the drug and
cause the desired enzyme substrate binding. The contents continued through into the next
section representing the rest of the small intestine. This section is composed of a hollow fiber
membrane to separate the slurry from the drug. The slurry passes out as the retentate along
with some buffer solution and can likely be recycled. The permeate is composed of the drug
and is sent to an affinity chromatography unit to separate out how much drug is left. By
29
determining how much drug permeates out it would be possible to determine how much is
adsorbed and how affinity changes with food interaction. This design can be seen in figure 18.
Assumptions: There are no outside interactions with the process not already mentioned.
Enzymes are not adsorbed into the blood stream but are used inside the small intestine.
Difficulties: There could be problems forcing the slurry through the packed column. It may not
flow through and could cause accumulation and fouling. The major limitation of this model is
that it will likely not be able to demonstrate food’s inhibitory effect upon Captopril in
decreasing adsorption. This design is also fairly unrealistic, as enzyme substrate binding
(Captopril and ACE) takes place in the blood stream, not the small intestines. This will change
the affinity of the enzyme to the drug. This is one area where design one succeeds, making it
our overall recommendation.
Mass Balances
According to the literature, the average human will adsorb roughly 75% of the overall drug
input into the body through the small intestines (Stewart et al, 2009). This was determined
through carbon-14 labeling. This effectively means that 25% of the drug will never be used and
exits the body as waste. To simulate stomach acid, two liters of 0.02 M HCl will be added into
the stomach bioreactor. This acid will be neutralized by the necessary buffer solution to raise
the pH to nearly 5. This work can be seen in the sample calculations section. A standard
dosage (50 mg) of Captopril will be added to the acid and will flow out of the reactor at 5 mL/s.
When Captopril is taken within 2 hours of a meal, the food greatly inhibits the adsorption of the
drug through the small intestines. To simulate the food in the stomach, 100 g of Carnation
instant breakfast will be mixed in making a slurry. When this is the case, adsorption of the drug
into the bloodstream is decreased to about 45%. Of the drug that enters the blood stream,
only roughly 60% of it will ever interact with the drugs targeted enzyme, Angiotensin
Converting Enzyme (ACE). When food inhibits the drugs adsorption into the body (one of the
effects our design can model), roughly 27% of the drug has any type of impact upon our body.
73% of the drug is wasted {Seem in conflict with the 25% waste}, and simply passes through our
30
body just like anything else that is indigestible. To show this the mass balance can be found in
figure 20.
However, this plays a major role in regulating the blood pressure of the patient, as only 27% of
the drug significantly decreases the apparent affinity of ACE to Angiotensin I. Captopril doesn’t
cease the conversion of Angiotensin I to Angiotensin II (two proteins that regulate our blood
pressure), it simply slows the process down. Angiotensin II is three times more potent in
constricting in our blood vessels then Angitotensin I, which is why this drug is effective (Stewart
et al, 2009.)
Our design is going to be judged a success if we can effectively simulate two steps. One is how
close our design’s mass balance is to a patient’s mass balance in the small intestines. The
second comparison in mass balances is made in the blood stream. When testing begins,
numerous parameters will have to be adjusted. This is a simulation, so we want to create a
scenario that is directly comparable to the human body. These are the parameters adjustments
that are forecasted.

Transmembrane pressure in perforate column membrane

Enzyme concentration in affinity chromatography column

Flow rates of the buffer solution and fluid exiting the stomach vessel
The transmembrane pressure will play a major role in how much of the drug is absorbed
through the perforated column membrane (the small intestines lining). Currently our system
runs on a pump system, and this is will create the pressure to affect the retentate and
permeate ratio.
Immobilizing the enzyme in the column for chromatography will be performed from the
company that the column is purchased from. If the enzyme concentration is not correct to
effectively simulate 60% of the drug interacting with the enzyme, the concentration may have
to be modified. This would be a very expensive modification to make as the cost of the
chromatography column is one of the major costs of this design project.
Economic Analysis
31
The focus of this project was to make a bioreactor that could simulate the adsorption through
the small intestine into the bloodstream and work with drug reactions. By simulating this, it
was hoped that information gathered from the testing would help in further testing on animal
and even humans. By providing the data produced from the design these animals and humans
could be kept safer. There is no real price that can be associated with a human life, but there is
a monetary equivalent to this design.
Building the artificial system is the main cost of the whole design. Giving a generous estimate
to compensate for any unforeseen problems that could arise, Table 4 shows the costs for each
of the major functioning parts and substances added on a per use basis along with an overall
estimate of the whole cost. The costs of the Captopril (Health Warehouse, 2009), the HCl
(Science Kit, 2009), and the Instant Breakfast (Drugstore, 2009) add up to very little compared
to the major costs. The major costs are shown to be the enzyme ACE, the vessel simulating the
stomach, the pumps needed, and the chromatography column. The ACE is very expensive,
because it is very difficult to localize and produce (Bachem, 2009). The Vessel simulating the
stomach is expensive because it contains all instrumentation and control features needed for
the design to work and further features that could be utilized for differing designs depending
on different drugs or enzymes or different experiments (Culotta, 2009). Three pumps will be
needed to keep the flow constant throughout the whole system (Boylan, 2009). The
chromatography column would cost about $8000 since it would need to be custom made for
the project (General Electric Healthcare, 2009). One other cost to account for is the
membrane. If the entire adsorptive surface area of the small intestine was to be accounted for
using this membrane it would cost over 1 million dollars by itself, but since only 1 m2 is being
used then the cost is reduced significantly (Fischer Scientific, 2009).
PROJECT EVALUATION
When 50 to 300 million dollars are spent in a clinical trial per drug, the design of a bioreactor
can be very expensive and still an advantage to the pharmaceutical company doing the drug
testing. That is with the assumption that your design can accurately model the small intestines
32
and stomach of the GI tract, showing how the drug is changed and adsorbed into the body.
Currently all bioreactors being developed for this purpose are still in their research and
development stage.
To establish if this project is a success is establishing a model that will successfully model the
drug chosen. The drug chosen, Captopril, works in the bloodstream. With the current model
we have chosen it successfully establishes all of the parts of the gastrointestinal tract the oral
drug would have to pass in order to get in the blood. This project did establish a relatively low
cost for most of the equipment. In all, this project established what it needed to set up for
future work.
DESIGN SCHEDULE
In designing this artificial stomach and small intestine bioreactor, there have been many
obstacles to overcome and many issues to work through. The original idea for the reactor was
to have a single chamber, representing the stomach with its chemical reactions, that would
account for the intake of all substances, the reactions that would take place, the adsorption and
absorption into the body, and the removal of wastes. With the discovery of enzymatic
reactions occurring in the duodenum and that most adsorption occurs later in the small
intestine the idea of keeping a single chamber reactor was thrown out. Also, with choosing
Captopril, this drug reacts in the bloodstream and this had to be properly represented.
Another issue that that is still troublesome is the fact that there is very little research on the
idea of a stomach/small intestine bioreactor. Lack of information in research made us look at
the basic levels of information on how the stomach and small intestine worked and develop our
own designs based on that.
Timeline
Task of: Neil Templeton (NT), David Morgan (DM), Kevin Richter (KR)
Fall 2008
33
Sep. 18 – Dec. 4
Learn background of how stomach and small intestines work (NT, DM,
KR)
Oct. 1 – Dec. 4
Meet with the faculty and outside researchers that are doing relevant
work to our project (NT, DM, KR)
Oct. 1 – Dec. 4
Learn of specific biological conditions that we will try to emulate in our
mechanical design of the stomach and intestines (NT, KR)
Oct 1 – Oct 8
Create Scope of Work and Cover Page (NT, DM, KR)
Oct. 9
Revise cover page and scope of work due (NT, DM, KR)
Oct 10 – Oct 22
Revise Scope of Work (KR) and Cover Page (DM). Begin Modeling and
Economic Considerations (NT)
Oct. 23
Cover page (DM), scope of work (KR), and resources (NT) due
Oct 23 – Nov 13
Work on Safety (KR), Regulatory (KR), and Environmental plans (NT) and
work plan (DM). Begin Report compiling (NT). Continue Modeling with
Alternate Models (NT, DM, KR)
Nov. 13
Cover page (DM), scope of work (KR), resources (NT), safety (KR),
regulatory (KR),environmental(NT), and work plan (DM) due
Nov 13 – Dec 4
Develop Presentation (DM) and continue working on Report (DM, KR, NT)
Dec. 2 & 4
Oral presentation due (NT, DM ,KR)
Dec 4 – Dec 15
Prepare Final Report (NT, DM ,KR)
Dec. 16
Revised final report due (NT, DM ,KR)
Spring 2009
34
Jan 19 – Feb 16
Change Final Report per Advisor’s recommendations and Prepare
Midterm Presentation (NT, DM, KR)
Jan 19 – May13
Establish By-weekly meeting with Dr. Mike (NT, DM, KR)
Feb 15
Choose between Enzyme inhibitory drug and Pro-drug (NT, DM, KR)
Feb 16
Mid Term Report due, choose drug (NT, DM, KR)
Feb 16
Revise Work Plan (NT, DM, KR)
Feb 28
Begin Modeling (Report Volume Flowrate Input and Output) (NT, DM, KR)
Mar 1
Choose membrane and verify specific enzyme interaction (NT, DM, KR)
Mar 2
Revised Work Plan due (NT, DM, KR)
Mar 3
Establish all alternative designs and choose the best design (NT, DM, KR)
Mar 4
Develop Block Flow Diagram for each design. (NT, DM, KR)
Mar 15
Determine Components for Mass Balance (NT, DM, KR)
Mar 20
Practice Presentation (NT, DM, KR)
Mar 20-Apr 10
Develop Michaelis-Menten Enzyme Kinetics model (NT)
Mar 22
Determine if enzymes follow Michaelis-Menten approach (NT)
Mar 23
Mid Term Presentation (NT, DM, KR)
Mar 23
Begin preparation of draft of Final Report. Establish and problems with
the chosen design (NT, DM, KR)
Mar 24
Determine enzyme inhibitors to be concerned with (NT)
Mar 25
Determine ideal substrate concentrations of drug to use (NT, DM, KR)
Mar 25
Finalize Mass and Energy Balance (Volumetric Flow rates) (NT, DM, KR)
35
Mar 30
Finalize Economic Plan (NT, DM, KR)
Mar 30
Design model with SuperPro (KR)
Apr 15
Create Poster and Practice Presentation (NT, DM, KR)
Apr 20
Print Poster (NT, DM, KR)
Apr 27
Present Poster (NT, DM, KR)
May 13
Final Report due (NT, DM, KR)
Meeting with Advisor Dr. Mike every two weeks (NT, DM ,KR).
Start and Finish Dates
This project requires a strict monitoring of when work is to be done. It is for this reason that
distinct start and finish dates are outlined in the timeline, as well as whom among the team will
be addressing the task. Some of the tasks are not given a specific date, and just a month rather,
because it is difficult to estimate just how much time that individual task is going to take. For a
general synopsis of the tasks to be completed, the Gantt chart can be viewed in the appendix
(Figure 12).
Non-routine and Routine Tasks
This is a research based project, and it is currently unfunded. Tests are not being taken out in
the field on a weekly basis, so the idea of a routine task is in inadequate way to define our
project. However, there is a degree of repetition that can be seen in this senior design project.
Routinely there are meetings with Dr. Mike Zhang, once every two weeks, to discuss progress
and address questions. Routinely meetings are to be held, at least once a week, to further
progress to our eventual goals.
As for non-routine tasks, a great deal of work has had to be done to make sure the background
knowledge is known to be able to adequately develop a design. It has been very challenging to
develop a design for the stomach and small intestines, because so much background knowledge
36
is required before design can begin. The majority of the work done this semester has been
non-routine tasks. This can be seen in the timeline.
Resource Constraints
A year of time has been given to complete this project. All of the team members are
undergraduates a part of Biological Systems Engineering. Since this is largely a biomedical
project, to be able to accomplish this project, several human resources have been established
that are outside of the department. These resources can be seen in the Appendix A of this
report. Currently, no equipment has been leased for this project, but this will likely change in
the design development coming in the spring semester. No outside funding has been enlisted.
Regarding funding, the work done in this project is in the expectation that a company (likely a
large pharmaceutical company) would purchase this design. Before that could happen, the
design would have to be built in-house, and tested appropriately. The actually building of our
design is a goal, but it may or may not be feasible based upon the funding of this project. One
of the larger constraints of this project is the funding, as most of the development will have to
take place without any actual testing. If a bioreactor was to be built, it would greatly improve
the team’s ability to judge the cost of production, as well as how accurately the bioreactor
modeled the small intestines and stomach. This would allow a standardized bioreactor to be
determined and give rise to possible changes that could make the device to work more
efficiently or work more specifically for whatever test it is used for. If a bioreactor was built, it
would quickly (relative to animal testing) allow for observation of differing drugs and biological
chemicals. It would give a more accurate representation of how adsorption would work by
allowing for testing. It would also allow the problems with possible high concentrations of
harmful chemicals to be observed and countered.
Accomplishments and Future Considerations
The main goal of the first semester was gaining knowledge about how and why the design of a
stomach bioreactor would be developed. Discovering the intricacies of the gastrointestinal
tract was vital to the report, and one of the first discoveries was that for adsorption to be
37
accounted for, the small intestine had to be incorporated. The application of the design was
found to be in drug delivery and adsorption, so general drug kinetics had to be understood.
With a general understanding of drug kinetics and possible drugs that could be utilized,
different modeling schemes were discussed and designed. Many considerations for how the
drugs could be administered, how the human GI tract would be affected, and what kind of
safety regulations had to be followed were all taken into account. Finally, the design would
have to be somewhat economically sound. When dealing with the enzymes in the GI tract,
expenses could become very high. Since the application of the design would ultimately be in
safety, the justification for the price would be acceptable.
The accomplishments of the second semester were extremely specific. Flowrates, materials,
and adsorption characteristics were chosen for the model. The drug Captopril was chosen,
which is a high blood pressure medication from a small molecule drug. It is established that this
is a feasible study to actually produce the model in this paper. It may be a pricy model, but
there is no price too high for safety.
For future work, the highest priority thing is to get funding for this project. The NIH is the
number one prospect for funding for this model. If design can produce the in vivo measured
rate constants already known, then the ability to predict a drug’s performance in the human
body exists. This could also establish better dosages to minimize harmful side reactions of the
drugs. This project should be continued with actually producing the unit designed or updated
from this paper to show drug adsorption in the human body. From this project a computer
simulation could be established prior to building the first reactor.
If funding is found there is potential to go beyond even the reaches of this project. The vessel
being used is used to control only a few aspects of the project like temperature and pH, but
there is potential to move into different fields of study using the added instrumentation. This
instrumentation includes DO probes and control and foaming control. Many different
experiments could be designed to with this in mind. One such example is the effects of
potential drug altering products from the addition of bacteria in the stomach.
38
CONCLUSION
The main focus of the fall semester was to focus on learning the information needed to make
this a feasible project. Learning the basics of the gastrointestinal tract, of enzyme kinetics, and
of drug delivery were all vital to creating a proper focus for the project. The change in focus
from a stomach bioreactor to a stomach and small intestine bioreactor and ultimately to a small
intestine and blood stream bioreactor was necessary to incorporate small molecule drug
adsorption. Picking the correct design was a major focus that required time and effort. The
different designs had varying benefits and drawbacks that made each possible choice good and
bad in specific ways. By incorporating the best of the each design the best choice was made.
The model chosen to properly represent Captopril being adsorbed in the small intestines and
reacting in the blood stream is the single enzyme model. The model first has a pretreatment
phase of the stomach where the drug will be mixed with Hydrochloric acid (HCl), Captopril, and
the food buffer. This will enter a mixing cylinder that will raise the pH to 4. Then the drug will
be filtered by a perforated column membrane. This membrane column will allow about 45% of
the drug to pass through, simulating small intestine adsorption, to be absorbed into the
bloodstream. The blood stream and the ACE inhibitor will be represented by a chromatography
unit. The chromatography unit will allow the drug to interact with the enzyme and proper
measurements for how the drug interacted with the enzyme.
The spring semester established design specifications for the model. The information in this
report should be sufficient enough to establish a computer-based model. If a computer model
could establish more information on this design, construction would be likely on this model.
More specifics were also found in the spring semester. The specific design was created and
along with the chosen drug, the proper amounts of each substance to be used was found, the
mass flow was found, and an economic analysis was formed.
With all of these parts of the project found, the implementation of the project became even
more of a reality. Compared to the monetary cost and the inherent risk associated with not
using this design, using the design would be the best idea all around. There will be less money
39
spent on getting necessary drugs to people who need them and less danger to people when
they find themselves in need of new medicines.
REFLECTIONS
During this design project the team learned time management skills, leadership roles, and
abilities to compromise. Time management and compromise went hand and hand in this
project. This meant we had to be reasonable with what we demanded from our team members
because our teams did have other activities going on besides senior design. This also made
scheduling team meetings at reasonable times and distributing fair amount of work evenly
throughout the team.
If we were to do something different, it would be establishing contacts prior to the project
beginning. If there was a solid base for where to begin this project it would have been a more
enjoyable process. It is not a bad thing to redirect the project when the original project is
infeasible but when it happens several times throughout the project it just is not fun. We also
probably could have done better on keeping track of everything. It would have been good to
be more clear on the importance of keeping to the dates established for carrying the project
through. While everything we really wanted to do was accomplished, they were not all at the
same times as our original timeline predicted.
For project management, it would be easier if we had more areas of interest we were pointed
to. Our group would need to establish a stronger literature review at the beginning of the
project. This would include an earlier primitive design in order to establish where the project is
headed at the beginning. It also would have been nice to actually build our design and have a
working model to both show the process more clearly and facilitate our own understanding of
how our objective could be accomplished. However, it was not within the scope of the project
time-wise to make this possible. I do not think it would have been possible with the amount of
research and understanding we had to incorporate into the project, but it would have been
nice.
40
Overall the experience was extremely beneficial in our own application toward our future
career and our lives in general. Learning how to deal with a team in good circumstances and in
good circumstances is vital to succeeding in industry. Applying research and engineering skills
together on this project makes the experience even more beneficial.
41
APPENDIX A
List of Figures
Input
Pressure
Retentate
Pressure
Permeate
Pressure
Figure 1. Diffusion simulation.
42
Oral Drug
Excretion
in air
Storage Sites
(Ex: fat)
Lungs
Blood
Peripheral
Tissues
Free Drug
Metabolism
GI Tract
Drug Plasmid
Protein Complex
Liver
Drug
Metabolites
Drug
Metabolism
Kidney
Metabolism
and
excretion
Metabolites
in bile
Drug and Metabolites
in Waste
Figure 2. Drug kinetics model.
The bioreactor design (and model) will incorporate the stomach, small intestines, and blood stream
which are highlighted in green in this figure.
Figure 3. Two compartment model of drug kinetics.
43
Figure 4. Gastrointestinal tract (Mariana Ruiz Villarreal, 2006).
44
Figure 5.Anatomy of the stomach.
45
Figure 6. Duodenum of the small intestines.
Figure 7. High pressurefiltration.
46
Figure 8. Hollow fiber membrane.
Figure 9. Double enzyme reactor.
Figure 10. Single enzyme reactor
47
Figure 11. Simplified bioreactor design.
48
Gantt Chart
9/9/08
Learn Biological Conditions of GI Tract
Create Scope of Work and Cover Page
Begin Modeling and Economic Considerations
Saftey, Regulatory, Environmental Plans
Work Plan and Alternate Models
Develop Presentation and Report
Prepare Final Report
Prepare Mid Term Presentation
Revise Work Plan
Modeling and Economic Consideration
Mass Energy Balance, Volumetric Flowrates
Finish Presentation
Draft Final Report and Economic Plan
Create Poster and Practice Presentation
Figure 12. Fall Semester Gantt chart.
49
10/29/08 12/18/08
2/6/09
3/28/09
5/17/09
08/31/04 10/20/04 12/09/04 01/28/05 03/19/05 05/08/05
Learn Biological Conditions of GI Tract
Create Scope of Work and Cover Page
Begin Modeling and Economic Considerations
Safety, Regulatory, Environmental…
Work Plan and Alternate Models
Develop Presentation and Report
Prepare Final Report
Prepare Mid Term Presentation
Revise Work Plan
Economic Plan
Mass and Energy Balance, Volumetric Flowrates
Drug and Enzyme Research
Membrane Implementation
Create Poster and Practice Presentation
Finalize and Edit Paper
Figure 13. Spring Semester Gantt Chart.
50
Figure 14. Decision Flowchart.
51
Nuclear
Integrins,
Receptors, 2%
DNA, 1%
1%
Other
Receptors, 2%
Miscellaneous,
1%
Ion Channels, 7%
Enzyme
Inhibitors,
47%
Transmembrane
Receptors, 30%
Figure 15. Breakdown of currently marketed drugs in the United States today (Copeland, 2005).
52
Gastric
Fluid
Stomach
Buffer
Solution
-Drug
-Buffer
Food
Slurry
Encapsulated Enzyme
Inhibitor Drug
Vessel
Hollow
Small Intestines Fiber
Membrane
Permeate
Retentate
-Slurry
-Drug
Filtration
Affinity Chromatography
w/ Immobilized Enzymes
Blood Stream
No EI
Interaction
SAMPLE
Figure 16. Design 1. Blood Stream Inhibition Reactor.
53
EI
Interaction
Affinity
Chromatography
Gastric
Fluid
Encapsulated
Pro-Drug
Stomach
Enzyme
Solution
Vessel
Interaction in
Small Intestines Vessel
Buffer
Solution
Adsorption in Hollow
Small Intestines Fiber
Membrane
-Pro Drug
-Products
Permeate
-Enzymes
Retentate -Pro-Drug
-Products
Ion Exchange
Chromatography
Ion Exchange
Chromatography
Figure 17. Design 2. Pro-Drug Non Immobilized Reactor.
54
Gastric
Fluid
Encapsulated Enzyme
Inhibitor Drug
Vessel
Stomach
Buffer
Solution
Interaction in Small
Intestines
Adsorption in
Small Intestines
-Drug
-Buffer
Permeate
Food
Slurry
Affinity Chromatography w/
Immobilized Enzymes
Hollow Fiber
Membrane
Retentate
Ion Exchange
Chromatography
-Slurry
-Drug
Ion Exchange
Chromatography
Figure 18. Design 3. Non-Food Enzyme Inhibitor Reactor.
Angiotensin I
ACE
Captopril
Uninhibited
Inhibited
Figure 19. Competitive enzyme inhibition.
55
Figure 20. Enzyme kinetics of Captopril.
This figure intentionally showcases interactions with colors. In the case of the enzyme (ACE) and the
substrate (Angiotensin I), blue and red combine to form the enzyme substrate complex, which is purple.
The same effect is seen for the enzyme (ACE) and inhibitor (Captopril).
Figure 21. Mass Balance.
56
List of Tables
Table 1. Design of adsorption models.
Design (adsorption)
Advantages
Drawbacks
High Pressure
-
Realism
-
Can’t represent plug flow
-
Hollow Fiber
Would probably have
higher yields of
adsorption
-
Cheap
-
Simple
-
Well understood
-
Realistic model of system
-
-
Modest energy
requirements
Little research done on
application
-
Expensive filters
-
No waste
-
Large surface area per
unit volume
-
Low cost of operation
Table 2.Decision matrix on alternate designs.
Weight (%) High
Hollow
Pressure
Fiber
Single
Enzyme
Double
Enzyme
Pig
Stomach
Feasibility
35
8/10
8/10
8/10
6/10
3/10
Realism
35
2/10
8/10
7/10
7/10
8/10
Cost
10
10/10
6/10
7/10
7/10
2/10
Longevity
10
9/10
7/10
8/10
7/10
2/10
Ease of
Use
10
8/10
6/10
8/10
6/10
1/10
Total
Score
100%
62%
75%
75.5%
65.5%
43.5%
57
Table 3. Design of enzymatic systems for stomach and small intestine.
Design (Enzymatic)
Advantages
Drawbacks
Double Enzyme
-
Strong model of
enzymatic reaction in
stomach and small
intestine
-
Enzymes in the
stomach do not play a
role in most small
molecule drugs
-
Higher cost than single
enzyme model
Single Enzyme
-
Cost
-
Neglects Enzymes in
Stomach
Pig Stomach
-
Very similar
components as human
stomach
-
Very difficult to reuse
pig stomachs after one
use
-
Allows for passive
diffusion to naturally
take place
-
Represents adsorption
in the stomach rather
than the small
intestine where most
adsorptions of small
molecule drugs occur
-
Ethical issues
58
Table 4. Economic Analysis. {References? Where did this come from?}
Product
Hcl (2 L)
Captopril (50 mg)
Instant Breakfast (60 g)
3 pumps
Chromotography Column
Price ($)
160.00
237.50
2.10
9,000.00
8,000.00
Membrane (1 m2)
ACE (100 mg)
Vessel
Mixing/Storage Tank
Millipore Sterile Syringe
Filter
2,000.00
305,000.00
25,000.00
400.00
Total
349,803.64
4.04
59
Qualifications
Required Prerequisite Courses:
BSE3524
Unit Operations
BSE3504
Transport Processes
CHEM3615 Physical Chemistry
CHEM1035 General Chemistry
BIO1105
General Biology
BIO2604
Microbiology
BSE3154
Thermodynamics
ESM3024
Fluid Mechanics
Required Co-requisite Courses
BSE4125
Senior Design
BSE4504
Bioprocess Engineering
BSE4544
Protein Separation
Recommended Courses
CHEM4544 Drug Chemistry
BSE4524
Bioprocessing Plant Design
Estimated Commitment from a 3 member student design team
About 4 hours a week per member
Skills to be developed for successful completion of this project
 Time management
 Communication and team work
 Basic principles of membrane separations
 Basic knowledge of the digestive system
 Considerations for economics
 Technical writing
Advisors
Dr. Mike Zhang
Dr. Robert Grisso
Human Resources
Sandra Daniel
John Weisbrod
Jack Evans
John Stefanyk
D. Foster Agblevor
VT Microbiology Professor
Pharmacist
VT Pre-Pharmacy Advisor
Millipore Representative
Biological Systems Engineering Professor
60
Sample Calculations
Acid-Base Calculations using Henderson-Hasselbalch Equation.
61
62
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