This document discusses a student research project aimed at developing new drug leads for Alzheimer's disease by targeting beta-secretase, an enzyme involved in the production of amyloid beta peptides. It provides background on Alzheimer's disease mechanisms and treatments. The project will use rational drug design methods to develop small molecule beta-secretase inhibitors, which will then be patented and licensed to a company for further development as a potential pharmaceutical.

1. Neuro Group: Discovery of New Secretase Inhibitor Drug Leads for Neurodegeneration
and Alzheimer’s Disease
Dorothy Du
Catherine Hutchinson
Melissa McCoy
Advisor: Gilbert Rishton, Ph.D.

2. The human population as a whole is living longer thanks in large part to the many
medical advances that have developed within the last several decades. The areas seeing the
greatest increase in life expectancy are the developing countries. Unfortunately, as a result of this
increase in our aging population we are also seeing a corresponding increase in the prevalence of
Alzheimer’s disease, the most common cause of dementia. According to the Alzheimer’s
Society, currently 14 million people world wide suffer from Alzheimer’s disease with that
number expected to increase to 40 million by the year 2050. Over 4 million of those individuals
are in the United States and annual total costs for caring for these individuals has reached 100
billion dollars. These statistics illustrate the need to begin aggressively searching for a way to
stop the progression of this disease rather then just temporarily treating the cognitive symptoms
associated with it.
The mission statement of our project is to address this unmet medical need in
Alzheimer’s disease by the discovery and development of safe and effective drug leads and,
ultimately, to help bring new mechanism-based medicines to the Alzheimer’s disease patient
population. The purpose of this paper is to expand upon that statement by providing a detailed
overview of Alzheimer’s disease and the current drug market, the potential disease mechanism
being targeted, the rational methods that will be employed when designing the inhibitor, and the
subsequent patent and licensing of the drug lead to a corporate partner for further development as
a pharmaceutical candidate. It is also important to emphasize how much of a need there is for
the continued development of novel drug leads for Alzheimer’s disease even as new treatments
are approved by the FDA.

3. Timeline:
I. Discovery
II. Rational Methods
III. Provisional Patent
IV. Development
Discovery
The discovery stage for developing small molecule drug leads for Alzheimer’s disease
begins by having a clear understanding of the disease history, pathology, and molecular
mechanism. From that understanding, a drug can be developed that targets the disease pathway

4. and methods can be designed that test the effectiveness of the molecule in inhibiting that
pathway.
Alzheimer’s disease (AD) was first reported in 1907 by Dr. Alois Alzheimer in Germany.
It was described in the patient Aguste D., as an early onset form of dementia starting before age
sixty and as being characterized by plaques and neurofibrillary tangles (NFTs) in the cerebral
cortex. The plaques and NFTs have come to be known as senile or Amyloid plaques and still
represent the only way to accurately diagnose the disease postmortem. Since the patient first
diagnosed with AD was only 54, the disease was at first termed as a pre-senile disease and was
considered separate from dementia associated with old age. After many advances in the last
several decades, it has become clear that the distinct pathological symptoms and cognitive
decline associated with AD are much more prevalent in the elderly and that the pre-senile form is
a much rarer genetic form of the disease (1).
The clinical features of AD include a progressive decline in memory, as well as cognitive
symptoms associated with impaired judgment, decision making, and orientation. There are also
physical signs which include aggression, psychomotor agitation, and psychosis (2). There are
several risk factors for the disease with old age being the number one factor. Others include
environmental reasons such as low education and reduced mental or physical activity later in life.
There are also some other diseases that are associated with AD such as vascular disease, high
cholesterol and smoking, but no clear link has yet to be shown (2).
The genetic link in the disease is made clear by the fact that both familial and sporadic
forms of AD occur. The familial form of the disease occurs early before age 65 and is associated
with mutations in amyloid precursor protein (APP), presenilin 1 and presenilin 2 genes. This

5. only accounts for 0.1% of the disease cases. There is also a gene associated with the sporadic
form of the disease known as the ApoE4 gene. This protein acts as a cholesterol transporter in
the brain and is also needed for amyloid β deposition in the brain and may promote plaque
formation in patients with AD. Other genes associated with the sporadic form of AD probably
play a much more minor role.
In order to better design treatments for AD it is important to have a clear
understanding of the molecular basis of the disease. Currently there are multiple competing
theories that try to explain the pathogenesis of AD. Two of the major ones that are being
targeted for development of therapeutics include the Amyloid cascade hypothesis and Tau
protein hypothesis (Figure 1 and Figure 2 in the Appendix).
The Amyloid cascade hypothesis is the target of the small molecule drug of our group
project. Aβ (β-Amyloid) is a peptide that is cleaved from APP (Amyloid Precursor Protein), a
protein with mutations associated with AD, and is the material that makes up the senile plaques
that are a hallmark of the disease. Aβ is cleaved by three different enzymes known as α-
secretase, β-secretase, and γ- secretase. Each of these enzymes cleaves APP at different
positions. The α-secretase activity represents a separate pathway that is non-pathogenic. It
cleaves APP within the region containing Aβ and releases a soluble fragment called sAPP (2).
The pathogenic pathway involves the other secretases. First, β-secretase
cleaves APP before the Aβ domain and releases another soluble APP fragment. The remaining
portion of APP is cleaved by γ-secretase which leads to the release of longer the peptides, Aβ-40,
Aβ-42, and Aβ-43 (1). The 42 form is thought to be the more pathogenic of the two because it is
stickier and tends to clump together to form soluble oligomers which in turn form insoluble
plaques. The Amyloid cascade hypothesis asserts that the imbalance between Aβ formation and

6. clearance is what causes neuronal degeneration and eventual dementia. The support for this
claim comes from familiar AD which is associated with mutations in the APP gene and in
presenilin genes which encode parts of the γ-secretase enzyme (2). These mutations lead to an
excess of Aβ production which is believed to cause neuronal dysfunction by destroying synapses
and dendrites (2)
The second major hypothesis associated with AD involves the protein Tau. Tau is a
protein that is normally associated with microtubules in the brain that are involved in the
transport of molecules along axons (1). Its phosphorylation is normally regulated by the kinases
like GSK3-β and CDK5 along with phosphatases (2). Tau is the main component of the second
diagnostic marker for AD, the neuro fibrillary tables (NFTs) seen in the brains of patients. The
tau that is observed in the NFTs, however, is hyperphosphorylated meaning that it has extra
phosphate groups attached to it. These extra phosphate groups cause tau to become insoluble
and aggregate to form NFTs (1). Tau also becomes sequestered along with other microtubule
associated proteins (MAPS) which leads to the disassembly of microtubules and
neuronal/synaptic dysfunction. (2).
In addition to the above hypothesis, there are several others including abnormal cell cycle
regulation, inflammation, oxidative stress, and mitochondrial dysfunction that could disrupt
metabolism necessary for neuronal function. It is not clear weather these pathways contribute to
the pathogenesis of the disease or are an end result of the other two pathogenic mechanisms
discussed above.
In addition to knowing the mechanism behind the disease, it is also important to have a
good understanding of the current treatment market. This includes having knowledge of the

7. current therapies that are already approved for treating AD as well as the therapies that are in
clinical and pre-clinical development. The current treatments on the market for AD focus on
treating the symptoms of the disease and not the progression of the disease itself. There are two
different classes of drugs available, Acetycholinesterase (AChE) inhibitors and N-methyl-D-
aspartate (NMDA) receptor antagonists. (Table 1)
Table 1. Current drugs on the market for treatment of Alzheimer’s disease (3)
Drug Company Mechanism Indication
Aricept (donepezil) Eisai/ Pfizer AChE inhibitor Mild to moderate AD
Exelon
(rivastigmine)
Novartis AchE inhibitor Mild to moderate AD
Razadyne
(galantamine)
Johnson and Johnson AchE inhibitor Mild to moderate AD
Namenda/ Ebixa
(memantine)
Lundbeck/ Forest NMDA receptor
antagonist
Moderate to severe
AD
AChE inhibitors function by preventing the neurotransmitter acetycholine from being
broken down in the synapse. It is believed that in AD, cholinergic neurons degenerate and that
this leads to loss of memory and other cognitive symptoms. One way to enhance transmission in
these neurons would be to increase the levels of acetylcholine which is the neurotransmitter
involved in transmitting signals between these neurons (2).
NMDA receptors target another neurotransmitter, glutamate, which is the major
excitatory neurotransmitter in the brain. In AD, there is an increase in glutamate activity that
results in low level activity of NMDA receptors. These receptors are important in learning and
memory and as a result their low activity results in impaired neuronal function. NMDA receptor
antagonists act by protecting neurons from overstimulation as a result of excess glutamate
activity (2).

8. Table 2. Current Alzheimer’s disease therapies in development (4)
Mechanism Potential
Drug/Molecule
Company Stage of
Development
Secretase modulators
-γ-secretase inhibitor
-γ-secretase modulator
-β/γ-secretase
inhibitors
-LY450139
Dihydrate
-Amgen, Bristol-
Meyer Squibbs,
Elan
Pharmaceuticals,
Scios Inc,
GlaxoSmithKline
-Eli Lilly
-Pre-clinical
-Phase II
-Flurazen -Myriad
Pharmaceuticals
-Phase III
Aβ immunotherapy
-Active Aβimmuno-
conjugate (AD vaccine)
-Passive Immunotherapy
-Humanized mAb against
Aβ (also passive)
-ACC-001 -Elan
Pharmaceuticals
-Phase I
-Intravaneous
Immunoglobulin
-Baxter Healthcare -Phase II
-AAB-001
(bapineuzumab)
-Wyeth -Phase II
Aβ fibrillization
inhibitors
-Alzhemed
-PBT2
-Neurochem, Inc
Prana
Biotechnology
-Phase III
-Phase II
Anti-tau CDK5/GSK-3β
inhibitors
UCSB Pre-clinical
Anti-inflammatory -Ibuprophen
-Naproxin
-Various
-Various
-Phase III
-Phase III
Cholesterol-lowering Lipitor (+ other
Statins)
Pfizer Phase II
Oestrogens
-May delay onset of AD -Estrogen -Various -Phase III

9. Several of the therapies currently in development work by targeting the potential
pathogenic mechanisms of AD and thus may provide hope in the future for stopping or reversing
the progression of the disease (Table 2).
One current major area of research involves secretase modulators and involves either
inhibiting or modifying the activity of β-secretase or γ-secretase. The idea would be to prevent
the production of the toxic Aβ-42 peptide which is prone to aggregation and makes up the
amyloid plaques. There are several pharmaceutical companies and research institutions involved
in pre-clinical work trying to design small molecule inhibitors for both secretases and there is
one γ-secretase inhibitor undergoing clinical trials from Eli Lilly. A potential issue that has been
seen with γ-secretase inhibitors is that this secretase also acts on the Notch protein. This protein
has an important role in cell fate, cell growth and cell differentiation. Studies in mice and rats
using γ-secretase inhibitors have shown that these animals experience goblet cell metaplasia in
the digestive system (5). In human subjects treated with LY450139, the inhibitor currently in
phase II studies, subjects experienced several gastrointestinal side effects such as vomiting and
diarrhea (4) indicating that the drug is disrupting Notch signaling in this region.
Another molecule currently undergoing clinical trials is called Flurazen. Instead of
inhibiting γ-secretase, Flurazen is believed to modify its activity by causing it to cleave APP
differently to produce shorter, less toxic peptides rather then the Aβ42 peptide (4). Since this
drug does not inhibit the enzyme it does not appear to affect Notch processing and thus may be a
safer alternative.

10. Another major area of research for AD treatment involves immunotherapy. There are
currently therapies in clinical trials that involve both active and passive immunization. Active
immunization involves injecting an Aβ immunoconjugate, which is the N-terminal portion of Aβ
conjugated to a virus like particle called a carrier protein. Passive immunization involves
injecting an antibody against Aβ. It is though that if these immunizations work they could
possibly function by inducing an immune response which would cause antibodies against Aβ to
bind to plaques and cause them to be cleared by neuronal immune cells such as microglia. The
problem with these treatments is the usual side effects associated with immune therapies such as
adverse immune responses. There was also one clinical trial that was interrupted because some
patients taking the therapy developed encephalitis (4).
Another class of compounds being looked at includes Aβ fibrillisation inhibitors. One
such drug candidate is Alzhemed which is a glycosaminoglycan mimetric that is thought to work
by interrupting the association of Aβ with glycosaminoglycans (5). These molecules are known
to bind Aβ and cause it to aggregate (2). Another drug candidate, PBT-1 works to reduce
aggregation by acting as a metal chelator. Copper and zinc ions have been linked to aggregation
and toxic effects in the brain in AD patients (2). Both of these drugs show few side effects but it
remains to be seen if they work in human AD subjects.
Another class of drugs targeting the second major disease pathway in AD is anti-tau
drugs. These drugs would seek to aid in the reduction of tau hyperphosphorylation by inhibiting
kinases like CDK5 and GSK-3β which are responsible for phosphorylating these proteins (2).
Unfortunately, tau consists of multiple isoforms and is controlled by multiple kinases and
phosphatases so it would be difficult to completely inhibit its phosphorylation by just targeting
one or two. Currently the potential tau drugs are in pre-clinical development (2).

11. Three other classes of drugs being looked at are already used widely to treat other
conditions. Anti-inflammatory drugs, specifically NSAIDs are thought to help treat AD by
reducing the inflammatory response that is associated with plaque formation or they may act by
preventing the disease by being taken early in life. There have been no positive results from
clinical trials that have looked at the use of various NSAIDs. The statins, which are cholesterol
lowering drugs, have also been looked at as a possible treatment method because positive results
have been seen in animal studies that show the levels of Aβ decreasing with treatment (2). No
positive correlation has yet to be seen in human subjects, however. A final treatment option
being considered, estrogen, is being looked at because of a link between a lower incidence of
dementia in postmenopausal women on estrogen therapy (2). Again, though, there have been no
positive results in clinical trials.
The reason it is important to understand the current drug market for AD before
undertaking the development of another therapeutic is to better understand the disease targets
under investigation as well as our future competitors. Even though there are already many
companies looking at secretase inhibition as a treatment option for AD, for every 1000 inhibitors
designed there may be only one or none that has the potential to become a drug. These
companies may also represent future corporate partners who could possibly carry our drug leads
to clinical trials.
There are several reasons that β-secretase makes an attractive drug target for AD. β-
secretase represents the first rate limiting step in the APP cleavage reaction and as a result it
makes an ideal candidate for designing inhibitors (6). Another important fact to consider is that
when transgenic mouse models of AD that were deficient in β-secretase were analyzed they
appeared healthy, fertile and anatomically normal and did not produce β-amyloid (7). The

12. results of the mouse study indicate that not only do β-secretase inhibitors prevent Aβ formation,
but they also appear to be non-toxic. This would make them a better candidate then γ-secretase
inhibitors and some of the other treatments currently being looked at. Finally, β-secretase
belongs to a class of enzymes known as aspartic proteases. The HIV protease also belongs to
this class and successful inhibitor drugs have already been developed that target it (6). The
model utilized for designing the HIV protease inhibitors can be used to develop similar inhibitors
for β-secretase.
As we develop our drug leads we will need to utilize various biochemical assays along
the way in order to screen candidate molecules. The screening process will involve measuring
the kinetic activity of the molecule(s), measuring the functional activity, and determining how
the drug is metabolized. The data that we generate from these assays will potentially be used to
convince a potential corporate partner to license our drug lead(s) and attempt to carry them
through clinical trials. As such these assays will need to be sensitive as well as selective to
provide a detailed biochemical profile of each drug candidate that gets developed.
The first type of assay that will need to be developed will be one in which kinetic
measurements can be determined. This type of assay will need to measure the biochemical
activity of the potential small molecule inhibitor. It will need to measure the ability of the
inhibitor to inhibit the β-secretase reaction:
β-secretase
APP sAβ fragments, Aβ40/42
There are several commercial assays available that can be purchased or whose methods
can be readily adapted for use in creating such a study. One type of commercially available

13. assay that has been utilized in many biochemical assays that look at β-secretase inhibitor activity
is the FRET assay (8, 9). FRET stands for Fluorescence Resonance Energy Transfer. The
theory behind the assay involves a peptide substrate containing the cleavage site for β-secretase
along with two fluorophores. One of the fluorophores acts as a fluorescence donor and is located
on one end of the peptide and one acts as a fluorescence acceptor and is located on the opposite
end.
The distance between the two fluorophores allows the donor’s fluorescent energy to be quenched
by the acceptor through resonance energy transfer when the molecules are excited by light. If
the peptide is cleaved by β-secretase, however, then the two groups are separated and the donor’s
full fluorescence is restored. Therefore, the activity of the enzyme is directly proportional to the
increase in fluorescence (9).
The assay kit includes all the necessary buffers as well as the peptide substrate and β-
secretase enzyme. In order to cut the costs it may be desirable to use in house available materials
such as β-secretase obtained by the structure group’s protein manufacturing facility. Many of the
other materials of the assay such as the peptide substrate can be purchased separately and buffers
could probably be made in house.

14. The important kinetic measurements that would need to be obtained from the assay
would be Km, Vmax, Ki, and IC50 . Vmax represents the maximal reaction rate of the APP cleavage
reaction and Km is a constant and is equal to the substrate concentration when the reaction has
reach half of the maximum velocity. Both of these measurements help to characterize the
reaction and are necessary for calculating other kinetic parameters such as Ki. Ki is the
inhibition constant and is needed to determine what type of inhibition is occurring by the
inhibitor being tested. IC50 is the inhibitor concentration at which 50% of the enzyme activity is
observed and is used to determine how well the inhibitor is inhibiting the reaction. The smaller
the IC50 value the more potent the inhibitor is (11).
In addition to utilizing the necessary biochemical assay to measure the kinetics of
potential drug leads for AD, it will also be important to measure the functional activity of these
molecules. Functional activity assays involve developing or utilizing a current model system of
Alzheimer’s disease to test the drug lead to see how well it eliminates the pathogenic process
being targeted. In the case of β-secretase inhibitors we are seeking to eliminate the production of
toxic Aβ. The assay will need to quantify the amount of Aβ peptide present in a sample obtained
in the presence and absence of the inhibitor.
There are several model systems that can be used for this type of assay, including both
cells and transgenic animals . It would be desirable to use both types of systems in order to
gather a broad spectrum of data on the activity of the inhibitors and whether there are any
differences in behavior seen in a particular system. Examples of cell systems would be
transfected cancer cell lines such as HeLa, CHO, HEK293, and M17. All these cell lines can be
stably transfected with the human APP gene. Usually, the cells are transfected with the Swedish
mutant form of the gene because this one is known cause greater Aβ production. The cells can

15. also be transfected with the human β-secretase genes. Transfected cell lines have been used in
several studies to determine the extent of inhibition of Aβ production in the presence of inhibitor
(12, 9).
Transgenic mouse models of AD are the animal model of choice for functional activity
assays. Mouse models are made by creating mice that express wild-type APP, APP fragments,
and FAD-linked mutant APP and PS1 (13). These mice don’t always exhibit all the associated
symptoms of Alzheimer’s, including NFTs, but they do produce excess Aβ and can be utilized to
measure the effectiveness of the inhibitor drug leads. They can also be used for blood brain
barrier (BBB) assays to determine how well the drug leads cross the BBB and also for
preliminary metabolism studies on the drug leads. Several types of transgenic AD mice are
available commercially.
There are several types of techniques and commercial assays that can be used to measure
how well the potential inhibitor drug leads inhibit the production of β-amyloid. Many of these
are immune-assays such as Western blot, immunoprecipitation, and ELISA. All of these assays
use antibodies that target the β-amyloid peptide to measure how much of the peptide is in the
sample, either cell lysate or mouse brain tissue (12). The lower the amount of Aβ present in the
samples the better the drug lead is working to inhibit β-secretase cleavage of APP. Several
assays such as β-Amyloid ELISA kits are available commercially and contain all the necessary
reagents. There are also several antibodies available suitable for immunoprecipitation or
Western Blot. An important distinction between these functional assays and the biochemical
assays that measure kinetics of drug leads is that the functional assays measure the activity of the
leads in a biological setting while the biochemical activity assays measure activity in an in vitro
setting.

16. In addition to the types of data that will be gathered by doing biochemical and functional
assays, it is also necessary to characterize the blood-brain barrier permeability of the drug leads.
If the drug leads are going to be able to target the neurons affected by AD they must be able to
cross the BBB. There are several ways in which to determine if a molecule is permeable to the
BBB. Examples include transgenic mice (measuring the amount of drug in the cerebrospinal
fluid) and in vitro methods that utilize Caco-2 cells or artificial membranes (14). The most cost
effective method of determining BBB permeability would be to use computational approaches.
These methods use mathematical algorithms derived from experimental data on BBB
permeability assays conducted on other nervous system drugs and can be used to analyze a large
number of compounds over a short period of time (15). It is important to determine the BBB
permeability of the drug lead prior to licensing because issues with permeability may need to be
resolved by altering the structure of the molecule.
In addition to BBB permeability there are a number of factors that need to be analyzed to
determine if the inhibitors that we generate are potential drug leads. These include
pharmacokinetic properties such as metabolic stability, metabolic liability, permeability and
protein binding. Other major drug properties are absorption, distribution, metabolism and
elimination (ADME) of the drug (16). The above properties maybe manipulated by the
medicinal chemist by altering the structure of the drug lead so must be considered early on. It
will probably be necessary to outsource many of the studies needed to determine these properties
to another facility because CSUCI does not have the resources or funding to carry them out.
There are several companies that will provide all the necessary screening to determine if the
inhibitors have all the characteristics of a good drug lead. The final major factor to consider is

17. the toxicology of the drug which involves a study with at least three different animal models and
will probably represent the stage in which a corporate partnership should be considered.
Rational Methods
Most of the drugs that exist and are used today were found by the historical method of
drug discovery. Drug discovery involves finding leads by chance, or by trial and error, then
testing these leads on animals, and matching the visible effects to treatments. However with
advances in technologies rational drug design methods have become the choice for creating lead
and therapeutic drugs. Rational drug design involves designing a drug that would be able to
block a part of a molecule that is causing a disease, thus leading to a treatment or cure. The
rational methods that will be used to create good drug leads for AD are medicinal chemistry, x-
ray crystallography, co-crystallization and computer-assisted drug design (CADD) (17).
Medicinal chemistry involves identifying a native protein, making it isoteric (looks like
and is shaped like a compound, but it can’t be cleaved) and developing it into a lead compound
that could be used for the treatment and prevention of AD. In designing a drug lead for AD we
start with the native wild type protein of APP that shows the cleavage site of β-secretase and then
compare it to the Swedish mutant APP. For drug lead designs the Swedish mutant APP will be
used because APP is cleaved more rapidly which leads to faster amyloid beta production. From
the Swedish mutant APP a truncated peptide β-secretase inhibitor is produced which leads to a

18. final low molecular weight non-peptide β-secretase inhibitor is created. To get to the final
inhibitor over 10 years of research and 1000s of analogs were created (Figure 3).
Figure 3. Structures of the wild type APP, mutant APP, truncated inhibitor and low
molecular weight inhibitor

19. After the inhibitor is produced by medicinal chemistry the three-dimensional structure
needs to be solved, showing the inhibitor bound to the molecule. X-ray crystallography, another
powerful tool that is used in rational drug design, is used to achieve this data. First, the molecule
is crystallized and then mounted on a diffractometer that is attached to a machine that emits a

20. beam of X-rays. Second, the crystal is bombarded with the X-rays and a diffraction pattern is
produced. Lastly, from this diffraction pattern data is produced and analyzed to produce the
structure (Figure 4).
Figure 4. Shown is an image of the X-ray crystal structure of the molecule bound to
memapsin 2, the enzyme responsible for amyloid plaque deposits in the human brain. The
bond disables the enzyme and prevents the plaques that cause Alzheimer's disease (19).
Another method that is used along with X-ray crystallography is co-crystallization. With
co-crystallization we can see the inhibitor binding to the active site and see the interaction
between the inhibitor and molecule (Figure 5).
Figure 5. Binding of AR-A014418 to GSK3β
. Left, surface representation of the inhibitor
binding pocket. Right, interactions between the ligand and the protein. Glycogen synthase
kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in pathological
conditions such as diabetes and Alzheimer's disease (20).

21. Finally, the last rational drug design method that could be used in this study is computer-
assisted drug design (CADD). CADD is using a computer to find novel drug leads. Data
generated from CADD can be used to develop predictive models in order to optimize ADME
properties of the generated lead molecules. However, it is important to remember that computers
cannot substitute for a clear understanding of the system being studied. CADD is an additional
tool to gain better insight into the chemistry and biology of the problem at hand (Figure 6).
Figure 6. New potent inhibitor for the Human Sirtuin Type 2 enzyme was found using the
CADD method (21).

22. After discovering drugs leads for the treatment of AD before moving into the patent and
development stages we need to ensure that we “good” drug leads. The characteristics of good
drug leads are that they have to be small organic molecules (< 400 molecular weight and
nitrogen in the structure), chemically stable, orally bioavailable, BBB and novel.
Provisional Patent
In order to produce a commodity of value, it is essential to protect new ideas. In the
pursuit of discovering a secretase inhibitor drug lead to treat neurodegeneration and Alzheimer’s
disease, methods, assays, antibodies, composition of matter, and other elements of the drug
discovery process are often recognized or created for the first time. These aspects qualify as
intellectual property (IP). For intellectual property to retain its values and for an individual or
groups to own their inventions, ideas need to be protected in the form of patents. A patent is
ownership that protects the patent holder. It gives him the ability to exclude others or prevent
wrongful copying. Novel intellectual property would be a bargaining tool once the project
reached the point of development, and partnerships and licensing with corporate sponsors were a

23. subject of consideration. It would be the chip necessary for a large pharmaceutical company to
even consider working as a partner. Protection is a means towards their drug production, sales,
and future revenue (18).
The value of owning IP comes from its sale or licensing, which is contracting for part of
the intellectual property rights. Also IP can be protected through lawsuits, injunctions, and
monetary penalties. Patents block competitors from copying. Infringement upon patented
intellectual property gives reason to sue and the ability to take the organization committing the
infringement to court. With a patent, the property rights could be retained for the specific term
of 20 years in which the original concepts can be protected; generic drug manufacturers would
not be able to compete for a share of the market with an identical item. The 20 year term begins
from the day the patent is first filed (22, 23).
It is important that drug companies, academic institutions or research laboratories work
as efficiently as possible because of the limited time of protection. A lot of the patent life is
spent in clinical trials to gain Food and Drug Administration (FDA) approval. Clinical trials
generally span seven to eight years. The clinical trials testing for safety and efficacy are
extensive studies in large populations for what can often feel like a grueling length of time.
However, it is important that the drug shows to be effective in humans. This time devotion for
clinical trials on the 20 year patent protection life clock may make it tough for drug companies to
get a return on their investment (23).
One way to stretch out the time length of protection to as long as possible is by filing a
provisional patent first. A provisional patent is almost like a preliminary patent. Provisional
patents are earlier patent filings that protect and keep intellectual property secret but do not

24. overlap into the 20 year term. During the term of a provisional patent, the filer can get protection
right away but will also be able to add new emerging data and variants of existing data to protect
further developments. The life of a provisional patent usually extends for one year.
Patents filed with the USPTO, or the United States Patent and Trademark Office, are
enforceable only in the United States. Patent Cooperation Treaty patents, or PCTs, are the
international patent application. This general application is filed, but in order to be granted
patents in different countries, the patents must be additionally pursued regionally. For example,
the PCT application can be filled out for worldwide submission; however, for a patent specific to
Japan, one would need to go through a Japanese patent office (22).
All of these dealings with intellectual property and patents are done with the expertise of
a patent attorney. He can deal with the procedures to obtain a patent. Working with the
scientific authority or inventor, the patent attorney can guide the patent applicant through the
involved process and make sure that the patent is written up in a strong fashion and is complete
and thorough. There needs to be documentation of the invention, such as with lab notebooks;
there also needs to be literature searches and patent searches to ensure novelty. PubMed for
medicine and SciFinder for chemistry are excellent database resources for literature searches.
And lastly, one needs to work on the preparation of the patent application. Components of the
patent application include the assignment of ownership, an inventor declaration, fees and forms,
paper or electronic filings, PCTs, references, disclosure of prior art, confirmation of dates and
inventorship, examiner reviews, applicant responses, publishing of the application, and patent
interference (23).

25. The average timeline for this entire process is approximately three years. An estimation
of the expenses for a patent involving biotechnology is in the hundreds of thousands. It costs
eight to ten thousand dollars for literature and data reviews and application preparation. It costs
ten to 30 thousand for prosecution for infringement for over three years in the United States.
Abroad, it costs 100 thousand dollars each for protection in Japan, Europe, and possibly Canada
and China. Other countries run up prices of roughly 30 thousand dollars each (23).
This particular project has close ties and relationships with the California State
University, Channel Island’s Alzheimer’s Institute. There are many university based research
laboratories pursuing exciting breakthroughs in technology and novel drug leads. Because of the
academic setting and hence academic regulations, it is important that there is compliance with
university policy.
CSUCI recently approved of a new policy on intellectual property. Key elements will be
outlined and highlighted as a model.
The introduction provides the scope of the policy. The university wants to encourage the
transfer of knowledge from academia to the private sector for the public good, yet it needs to be
a good steward of public resources and safeguard against the use of public funds for private gain.
The rights, interest, protection, and transfer of intellectual property by staff, faculty, and students
are addressed.
In terms of ownership, if the university initiates and funds a project, the university owns
the intellectual property unless it agrees to share. When a faculty member develops intellectual
property without university support, the intellectual property solely belongs to the faculty
member.

26. It is the responsibility of the university President and the Office of Provost to implement
this policy and to work out negotiations in regards to IP.
When the university owns intellectual property or has equity interests in copyright
material, the proceeds go towards the funding of future creative efforts by the university. It can
be used in the creation of a commercialization fund to protect existing intellectual property or a
research fund to develop new IP.
And lastly, this policy is reviewed and revised every five years so that it may stay current
with the times.
As one delves further into the process of patent application, once one understands the in-
house rules of working within a university facility and when heading in the direction of
protecting intellectual property, there needs to be a check for novelty—the literature and patent
search. As addressed earlier, patent searches are done with patent attorneys. But in order to gain
some perspective on how patent searching is done, quick searches can be done at the USPTO
website. The website contains a database in which all filed patents are recorded. The
applications are in an array of stages, from just in the beginning steps to approval or
abandonment.
On a given April 2007 day, with a simple search phrase like “Alzheimer’s disease,” the
USPTO database was able to pull up 694 hits. With a search phrase of “beta-secretase inhibitor,”
the search showed 13 hits. The hits for “beta-secretase inhibitor” included patents on
composition of matter, assays, and antibodies.
Searches allow one to compare and check what’s out in the competitive market.
However, quick search results can be somewhat misleading, because certain patent descriptions

27. may not include exact search phrase. For example, instead of utilizing the term “beta secretase
inhibitor,” a patent may be called an “amyloid lowering agent,” in order to hide the discovery.
Also, it is a broader term, so if it is later discovered that a lead molecule has a different
mechanism of function, the patent will not be lost based upon the wrong information. This
further confirms the necessity of a patent attorney who could aid through the sifting of this
extensive material.
In the rational drug design section, the application of medicinal chemistry is
demonstrated through the identification of the cleavage site of beta secretase, a study of the
variations of the Swedish mutant and finally, the arrival of truncated synthetically formed small
molecules. One of the most recently discovered, published and patented small molecules is
from Elan Pharmaceutics. It can serve as a good sample lead, an example towards the drug
discovery and intellectual property protecting process. The patent for this particular molecule is
numbered U.S. Patent 7,119,085 and entitled ‘Methods to treat Alzheimer’s disease.’
The molecule is a low molecular weight, non-peptide, beta-secretase inhibitor. The
abstract of the patent describes the patent as protecting substituted hydroxylethylene compounds
for treating Alzheimer’s disease and other similar diseases. The patent thoroughly and fully goes
into depth about the composition of matter, describing the structure of the small molecule. The
most important section, the claims section has the molecular formula and modifications or
analogues of the formula, along with various ranges and concentrations of activity. It discloses
known information to protect, yet it makes both dependent and independent claims to keep the
scope of the patent as broad as possible to protect a larger field. The formula and its analogues
address the character of the molecule. It addresses its composition: the secondary alcohol that
cannot be cleaved, that it is less peptidic, and that it contains an amine group for the ability to

28. work as a central nervous system (CNS) drug, and that it is blood brain barrier (BBB) permeable.
The patent also includes thorough descriptions into the field of invention, background, summary,
additional disclosure, detailed descriptions, definitions and conventions and examples (22).
The scientific paper correlating with the Elan patent was published by the Journal of
Medicinal Chemistry in 2007. It was entitled ‘Design, synthesis, and crystal structure of
hydroxyethyl secondary amine-based peptidomimetic inhibitors of human beta-secretase.’
The contents of the article are as the title describes. The article starts off with an
introduction to the amyloid cascade hypothesis and how strategies for lowering the concentration
of neurotoxic amyloid protein have been a goal for clinical treatment. Beta secretase enzyme
plays a big role in a rate-limiting step in the amyloid cascade towards the production and
aggregation of amyloid protein. Targets had been isolated in laboratories with the use of BACE
knockout animals. The article further explains the design and synthesis of a novel series of
potent and cell permeable non-peptide but peptide-like inhibitors of beta-secretase or BACE.
The article includes elements that were included in the patent. The composition of matter: the
hydroxyethyl secondary amine isotere and novel aromatic ring is in the article. This particular
molecule of interest would need to be bioavailable and CNS active. The molecule was designed
using rational drug design. Using information about previously existing molecules with
unfavorable potency ratios and an isostere in HIV protease, scientist were able to make chemical
modifications to make this effective drug lead. Replacement and side group changes were made
for the greatest binding and activity. Former studies with statine and hydroethylene transition
state isosteres had unfavorable cell-to-enzyme potency ratios. Based off of existing structures,
chemical modifications were made to improve the potency (25).

29. One of the images in the article shows the x-ray crystal structure of the inhibitor
compound co-crystallized with BACE overlaid with a previously understood molecule that had
the unfavorable potency. The crystallized complex and solved structure had a resolution of 1.7
Angstroms. The Elan molecule has a slightly different composition, is shorter and has better
binding, than its predecessors.
The crystallization of BACE in the presence of the novel small molecule allowed for x-
ray crystallography to characterize how binding occurs. A noteworthy feature of the binding is
the molecule’s position, sitting at the active site between the protein’s catalytic aspartates and the
existence of seven hydrogen bonds between the inhibitor and the protein. Hydrophobic
interactions are a major component of the binding affinity. These interactions may explain
increased activity with the new molecule over older ones (25).
Another patent found in the quick search off of the USPTO website patent database was a
patent for an assay related to beta-secretase inhibition. U.S. Patent number 7,196,163 can be
used as a model for an assay patent. It is entitled ‘Assays using amyloid precursor proteins with
modified beta-secretase cleavage sites to monitor beta-secretase activity.’ The inventor of this
patent is D.J. Hazuda and her team. The assignee of this patent is the pharmaceutical giant
Merck.
The abstract of Hazuda’s patent describes ways to identify inhibitors of beta secretase
that employ modified beta secretase substrates. The modified substrates have cleavage sites that
are changed from the wildtype. Many of them are more susceptible to enzymatic breakdown
than wildtype sequence containing substrates. Recombinant polynucleotide molecules encoding
the modified substrate, antibodies that recognize the cleavage products, stable cell lines

30. expressing the altered substrate, and transgenic animals expressing the modified beta secretase
substrate are all provided. The patent references existing U.S. and foreign patents and scientific
articles. It includes a parent case text to cross-reference to related applications and gives its
claims and description, including the highlighted amino acid sequence of the NEFV modified
beta secretase substrates. It is much like the Elan molecule patent (22).
A corresponding journal article recognizing J.D. Hazuda as a contributor is ‘Biochemical
and cell-based assays for characterization of BACE-1 inhibition,’ published in the journal
Analytical Chemistry. The article gives brief insight into how beta amyloid peptide is deposited
and the scientists’ goal of creating assays for characterization of BACE-1 inhibitors for
identifying a primary therapeutic target for Alzheimer’s disease. The assays include a 96-well
HPLC biochemical assay that contains the unique substrate with the optimized peptide cleavage
sequence NFEV. NFEV is the same sequence protected by the before mentioned patent. This
substrate is processed by beta secretase ten times more efficiently than the substrate containing
the Swedish mutation sequence. Therefore, this assay can be run using a smaller enzyme
concentration. The second assay is a homogeneous electrochemiluminescence assay for the
evaluation of beta secretase inhibition in specific cell lines transfected with amyloid precursor
protein with the NFEV sequence. The scientific journal articles and the patents line up in terms
of materials contained in each; just as the patent and article for Elan’s beta secretase inhibitor
molecule is a pair. They served as a good model of intellectual property content for both the
composition of matter patent and assays patent in Alzheimer’s disease drug discovery (24).
Development
Once a good drug lead has been established and novel IP has been developed and a
provisional patent describing the composition of matter has been filed, it is time to move into the

31. concluding phase within the scope of the project’s timeline scale, the development phase.
Development will be entered with a molecule that is lead-like, drug-like, has low molecular
weight, has oral bioavailability, and that is blood brain barrier permeable. With this molecule,
outside resources will be utilized, since the facilities for the large scale of development will not
be available within the given academic setting. Contracted labs can be used for in vivo work.
Lastly, partnering with a drug company is an option to further pursue development of the lead.
On a more personal, local level, there are very exciting future projections for the CSUCI
campus. It is applicable to the Alzheimer’s Institute along with other existing or soon to be start-
up biotechnology companies and organizations. The university has great aspirations and
ambitions for biotechnology. The area around CSUCI and Ventura County has the expertise of
the existing scientific community, with biotech companies such as Amgen and Invitrogen
neighboring the university campus. There is already biotech companies established on campus.
These companies include Alliance Protein Laboratories, Integrity Biosolutions, and AmProtein.
However, there is room for growth. The university envisions developing a research and
development (R&D) park that can be the new hub for discovery. It will be important for the
R&D Park to have wet labs for biological and chemical research to facilitate the goals of projects
like the Alzheimer’s Institute. There are very good prospects and a positive outlook for the
future of research and development of drug lead candidates and biotechnology as a whole at
CSUCI.
Conclusion
In summary, the recognition of Alzheimer’s disease started with the discovery of
neurodegeneration and observation of presenile dementia at the beginning of the twentieth
century and is currently progressing towards affecting millions of more individuals in the United

32. States and worldwide in the near future. This widening medical burden will impact families
along with federal health care systems, with costs up to 100 billion dollars annually in the U.S.
alone. There is an unmet need for discovery and development of safe and effective drug leads
for mechanism-based medicine for the Alzheimer’s disease population. A timeline from
discovery, rational methods, patent protection, and development, with an emphasis in the first
three stages, has been established to pursue this goal. Discovery looks into laboratory chemistry
and enzyme or in vitro assays. Rational methods unfold through drug design and
crystallography. Patent protection highlights composition of matter and intellectual property.
Finally, development will be coupled with a corporate partner. With a persistent drive towards
the growth of biotechnology and the pursuit of these presented phases towards drug leads against
neurodegeneration, there is a positive outlook for medicine and the treatment of Alzheimer’s
disease.

33. References:
1. Altman J. Alzheimer: 100 Years and Beyond. Alzforum-Print News. The
Alzheimer’s Research Forum. Available at:
http://www.alzforum.org/new/detailprint.asp?id=1518. Acessed: March 30, 2007.
2. Blennaw K, de Leon MJ, Zetterberg H. Alzheimer’s Disease Seminar. The Lancet
2006; 368: 387-403.
3. Mount C, Downton C. Alzheimer’s Disease: progress or profit? Nature Medicine
2006; 12: 780-784.
4. Alzforum: Drugs in Clinical Trials. Available at:
http://www.alzforum.org/drg/drc/default.asp. Accessed: February 13, 2007
5. Milano J, McKay J, Dagenais C, Foster-Brown L, Pognan F, Gadient R, Jacobs RT,
Zacco A, Greenberg B, Ciaccio PJ. Modulation of Notch Processing by γ-Secretase
Inhibitors Cauases Intestinal Goblet Cell Metaplasia and Induction of Genes Known
to Specify Gut Secretory Lineage Differentiation. Toxicological Sciences 2004; 82:
341-358.
6. Maillard MC, Hom RK, Bension TE, Moon JB, Mamo S, Bienkowski M, Tomasselli
AG, Woods DD, Prince DB, Paddock DJ, Emmons TL, Tucker JA, Dappen MS,
Brogley L, Thorsett ED, Jewett N, Sinha S, John V. Design, Synthesis, and Crystal
Structure of Hydroxyethyl Secondary Amine-Based Petidomimetic Inhibitors of
Human β-Secretase. Journal of Medicinal Chemistry 2007.
7. Luo Y, Bolon B, Kahn S, Bennet BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang
J, Gong Y, Martin L, Louis J, Yan Q, Richards WG, Citron M, Vassar R. Mice
deficient in BACE1, the Alzheimer’s β-secretase, have normal phenotype and
abolished β-amyloid generation. Nature Neuroscience 2001; 4: 231-232.
8. Murray CW, Callaghan O, Chessari G, Cleasby A, Congreve M, Frederickson M,
Hartshorn MJ, McMenamin R, Patel S, Wallis N. Application of Fragment Screening
by X-ray Crystallography to β- Secretase. Journal of Medicinal Chemistry 2007.
9. Stockley JH, Ravid R, O’Neill C. Altered β-secretase enzyme kinetics and levels of
both BACE1 and BACE2 in the Alzheimer’s disease brain. FEBS Letters 2006; 580:
6550-6560.
10. BACE1 (β-Secretase) FRET Assay Kit Protocal. Available at:
http://www.invitrogen.com/content/sfs/panvera/L0724.pdf. 2002.
11. Assay Guidance Manual Version 4.1, 2005, Eli Lilly and Company and NIH
Chemical Genomics Center.
Available at: http://www.ncgc.nih.gov/guidance/manual_toc.html (last accessed
[3/13/07])

34. 12. Chang W, Koelsch G, Wong S, Downs D, Da H, Weerasena V, Gordon B,
Devasamudram T, Bilcer G, Ghosh AK, Tang J. In vivo inhibition of Aβ production
by memapsin 2 (β-secretase) inhibitors. Journal of Neurochemistry 2004; 89: 1409-
1416.
13. Won, PC, Cai H, Borchelt DR, Price DL. Genetically engineered mouse models of
neurodegenerative diseases. Nature Neuroscience 2002; 5: 633-639.
14. Yoon CH, Kim SJ, Shin BS, Lee KC, Yoo SD. Rapid Screening of Blood-Brain
Barrier Penetration of Drugs Using the Immobilized Artificial Membrane
Phosphatidylcholine Column Chromatography. Journal of Biomolecular Screening
2006; 11(1).
15. Rishton GM, LaBonte K, Williams AJ, Kassam K, Kolovanov E. Computational
approaches to the prediction of blood-brain barrier permeability: A comparative
analysis of central nervous system drugs versus secretase inhibitors for Alzheimer’s
disease. Current Opinion in Drug Discovery & Development 2006; 9(3): 303-313.
16. Pajouhesh H, Lenz GR. Medicinal Chemical Properties of Successful Central
Nervous System Drugs. NeuroRx 2005; 2: 541-553.
17. Bachurin SO. Medicinal Chemistry Approaches for the Treatment and Prevetion of
Alzheimer’s Disease. Medicinal Research Reviews 2003; 23(1): 48-88.
18. Figure 3.
19. Ghosh AK, Kumaragurubaran N, Hong L, Kulkarni SS, Xu X, Chang W, Weerasena
V, Turner R, Koelsch G, Bilcer G, Tang J. Design, Synthesis, and X-ray Structure of
Potent Memapsin 2 (-Secretase) Inhibitors with Isophthalamide Derivatives as the P2-
P3-Ligands. J. Med. Chem. 2007; 10.1021/jm061338s.
20. Bhat R, Xue Y, Berg S, Hellberg S, Ormö M, Nilsson Y, Radesäter AC, Jerning E,
Markgren PO, Borgegård T, Nylöf M, Giménez-Cassina A, Hernández F, Lucas JJ,
Díaz-Nido J, Avila J. Structural Insights and Biological Effects of Glycogen
Synthase Kinase 3-specific Inhibitor AR-A014418. J. Biol. Chem. 2003; 278(46):
45937-45945.
21. Computer Assisted Drug Design. Available at:
http://www.uku.fi/farmasia/fake/modelling/pdf/mallinnusposteri_valmis.pdf
22. U.S. Patent and Trademark Office Homepage. Available at: http://www.uspto.gov/
(February-April 2007).
23. Pessin K. Lecture Slides from Biol 503, CSUCI. Biotechnology Law and Regulation
Course. (Spring 2006).
24. Pietrak BL. et al. Biochemical and cell-based assays for characterization of BACE-1
inhibitors. Analytical Biochemistry (2005); 342: 144-151.

35. 25. Maillard M.C. et al. Design, synthesis, and crystal structure of hydroxyethyl
secondary amine-based peptidomimetic inhibitors of human beta-secretase. Journal
of Medicinal Chemistry (2007); 50: 776-781.

36. Appendix:
Figure 1: Outline of Amyloid Cascade Hypothesis (Taken from Presentation)

37. Figure 2: Outline of Tau Protein Hypothesis (Taken from Presentation)
PartnerCompositio
n of matter
Intellectual
property
Drug
design
Crystallogr
aphy
Laboratory
chemistry
Enzyme
assay or in
2
years
Developm
ent
Provisiona
l Patent
Rational
Methods
DiscoveryDiscovery