Michael Hicks

BIO399
Dr. Ingram
Experiment Design

Research Question: Does cat’s claw bark reduce breast cancer.

Possible Answers
1. The cat’s claw bark could prove to be ineffective in treating instances of breast cancer or
cancer in general. With the literature on cat’s claw bark being anecdotal at best, its
mechanism of action and associated efficacy in treating cancer remains relatively unclear.
From potentially reducing the availability of adenine in cancer cells by an unknown
method to mitigating inflammation, highly variable accounts of its biological effects do
little in inspiring confidence in the treatment.
2. The purported anti-inflammatory effects of cat’s claw bark could lead to it being effective
in reducing breast cancer. As noted by the literature, cat’s claw bark inhibits tumor
necrosis factor alpha and nuclear-factor kappa beta, both of which are inflammatory
molecules associated with cancer development. Consequently, the collective impediment
of these molecules could promote a proper immune response to the breast cancer, leading
to an overall reduction.
3. Cat’s claw bark might have a deleterious effect on the subjects as the compound
accumulates in the body. Despite individuals having utilized the compound before, the
reference range for effective dosages has not been clearly established. As such, it is
possible that dosage required for an effect to be observed is ultimately lethal to the
subject.
4. Cat’s claw bark might promote a cancer immune response by increasing the number of T-
cells in the tumor microenvironment. Although the mechanistic process is not formally
established, there is anecdotal evidence that treatment with cat’s claw has increased the
number of circulating T-cells in patients. Should this be true, the augmented immune
system may be better able to respond to cancerous growths, thereby leading to a
reduction in breast tumor size and/or number.

Approach – Direct Testing of Acquired/Retained Subjects

Justification:
1. Whereas externally collected data would expedite the study, there is no guarantee that the
data itself has not been altered to yield statistically significant results. In the current
research environment, experimenters engage in questionable research practices with an
alarming frequency as a means of maintaining status and employment. By directly testing
subjects, the likelihood of acquiring unrepresentative data is significantly lowered.
Additionally, the conditions to which they are exposed to over the course of the study can
be standardized and maintained. As a result, the influence of potential confounding
 variables on the research outcome(s) is minimized, thereby increasing the overall power
of the experiment.
2. In assessing cat’s claw bark, mice or another animal could be utilized as a substitute for
human experimentation, especially in procedures that place the subject at risk of death.
For example, experiments with BALB/c mice have been used to provide evidence for the
preclinical significance of novel chemical agents. However, the potential effects of cat’s
claw bark in mice or another animal may not translate to human subjects and their
respective instances of breast cancer. Rather, it is distinctly possible that the treatment
has a neutral or negative effect in a non-human subject, thereby wasting time and
resources. By directly testing human specimens, the potential effect of cat’s claw bark on
breast cancer can be readily ascertained while the generalizability and/or applicability of
the results is increased.
3. A survey represents a relatively inexpensive and quick alternative to the direct testing of
the treatment. With the consent of hospitals, electronic forms could be distributed to
breast cancer patients urging them to take cat’s claw bark and report any change in their
prognosis. Following this, the data could be adjusted for various factors and compared to
previously established breast cancer outcomes to determine a possible effect. Although
this approach appears sufficient, it fails to guarantee a representative sample population
or a sample size large enough to achieve the necessary power. The former withstanding,
this approach invites a myriad of confounding factors that could not be properly
addressed by a questionnaire. In contrast, direct testing of acquired individuals increases
the likelihood of acquiring a representative sample as the background of the subjects can
be properly accounted for. Similarly, potential bias is reduced as a more representative
sample better reflects the range of physiological and environmental factors that can
influence the research outcomes.
Fair Test: In general, the direct testing of acquired subjects accounts for any and all
reasonable conclusions that may be associated with cat’s claw bark treatment. Although
tumor size and indicator molecules are the primary endpoints, there are not any measures in
the study that can restrict or bias the potential outcomes. Furthermore, the data being utilized
(indicator molecules and tumor size) is interdependent of the literature data (T-cells, adenine,
TNF alpha, etc.) that was used to establish the potential efficacy of cat’s claw for cancer.
Thus, this approach qualifies as a fair and reasonable test to assess the research question.

Design/Details
Sampling: Using social security numbers and governmental data, random sampling will be
applied to the entirety of the United States until 200 viable subjects have been acquired for the
study. For each participant, they will be informed about their selection for the study and
promised a significant monetary compensation in return for their involvement. Following this,
personnel will be sent to retrieve the individual and bring them to a central testing site, at which
their patient history and current health will be assessed. Individuals with chromosomal
abnormalities, autoimmune disorders or a current or past case of cancer, and less than 18 years of
age will be barred from the study. Women that are pregnant or breastfeeding, people allergic to
cat’s claw bark, people taking AIDS or HIV medications and transgenders will be similarly
excluded. Once accepted into the study, participants will be assigned a number. A computer will
then randomly allocate each of the numbers to one of the two treatment groups. A similar
process will occur within each treatment group to assign participants to a specific factor.
By applying random sampling to the United States at large, a significant amount of biological
and environmental variability is accounted for when selecting subjects. However, the limited
selection of 200 subjects may skew the actual sample composition in favor of more densely
populated areas, leading to a potential source of subject bias. Furthermore, the implementation of
exclusion factors similarly detracts from the overall representativeness of the study results as
certain individuals are actively excised from the experiment. Nonetheless, the sample can be
expected to be fairly representative of the entire range of biological and environment factors that
could influence the study outcome(s). The use of a computer to randomly assign participants
significantly reduces the likelihood of bias in the sampling distribution, thereby increasing the
validity of the results. Despite its effect on representativeness, the decision to include various
exclusion factors reduces the number of extraneous health conditions that could distort the
collected data. Overall, this sampling scheme has a moderate degree of efficiency as the
collection of subjects would take a week or two and may incur significant resistance.
Treatments, Factors and Levels:
Treatment – the injection administered to the subjects (cat’s claw bark)
Factor 1: Cat’s claw bark injection
Level: 300mg solution given once daily
The injection of a cat’s claw bark solution allows for any active ingredients/components to
quickly enter the bloodstream and be dispersed around the body, bypassing potential breakdown
by the digestive tract. Relative to the oral route, the injection method prevents the potential loss
of active ingredients through the intestines, thereby increasing the efficiency of administration.
Previous studies have loosely established the tolerable range of cat’s claw administration to be
between 30-300mg. By injecting patients with 300mg, the maximum possible effect of cat’s claw
bark may be observed without inciting adverse effects in the majority of the subjects.
Consequently, the loss of individuals and the respective data as the sole result of treatment is
avoided over the course of the experiment.
Controls:
1. Cancer group with placebo – Implementing a negative control group that receives saline
solution allows us to assess the potential effect of an injection on the development of
breast cancer. When compared to the data generated by the experimental group, stronger
evidence for or against the efficacy of cat’s claw bark may be generated. Such is the case
as the placebo group will permit us to determine if the effect of the cat’s claw bark
exceeds any changes in physiology that may be solely attributed to the mind of the
person.
 2. Cancer group with nothing – A neutral control group will be crucial in establishing a
natural metric for the primary endpoints of the study (indicator molecules and tumor
size). Bereft of any external influence, the development of breast cancer in these subjects
will ideally reflect the progression of the disease in the absence of any intervention. As
such, any statistically significant difference between this group and another may be
attributed to treatment differentials, thereby providing a clearer determination as to the
effect of cat’s claw bark.

3. Cancer group with sedative – With a sedative being routinely utilized to gather data, it is
imperative that the degree of its inertness be assessed. In solely being treated with
sedative, any difference in the development of breast cancer versus the control group may
be attribute to this chemical. Consequently, the data generated by this group allows us to
gauge whether the sedative could have distorted the research outcome(s) and by what
degree. Additionally, such data could facilitate statistical determination of the sedative as
a potential confounding variable, ultimately increasing the accuracy of the experiment
results.

4. Cancer group with health monitor and tracking device – Throughout the experiment,
participants will possess a health monitor and tracking device as a means of continual
monition. As such, it is necessary to determine if the functions of these devices are
capable of influencing the development of breast cancer and thereby distorting the
observed effect of cat’s claw bark. With the sole treatment being the inclusion of the
health monitor and tracking device, any appreciable difference from the control may be
attributed to these instruments. Thus, this control group is crucial in assessing if the
health monitor and/or tracking device are capable of altering the primary endpoints of the
study (indicator molecules and tumor size).
Replication: With the experiment paralleling a phase 1 clinical study at its highest level, it can be
assumed that a sample size of approximately 100 individuals would be sufficient. By using 200
subjects, the power of the experiment is practically guaranteed, and a much greater range of
potential variability is accounted for in the study. Upon entering the study, participants are
assigned a number that is entered into the database of a computer. For allocation, the computer
randomly selects a number (participant) and then randomly assigns them to a control group or
the experimental group. Among the sample size of 200, the replicates consist of the individual
participants within each of the control and experimental groups. Thus, each group can be
expected to possess approximately 50 replicates when participants are allocated.
Potential Sources of Bias:
1. With a total of 200 subjects being drawn from the entirety of the United States, the
sample used in the experiment is a potential source of bias. When gathering such a
limited number of subjects on the basis of social security numbers, those numbers
corresponding to individuals in highly populated areas possess a greater probability of
 being selected. As such, the biological (genetic) and environmental (SES, gender, etc.)
factors included in the final sample may not represent the expected degree of variability.
Furthermore, the occurrence of certain factors may be amplified among the final sample
population, thereby yielding research results that are more reflective of these individuals
than the entire population.
2. Coupled with the limited sample size, the disparity in cancer rates between men and
women could result in a sample group that is primarily comprised of female subjects. As
such, the potential effect size relative to the males may be insufficient in establishing the
effect of cat’s claw bark. Alternatively, the results of such a study will be more aligned
with the female physiology than that of males. Thus, the observed effect of cat’s claw
bark could be significantly skewed and produce unrepresentative conclusions.
3. By only including subjects that possess social security numbers, the myriad of
undocumented individuals present in the United States are not accounted for in the
sampling procedure. As the pertinent biological (genetic) and environmental factors of
these individuals can deviate significantly from official citizens, the outcomes of the
study may be skewed towards a subsection of the total population.
Reduction of Bias:
The implementation of a random sampling procedure confers an equally likely probability of
each person (social security number) being selected for the study. As such, the likelihood of
generating a biased sample population that is reflective of a distinct subpopulation is minimized.
Likewise, the random allocation scheme conducted by a computer reduces the probability that a
group’s composition is skewed in favor of a particular population demographic. In doing so, each
potential/reasonable outcome is given a fair chance to occur in the results. Relative to the
subjects, each participant will be blinded as to true nature of the study and what they are
receiving over the course of the experiment. As a result, the potential for subject reactivity to
influence the research outcomes is minimized. Similarly, the blinding of the researchers
associated with the experiment (data curators, biostatistician, etc.) prevents any expectancy
effects from influencing the conclusions of the study. In particular, the inclusion of an external
and blinded statistician negates the possibility of the data being processed or analyzed in a
manner that supports one conclusion over another. Relative to the data collection, the
standardized use of an MRI and indicator specific blood tests reduces the capacity of the
researchers to manipulate the data in order to yield a significant statistical outcome.
Data Collection: Prior to allocation, a baseline blood sample will be collected and screened for
CA 27.29, a breast cancer antigenic marker. A baseline MRI of the breast tissue will also be
collected. Birth control method(s), hormone supplements and the presence or absence of
BRCA1/2 will be similarly determined prior to assignment. Once the experiment has begun,
individuals will receive daily MRI screenings until it is determined that a stage 1 breast cancer
tumor has developed. At this point the participant will be randomly assigned to a control group
or the experimental (cat’s claw) group. Starting from the date of allocation, blood samples and
MRI scans will be collected every three days for the next three months. Throughout this process,
general health assessments will be conducted to determine if external conditions are affecting the
status of the subjects. Once the three months have elapsed for each subject, the final stage of the
tumor will be determined through MRI screening and the subject released from the facility.

The Plan
1. Construct a treatment facility, ideally in a remote area, that is capable of housing 200
subjects and a similar number of research staff for at least five months.
2. Acquire BT-549 cancer cells from the ATCC or American Type Culture collection
3. Cultivate the BT-549 in an RPMI 1640 medium containing 2mM of L-glutamine, 1.5 g/L
of sodium bicarbonate, 4.5 g/L of glucose, 1 mM of sodium pyruvate and 10mM of
HEPES with the entire medium supplemented with 0.023 IU/ml of insulin and 10% fetal
bovine serum. Ensure that enough cells are cultured to inject 200 subjects with 5 x 10^6
breast cancer cells.
4. Attain a digital register of all the social security numbers in the United States. Using a
random sampling algorithm, select and acquire 200 subjects for the study.
5. Collect 10 milliliters of plain blood from the subject with a scalped winged infusion set.
Allow the blood to clot at room temperature and then immediately run a CA 27.29 blood
assay on the sample.
6. Perform a full body MRI with contrast and record the results.
7. Perform a physical on the subject.
8. Sedate each subject with aerosolized Propofol, with the exposure corresponding 0.5
mg/kg over the course of 3-5 minutes.
9. While the subject is anesthetized, use an automated injection device to administer 5 x
10^6 of BT-549 cells into the adipose tissue of the gluteus maximus. Ensure that the
injection location at the center of the left glute for each subject.
10. Insert the health monitor and tracking device into the center of the right glute.
11. For each day following the BT-549 injection, perform full body MRI and mammogram to
assess if a stage one tumor has developed.
12. Once a stage one tumor has been detected, the subject is assigned a number and randomly
allocated to one of the experimental groups through a randomization algorithm.
13. Every three days after the detection of a stage one tumor, the subject is anesthetized with
aerosolized propofol while they are sleeping.
14. An automated propofol distribution apparatus is affixed to the face of the subject, keeping
them sedated and unconscious until the following tests are completed.
15. Immediately following this, 10 milliliters of the subject’s blood should be drawn with the
winged infusion set previously utilized in the study.
16. After the blood draw, the subject is transported to an available MRI, taking care to avoid
any other subjects in the facility.
17. Run a full body MRI with contrast and record the results, noting tumor size.
18. Determine if the health monitor and tracking device are working properly through the
built-in ping and response system.
19. Return the subject to their room and remove the propofol application apparatus.
20. Repeat steps 13-19 on each subject until three months since the detection of stage 1
tumor has elapsed.
 21. Once the three months has elapsed, the subject is released from the treatment facility and
given a monetary compensation of $10,000,000 for their participation.
22. Following the release of the final subject, the data is collated on the basis of tumor size
and CA27.29 expression levels.
23. The data is then digitally transferred to several independent biostatisticians.
24. The biostatisticians process the data using a causal and descriptive analytical approach.
Risk Minimization:
1. Throughout the course of the study, participants will be fed a standardized diet that meets
the daily consumption recommendations of the USDA. All subjects will be provided with
enough fruits, vegetables, grains, meats, and fluids to meet a minimum caloric intake of
2,00 calories each day. Individuals that possess food allergies will receive substituted
ingredients to avoid allergen exposure.
2. The room of each subject will be furnished with a bed, television, miniature library and
basic utilities, such as a shower and toilet. In addition, subjects may request recreational
items that cannot interfere with the study or be used to harm other participants. Similarly,
subjects will be several hours of socialization and general time each day in which they
can use the recreational facets of the facility.
3. As the blood draw represents a source of potential contamination, all winged infusion sets
will be kept in a sterile environment and treated with antiseptics before use. Additionally,
the individuals in charge of the blood draw will wear gloves and a mask to mitigate
spread of possible pathogens. Similarly, the MRI machine will be thoroughly sterilized
after each use.
4. Before and after each subject utilizes the MRI machine, a full diagnostic will be run to
ascertain any possible sources of harm to the subject. Likewise, subjects will be screened
for any and all metallic objects before entering the MRI housing area in order to prevent
bodily harm. Prior to the baseline MRI, the medical history of each subject will be
reviewed for metallic apparatuses, such as pacemaker, that can be affected or deactivated
by the machine.
5. The propofol distribution device affixed to the face of each subject will be cleaned before
and after each use to prevent the spread of pathogens. While they are exposed to the
propofol, the blood concentration of the molecule will be assessed by the health monitor
to prevent fatal or adverse levels from accumulating.
6. By choosing an MRI, subjects are not exposed to the radiation associated with CT or X-
Ray scans. In doing so, the potential for radiation sickness or significant mutations in the
DNA of the subjects’ cells are avoided. Thus, the decision to utilized an MRI reduces the
likelihood of incurring bodily harm from a necessary scanning protocol.
Data Collection:
All data acquired throughout the duration of the study will be uploaded and stored to Benchling.
The MRI machines, connected to the Benchling server before the experiment, will automatically
upload their images without external input. CA 27.29 assay tests, conducted by a machine where
possible, will be similarly uploaded through a preestablished connection with the Benchling
server. To ensure that the machines are uploading the respective data, blinded technicians will
monitor the data streams from each device and troubleshoot when they fail to transfer the
information. Likewise, both a human and artificial data manager will oversee the entry of data
onto Benchling, alerting the others when information is not uploaded but being unable to alter
the larger data store. To protect the information, all data will be encrypted upon transmission and
the Benchling repository will possess a rigorous array of passwords that are only revealed to the
data manager(s). Physical backups (hard drives and/or disks) will be generated every three days
and a secondary Benchling backup will be used until the conclusion the study. In verifying the
data, the specific encryptions serve as a preliminary tag denoting the content of each
transmission. During and after the study, random samples of the data will be exported to
biostatisticians or other experts that will assess the validity of the measurements relative to the
literature and previous experience.