Conditions: Traumatic Brain Injury
Keywords: cognition seizures electroencephalography transcranial magnetic stimulation huperzine chinese club moss
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 14
Healthy volunteers: no

Brief summary:
We will explore the use of Huperzine A in patients who have sustained a moderate to severe Traumatic Brain Injury. We aim to determine whether Huperzine A, as compared with placebo, would have an effect on memory function after TBI. Additionally, we aim to determine whether use of Huperzine A in these patients can change brain activity (as indexed by EEG and Transcranial Magnetic Stimulation - TMS), and reduce prevalence/frequency of post-traumatic seizures. We also aim to evaluate the safety of Huperzine A in this population as compared with placebo.

Detailed description:

Arms:
arm 1: Huperzine A will be administered to patients, titrating dose up from 100mcg/day to 600mcg per day over the course of 20 days - and remaining on the dose of 600mcg/day for the remainder of the drug phase (64 days) - for a total of 12 weeks on Huperzine A. arm 2: Placebo will be administered to patients at the same frequency/intervals as the experimental arm (Huperzine-A).

Interventions: intervention 1: Huperzine A intervention 2: Placebo
Intervention descriptions: intervention 1: Huperzine A will be administered for 12 weeks as outlined in the Arm Description intervention 2: Placebo Arm (blinded randomization) for Huperzine A Intervention

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
COVER PAGE 
 
 
 
 
Study Official Title:  Effects of Huperzine A in Treatment of 
Moderate to Severe TBI 
 
 
 
NCT #: 01676311 
 
 
 
 
Version Date of the Document: 4/11/19 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Detailed Protocol 2 
ORGANIZATION OF DETAILED PROTOCOL 
 
Title: Huperzine A for the Treatment of Cognitive, Mood, and Functional Deficits after Moderate 
and Severe TBI 
Protocol #: 2012-p-002490 
Date: 04/11/19 
 
 
I. 
BACKGROUND AND SIGNIFICANCE 
 
Traumatic Brain Injury (TBI) is a major public health issue, with 1.7 million new cases reported 
annually in the US [1, 2]. Public awareness of TBI is further heightened by the increase in combat-
related TBI (cTBI) in soldiers returning from Iraq and Afghanistan [3]. However, many of the 
cognitive and psychological sequelae following TBI, especially combat related TBI, are difficult 
to differentiate from other disorders, and the chronic symptoms remain a vexing problem for those 
affected and for the military. Indeed, the most frequent problems of military personnel with TBI 
and their families relate to cognitive and executive disorders, pain, behavioral deficits, and post-
traumatic seizures, which in the “real military world” present as a constellation of symptoms. Thus, 
we plan to evaluate symptoms as such. In order to meet the unmet medical needs in combat-related 
TBI, an ideal agent would provide multiple mechanisms of action relevant to the pathophysiology 
of TBI. Further, therapies for those with TBI should have low risk and side effect profiles and 
should preferably be already approved or available. Unfortunately, there are few if any such 
therapies in development.  
 
Based on Huperzine-A’s multiple mechanisms of action, we believe it will benefit patients with 
significant TBI. In this study, we will focus on Huperzine A’s potential to improve cognitive and 
functional deficits in the chronic setting since this is a gap, and a necessity for the military. We 
therefore propose a dose-escalating phase II study. In this study, we also plan to link treatment to 
neurophysiologic metrics, which may help guide future work for the military. 
 
Huperzine A 
Huperzine A (see figure 1) is purified from Chinese club moss 
and has been traditionally used in China for the treatment of 
swelling, 
fever, 
inflammation, 
blood 
disorders, 
and 
schizophrenia,[4]. In China, it is approved for use in the 
treatment of Alzheimer’s disease (AD). Huperzine A was 
classified as a dietary supplement by the FDA in 1997. As a 
nutraceutical, it is available in American health food stores or 
via the Internet, labeled as a memory aid.  
 
Huperzine A has multiple mechanisms of action that are relevant to the underlying 
pathophysiology of TBI. Huperzine A has been shown to be neuroprotective in several models. It 
is a non-competitive antagonist of N-Methyl-D-aspartate (NMDA) receptors at one of the 
polyamine binding sites [5] at or near the PCP and MK-801 ligand sites but without the 
psychotomimetic side effects of PCP or MK-801. [6] In addition to neuroprotection, this 
mechanism of action may also have importance for treating symptoms of depression within this 
Figure 1: Structure of Huperzine A 


Detailed Protocol 3 
patient population. Furthermore, Huperzine A attenuates oxidative stress; regulates the expression 
of apoptotic proteins Bcl-2, Bax, P53, and caspase-3; protects mitochondria; upregulates nerve 
growth factor and its receptors; and interferes with amyloid precursor protein metabolism [7] [8] 
with favorable pharmacokinetics [9] [10].  
 
Huperzine A produces dose-dependent increases of norepinephrine and dopamine in rat cortexes 
when administered chronically via the intra-peritoneal (i.p.) route, or locally through a 
microdialysis probe [11], which may have relevance to frontal lobe functioning in the TBI 
population. 
 
Huperzine A inhibits acetylcholinesterase, which suggests a potential benefit for memory 
dysfunction in patients with TBI. Indeed, short-term memory loss is a frequent complication of 
TBI and often a barrier to returning to work. Because there are no specific pharmacological 
strategies for treating memory dysfunction in patients with TBI, other than avoiding drugs that 
worsen memory, studies are beginning to explore the potential usefulness of acetylcholinesterase 
inhibitors with promising results [12]. Huperzine A is a potent, highly specific, and reversible 
inhibitor of acetylcholinesterase, with comparable potency to physostigmine, galantamine, 
donepezil, and tacrine [4].  
 
Therefore, given its multiple mechanisms of action, Huperzine A might be ideal to modulate the 
primary as well as secondary injury mechanisms that occur during the first several weeks following 
TBI as well as chronic phases. Thus, a link to neurophysiologic data could help us develop a 
biomarker for the role of Huperzine A in chronic recovery. 
 
Experience in humans: 
 
Huperzine A has been administered to humans orally, intravenously, and intramuscularly, and has 
been well studied in dementia. There is one published study on the use of Huperzine A for mild to 
moderate TBI, conducted in China and published in Chinese [13]. According to Wang et al [14], 
who discussed this TBI study in English, thirty patients were treated with standard therapies (0.8 
gram piracetam and 20 milligrams nimodipine, twice per day, combined with functional 
rehabilitation), and another 30 patients were treated with 100 micrograms Huperzine A BID in 
addition to standard therapies. Both groups demonstrated significant improvement in memory and 
cognition after both 1 and 3 months, but the improvement in memory and cognition in patients 
treated with Huperzine A was more dramatic than was the improvement in patients treated with 
standard therapies only. As an example of a study in AD, Xu and colleagues [15] evaluated the 
efficacy and safety of Huperzine A in patients with AD using a multicenter, prospective, double-
blind, parallel group, placebo controlled, randomized design. Fifty patients received 200 
micrograms of HUP A for 8 weeks and a well-matched group received placebo. Statistically 
significant improvements in memory (P < 0.01), cognitive functioning (P < 0.01), and behavioral 
measures (P < 0.01) were noted in Huperzine A-treated subjects compared to placebo. No severe 
side effects were reported.  
 
Regarding pharmacokinetics, published data in humans are limited but suggest rapid absorption 
and widespread distribution. The pharmacokinetics of a single 0.99 milligram oral dose of 
Huperzine A in six Chinese volunteers are consistent with a one-compartment open model with a 


Detailed Protocol 4 
first order absorption, with T 1/2ka = 12.6 minutes, T 1/2ke = 288.5 minutes, Tmax = 79.6 minutes, 
Cmax = 8.4 micrograms L-1, and AUC = 4.1 milligrams L-1 minutes [16]. A plasma steady state 
would likely be established within 2 days. Interactions with other drugs are unlikely, with the 
possible exception that CYP1A-inducing drugs, which may increase Huperzine A clearance.  
 
Other Huperzine properties of interest: 
 
Antinociception  
Nociception is the ability to feel pain and antinociception is reduction in pain sensitivity. In the 
mouse formalin pain model, Huperzine A 1 mg/kg i.p. produced complete inhibition of pain 
behavior in all treated animals at all time points, and 0.5 mg/kg i.p. (60% of the TD50) produced 
near complete inhibition [17]. Both sets of results demonstrated statistically significant differences 
from controls (P < 0.01). Similar results were obtained with Huperzine A 1 mg/kg i.p. in the sciatic 
ligature model of pain [17]. 
 
The ED50 of HupA when given intrathecally (IT) in the rat thermal escape model was 0.57µg/kg 
(95% CI [0.25, 1.30]) [17]. Likewise, IT Huperzine A resulted in a dose-dependent decrease in 
flinching in both phase I and phase II of the formalin test in rats. The observed antinociceptive 
effects of IT Huperzine A in both rat models were largely blocked by pre-treatment with IT 
atropine (15 µg/µL). Thus, this suggests the potential efficacy of this agent in combating 
nociception, a common phenomenon in our nation’s heroes with TBI. 
 
Anticonvulsant profile 
Huperzine A was active against subcutaneous (s.c.) pentylenetetrazole, but not maximal 
electroshock-induced seizures, following oral (p.o.) administration to Swiss-Webster mice, with 
peak anticonvulsant activity at one hour [18]. At doses of 1, 2, and 4 mg/kg, a maximum of 62.5% 
protection was observed. Impairment on the rotarod test was observed in 75 and 100% of mice 
tested at doses of 2 and 4 mg/kg, respectively. The TD50 was 0.83 mg/kg.  
 
In the 6-Hz model, ED50 values for i.p. Huperzine A were 0.28, 0.34 and 0.78 mg/kg for 22, 32, 
and 44 mA, respectively, suggesting a possible advantage over phenytoin, carbamazepine, 
lamotrigine and topiramate, each of which display limited efficacy in this model at doses devoid 
of behavioral toxicity [19]. The less than 2-fold ratio of dosages effective across the range of 
stimulations suggests a further possible advantage over other drugs active in this model such as 
levetiracetam. Atropine 30 mg/kg i.p. completely blocked the anticonvulsant effect in the 6-Hz 
model (32 mA) and nearly completely blocked the toxic effects of Huperzine A 1 mg/kg i.p. When 
given intravenously (i.v.) the ED50 of Huperzine A in the 6-Hz model at 32 mA was 0.21 pmol / 5 
µL injection volume and the TD50 was 36.15 pmol / 5 µL (TD50: ED50 = 172). 
 
There are no available published data of efficacy or tolerability in patients with epilepsy or chronic 
neuropathic pain. Huperzine A has been studied in patients with myasthenia gravis and AD [4]. 
Efficacy was favorable and cholinergic side effects, such as dizziness, diarrhea, and nausea were 
generally infrequent and mild in intensity. Additionally, a multicenter, NIH-funded, placebo-
controlled Phase II trial of Huperzine A has recently been published, testing 200 and 400 
micrograms twice daily in patients age 55 and older with mild-to-moderate AD [20].  


Detailed Protocol 5 
In this current study in patients with TBI, Huperzine A’s pleuripotential mechanisms will be of 
great value. Specific to this study, Huperzine A’s potency of ACh inhibition has been found to 
rival those of tacrine and donepezil [21]. Huperzine A appears to have no tolerance effect, as 
repeated doses appear to demonstrate similar AChE inhibition as that of a single dose in a rodent 
model [22]. Compared with agents such as tacrine and physostigmine, Huperzine A appears to 
result in the longest lasting boost in AChE levels. The dosage selected for this study is based on 
safety and efficacy data noted in several studies of AD in China. In these studies, the side effects 
are reported as being comparable to placebo. Subjects will receive study medications (either 
Huperzine A or Placebo) for 12 weeks while inpatient. They will undergo evaluations while on 
study drug (6 and 12 weeks), and post-dosing (24 and 52) weeks. 
 
 
II. 
RATIONALE FOR EXPANDING THE STUDY TO INCLUDE OUTPATIENTS 
The process of brain injury after a traumatic insult occurs in three different stages: an acute phase, 
characterized by a progressive path of cell degeneration, initiated by unrestrained neuronal 
depolarization. This initial phase is followed by the subacute and chronic stages of brain injury, 
which share common pathophysiologic mechanisms: cell repair, resolution of edema and 
inflammation, excess GABA-mediated inhibition in networks, changes in synaptic strength in 
networks: dysfunctional long-term potentiation (LTP) and long-term depression (LTD), structural 
modifications, and plastic changes and new connections [23].  
Recovery of function after TBI can also be divided into three stages. During the initial phase, 
activation of cell repair is the main compensation mechanism, leading to resolution of edema and 
inflammation. This stage takes place mainly over the first three weeks after the injury. The second 
stage is characterized by substantial changes in previously existing neuronal networks that are 
reflected both in functional and anatomical cell plasticity leading to the formation of new 
connections. Plasticity and remyelination are the most important mechanisms governing the final 
stage of recovery and are usually most prominent within the first three months after the insult. 
Therefore, it is clear that most changes and recovery occur in the subacute and chronic stages, 
which share a common underlying physiologic pathway [23, 24].  
It is clear that neuroplasticity plays a crucial role in promoting recovery after brain injury, and it 
can be viewed as a mechanism to compensate for the injury and reestablish function. Thus, 
understanding the functional and dysfunctional aspects of this ongoing process, as well as the main 
pathophysiologic events occurring after a TBI, is necessary to create optimal therapeutic 
approaches [23]. Due to the similar mechanisms subacute and chronic stages share for cell 
reparation and plasticity, strategies that promote reorganization of neural networks should allow 
for improvement of cognitive and functional deficits for patients in either phase of recovery [24].  
The ability to assess both inpatient and outpatient cases will allow for a broader understanding of 
the effects Huperzine A has across various stages of recovery. Until now, the majority of studies 
assessing the effect of Huperzine A have been in outpatient settings, although only in mild to 
moderate TBI [25]. Thus, by evaluating Huperzine A in both an inpatient and outpatient setting 
we will have a comparative base by which to determine whether functional and cognitive recovery 
is in fact similar in subacute and chronic patients. Given that Huperzine A has been tested mainly 
in the outpatient/chronic setting and that the pathophysiology of the subacute and chronic stages 


Detailed Protocol 6 
encompasses similar recovery mechanisms, we expect that benefits will be equally significant in 
these two populations. Expanding our study to outpatient population will also have clear benefits 
on enrollment, given that it will allow us to recruit TBI patients who did not receive direct care 
from our institution that would potentially otherwise be eligible.  
 
 
III. 
OVERALL GOALS 
 
We plan to conduct a phase II, 52 week, randomized, double-blind, placebo-controlled clinical 
trial that investigates the effect of Huperzine A on memory function in subjects who sustain 
moderate to severe traumatic brain injury. We will also explore the effects of Huperzine on 
transcranial magnetic stimulation (TMS)-induced neurophysiologic markers, EEG event related 
potentials (P50 and P300), seizure frequency/prevalence and incidence of adverse effects as 
secondary aims. Measures of mood, pain and subjective complaints of concussive symptoms, and 
length of time post-injury, will serve as covariates to address potential treatment confounders. The 
data generated from this study will be used to plan a subsequent phase II/III trial with Huperzine A.   
 
 
IV. 
SPECIFIC AIMS 
 
Primary Aim: 
• To determine whether Huperzine A, as compared to placebo, has a differential effect on learning 
and memory functions after moderate to severe TBI. We hypothesize that the changes in mean 
scores on tests of learning and free recall as measured by the California Verbal Learning Test- 
II (CVLT-II) will be significantly greater after 12 weeks of treatment with Huperzine A in 
individuals with moderate to severe traumatic brain injury relative to those treated with placebo. 
Secondary Aims: 
• To determine whether administration of Huperzine A produces significant differences in 
neurophysiologic markers (as indexed by EEG event related potentials (P50 and P300) and TMS-
indexed cortical excitability (cholinergic activity)) associated with cognition relative to a placebo. 
We expect that patients who receive Huperzine, as compared to placebo, will have decreased 
latency and increased amplitude for P50 and P300 and, in addition, they will have increased 
short latency afferent inhibition (as indexed by TMS measurement), thus indicating increased 
cholinergic activity and cognitive processing at 12 weeks post-enrollment.  
• To determine whether Huperzine A reduces the prevalence/frequency of post-traumatic seizures 
after moderate and severe TBI as compared to placebo. We hypothesize that the prevalence of 
seizure will be significantly lower at 12 weeks post-enrollment (immediate seizures prevalence) 
in subjects treated with Huperzine A relative to those treated with placebo.  
• To evaluate the safety and tolerability of Huperzine A in this patient population as compared to 
placebo. Safety and tolerability will be assessed by a comparison of the frequency and intensity 
of adverse effects. We hypothesize that the incidence of clinically significant adverse effects will 
be no different in subjects treated with Huperzine A relative to those treated with placebo. 
 
 


Detailed Protocol 7 
V. 
INVESTIGATORS 
Ross Zafonte, DO Spaulding Rehabilitation Hospital 
Principal Investigator 
Felipe Fregni, MD, PhD Spaulding Rehabilitation Hospital 
Co-Investigator, recruitment, screening and consenting, data collection and analysis 
Joseph Giacino, PhD Spaulding Rehabilitation Hospital 
Co-Investigator, recruitment, screening and consenting, cognitive assessment, outcome 
measurement and analysis 
Nancy Boudreau, RN Spaulding Rehabilitation Hospital 
Research Nurse Coordinator 
Seth Herman, MD Spaulding Rehabilitation Hospital 
Co-Investigator- Study physician 
Timothy Young, MD Spaulding Rehabilitation Hospital 
Co-Investigator- Study physician 
 
 
VI. 
SUBJECT SELECTION 
 
We plan to enroll 30 subjects with moderate to severe TBI (Huperzine = 15, Placebo = 15). 
 
Inclusion criteria: 
 
• Males and females aged 18 to 65 
• Meeting at least one of the following criteria for moderate or severe TBI:  
- 
PTA > 24 hours 
- 
Trauma related intracranial neuroimaging abnormalities  
- 
Loss of consciousness exceeding 30 minutes (unless due to sedation or 
intoxication) 
- 
GCS in the emergency department or ICU of less than 13 (unless due to intubation, 
sedation, or intoxication). If there is no GCS score available from the ED or ICU, 
the GCS from the scene can be documented. 
• All subjects will be no more than 1 year post-injury, and will be symptomatic at enrollment 
(i.e. all subjects will exhibit evidence of ongoing posttraumatic amnesia via the Galveston 
Orientation Amnesia test (GOAT), OR score at least 1.5 SD below the mean for 
completion time on Part B of the Trail Making Test, OR score at least 1.5 SD below the 
mean on either the Learning and Memory short form of the Traumatic Brain Injury Quality 
of Life (TBI-QOL) scale or the Ruff Neurobehavioral Inventory (RNBI) Postmorbid 
Cognitive Domain scale. 
• Agreement to undergo no changes in concomitant medications (including dietary 
supplements) or therapeutic interventions during the first 12 weeks of the study (that is, 
the 12 weeks of dosing with study drug), except where medically indicated. Intact enteral 
route 
• English-speaking (since not all of the outcome metrics are normed outside of the English 
language) 
• Patient can be on seizure medication. 
 


Detailed Protocol 8 
Exclusion criteria: 
 
• Patients taking acetylcholinesterase inhibitors and other cholinergic and anticholinergic 
drugs (e.g., tacrine, physostigmine, velnacrine, donepezil, rivastigmine, metrifonate) and 
CYP1A inducing/inhibiting drugs (ex. ciprofloxacin, enoxacin, fluvoxamine, methoxsalen, 
mexiletine, oral contraceptives, phenylpropanolamine, thiabendazole, zileuton, acyclovir, 
allopurinol, caffeine, cimetidine, daidzein, disulfiram, Echinacea, famotidine, norfloxacin, 
propafenone, terbinafine, ticlopidine, verapamil, montelukast, phenytoin, moricizine, 
phenobarbital). 
• Evidence of more than 1 seizure in the past 4 weeks prior to enrollment: Patients may 
not be enrolled if there is evidence of more than one seizure (clinical or electrographic, but 
not including epileptiform or other irritative discharges) during the 4 weeks prior to 
enrollment.  
• Premorbid history of epilepsy with seizure frequency >1 per month: Patients with a 
history of idiopathic epilepsy may not be enrolled if their seizure frequency was > 1 per 
month in the 3 months prior to injury. If pre-injury seizure frequency was < 1 per month 
but there is documented evidence that post-injury seizure frequency is > 1 per month or 
there is documented evidence of an increase in the severity or duration of a single seizure 
relative to the premorbid history, the patient must be excluded.  
• Evidence of premorbid major CNS disorder, developmental disorder, psychiatric 
disorder or substance abuse: Prior to sustaining TBI, patient was diagnosed and/or 
treated for a major neurologic condition, pervasive developmental disorder (e.g., mental 
retardation, autism), psychiatric disorder or substance abuse that continued to produce 
functional disability up to the time of injury. This would be demonstrated by active 
treatment, hospitalization, and/or documentation of active withdrawal within the 12-month 
period prior to enrollment. The PI will determine if the patient was a chronic substance 
user or simply had an overindulgence that was not maintained. If in the judgement of the 
PI and/or the study physician the patient exhibits no present evidence of substance 
withdrawal and has demonstrated a commitment to substance abstinence, the patient can 
be enrolled.  
• Individuals with disorders of consciousness, as defined at the time of screening of having 
vegetative and/or minimally conscious state, will not be enrolled. However, these patients 
may be followed until they meet eligibility criteria 
• Pregnancy, as determined by serum hCG testing before randomization 
• Breast feeding females 
• Significant hematologic, renal or hepatic dysfunction [Hepatic/renal dysfunction is 
generally identified as lab results > two times the upper limits of normal (ULN), and 
hematologic dysfunction is determined by clinically significant abnormal lab results], on 
baseline laboratory examination. 
• Slow heart rate (bradycardia) or other heart conditions related to rate 
• History of peptic ulcer disease 
• History of asthma or emphysema as exemplified by active treatment with consistent use of 
disease modifying drugs within the 12-month period prior to enrollment 
• History of GI/urinary tract blockages (i.e. ileus, IBS) 
• History of glaucoma 
• Current smoker (have smoked within 1 week of enrollment).  


Detailed Protocol 9 
 
 
VII. 
SUBJECT ENROLLMENT 
 
Potential subjects will be identified by the following sources: 
 
1. Attending physicians may refer their TBI patients to the study. We will provide physicians 
with study information sheets and flyers.  
2. Medical record review by physician on clinical treating team - of inpatients in Spaulding 
Rehabilitation Network facilities (Boston/Cambridge). If the patient is found to be eligible 
based upon this search, we will contact the primary rehabilitation physician to approach 
the patient and/or family about participation. 
3. Outpatients will be referred from other healthcare facilities, the community, or the treating 
physician.  
 
Once referred, study staff will approach subjects who retain decision-making capacity, or the 
authorized representative of a patient who lacks decision-making capacity, for permission to 
complete a screening for possible enrollment. Please note that clearance for consent will follow 
standard clinical procedures. A co-investigator/coordinator will review with the potential 
subject/authorized representative the informed consent form, and clearly explain all the procedures 
and risks of the testing. The subject/authorized representative will be encouraged to ask questions, 
both during the initial interview and throughout the study. The PI or a co-investigator/coordinator 
will answer any questions regarding the study during the consent process. During the enrollment 
interview, the subject/authorized representative will be instructed not to take any prescribed or 
over-the-counter herbal supplements or nutraceuticals during participation in this study to avoid 
potentially confounding study results. Once enrolled, the subject may terminate his/her 
participation at any time during the study.  
 
 
 
VIII. STUDY PROCEDURES 
 
Study Design: 
 
This is a randomized, double-blind, placebo-controlled phase II study. We plan to enroll 30 
subjects with moderate to severe TBI (Huperzine = 15, Placebo = 15). Subjects may be inpatients 
or outpatients. Subjects will receive study drug or placebo for 12 weeks, and follow-up will 
continue through Week 52. Subjects enrolled in this study will receive standard acute and 
rehabilitative care. All subjects will be enrolled at Spaulding. Spaulding inpatient subjects who are 
discharged from Spaulding prior to closure of the active treatment window (at week 12) will 
continue to be followed either by telephone or in-person. Weekly phone calls between the study 
coordinators and subjects (or their authorized representative) will ensure that, for each subject 
actively enrolled, the dosage of study medication is correct, any new adverse events (including 
seizure) have been captured and any concerns raised by the subject or study staff have been 
addressed. 
 
Recruitment and Consent 
 


Detailed Protocol 10 
SRH Inpatients 
Subjects will be recruited from the inpatient brain injury rehabilitation units at Spaulding 
Rehabilitation Network facilities. Medical records will be pre-screened by study staff to identify 
potentially-eligible subjects and relevant inclusion/exclusion criteria. The patient or patient’s legal 
surrogate will be approached for consent after basic information about the study is provided. The 
patient/authorized representative will review study information and discuss the study with the 
coordinator/co-investigator. 
During 
the 
enrollment 
interview, 
the 
subject/authorized 
representative will be instructed not to take any prescribed or over-the-counter herbal supplements 
or nutraceuticals during participation in this study to avoid potentially confounding study results. 
The coordinator/co-investigator will answer all questions and provide all study information to the 
patient/authorized representative prior to obtaining consent. After consent has been provided, the 
screening process will take place. 
 
Community-Dwelling Outpatients 
Potential participants will be recruited from the Spaulding Rehabilitation Hospital Network, and 
MGH psychiatry, and Home Base clinics. Clinic staff will be informed about the study with an 
information sheet and asked to speak to patients fitting the inclusion/exclusion criteria. If a 
potential participant gives permission to be contacted, the research staff will review available 
medical records to determine preliminary eligibility prior to contacting them to discuss the study 
and their potential participation. During the enrollment interview, the subject/authorized 
representative will be instructed not to take any prescribed or over-the-counter herbal supplements 
or nutraceuticals during participation in this study to avoid potentially confounding study results. 
 
Additional recruiting will be done through local healthcare providers, PatientsLikeMe, BIA 
chapters, mailings to databases such as RPDR, SHIP and TBIMS, support groups, health fairs, 
community programs for TBI patients and families, treating physician letters to patients, flyers 
posted in clinics, newsletters and local and regional presentations. Subjects will also be recruited 
through postings to electronic bulletin boards, internet websites (i.e. ClinicalTrials@Partners), and 
Spaulding’s intra/internet web pages. Newspaper, TV, magazine, and radio ads as well as press 
releases will also be utilized. 
 
A recruitment log of potential subjects screened using the study’s inclusion/exclusion criteria will 
be maintained.  The log will contain PHI such as the potential participant’s name, date of admission 
and, if excluded, the rationale, which may be medical.  Persons on the log include individuals that 
are being followed by study staff, pending consent, participating, refusing to participate or have 
been screened out. The log will be maintained during the study for tracking purposes to avoid re-
approaching or re-screening potential participants, thereby, decreasing potential participant 
burden. This recruitment log will be destroyed at study completion. 
 
For all potentially eligible patients, the patient, or authorized representative of a patient who lacks 
decision-making capacity, will be approached for consent. If the decision-making capacity of a 
subject is in question, the subject will be evaluated by a member of the clinical staff to determine 
if the subject understands the difference between treatment and research, the risks and benefits of 
the research protocol and its procedures, and the consequences of acting (or not acting), and can 
make the choice to participate. If the subject is determined to lack decision-making capacity, 
persons who might be proposed as appropriate authorized representatives are, in order of general 


Detailed Protocol 11 
preference: (1) court-appointed guardians with authority to consent to participation in the proposed 
research or authority to make decisions for a class of health care decisions inclusive of the proposed 
research, (2) health care proxy with authority to make decisions for a class of health care decisions 
inclusive of the proposed research, (3) durable powers of attorney with authority to make health 
care decisions inclusive of the proposed research, or (4) a spouse, adult child, parent, adult sibling, 
or other close family member. If an authorized representative signature is required due to lack of 
decision-making capacity, the authorized representative will sign the consent form on the Guardian 
or Legal Representative for Adult line and the subject may also sign the Assent line. 
Documentation of the informed consent process will be recorded for each subject with the Partners 
QI Documentation of Informed Consent document. 
 
If, in the opinion of the clinical staff or research team, a participant who was originally enrolled in 
the study by an authorized representative subsequently regains decision-making capacity (e.g., 
emerges from post-traumatic amnesia), he or she will be re-consented to determine the 
participant’s preference for continued study participation. 
 
During the consent process, participants/authorized representatives will be asked if they would 
like to be contacted regarding future research opportunities. If the participant/authorized 
representative chooses not to decide either “yes” or “no” to contact for future research at the time 
of signing, the co-investigator/coordinator will ask at subsequent follow-up visits, and their 
pending decision will be tracked. Completion of the study before obtaining an answer will result 
in that participant not being contacted regarding future research opportunities. 
 
Screening and Baseline Assessments: 
As part of the screening process, we will complete the following assessments: 
• Neuropsychological examination (i.e., GOAT, Trails B) 
• Learning and Memory short form of the Traumatic Brain Injury Quality of Life (TBI-QOL)  
• Ruff Neurobehavioral Inventory (RNBI) Postmorbid Cognitive Domain 
• Required lab studies (pregnancy test via serum hCG, hepatic, hematologic, and renal 
function) and physical examination (including vital signs and EKG to rule out cardiac 
arrhythmias)  
• A medical history including seizure history will be obtained via the patient’s medical 
records 
• Baseline symptom checklist 
• TMS screening checklist 
 
If all eligibility criteria are met and the patient is enrolled in the study, randomization will take 
place. We will employ a computer-generated randomization scheme to determine whether the 
subject receives placebo or Huperzine A. Randomization will be performed in permuted blocks of 
four or eight with random variation of the blocking number to prevent potential unblinding. The 
randomization code will be kept by a third party with no access to subjects or study data.  
 
Within 7 days of randomization, the following additional baseline assessments will be completed: 
• Neurophysiological baseline measures (i.e. P50, P300 and SAI)  
• Neuropsychological baseline measures (i.e. CVLT-II,) 
• Learning and Memory short form of the Traumatic Brain Injury Quality of Life (TBI-QOL)  


Detailed Protocol 12 
• Ruff Neurobehavioral Inventory (RNBI) Postmorbid Cognitive Domain  
• Beck Depression Inventory 
• Modified Brief Pain Inventory 
• British Columbia Post Concussion Scale 
• Eye Tracking baseline measures  
 
Study Visit Timeline: 
 
All study visits for inpatients and outpatients will take place in-person at Spaulding. During the 
course of this study, the subjects will be assessed at predetermined time points. These time points 
are: Screening/Baseline, Week 6, Week 12, Week 13, Week 24 and Week 52. In the table below, 
the study visits and assessments are outlined.  
 
As we are investigating patients with Traumatic Brain Injury, there may be instances where the 
study timeline will need to be adjusted. This may occur if the patient is transferred during his/her 
hospital stay through clinical treatment, or during follow-up scheduling. Therefore, we will employ 
a window of +/- 2 weeks to complete the study visits, in order to accommodate for scheduling 
issues and changes in the subject’s care. 
 
Study Visit Summary: 
 
Table 1 provides a summary of the study visits and study-related activities. 


Detailed Protocol 13 
Table 1: Study Visit Summary 
 
Hup-A Dose 
Administration 
Period 
 
 
Screening Baseline 
6 Wks 
12 Wks 
13 
Wks 
24 
Wks 
52 
Wks 
History and Physical 
 
 
 
 
 
 
 
Baseline Symptom Checklist 
X 
X 
X 
X 
X 
X 
X 
Pregnancy Test (Serum hCG) 
X 
- 
- 
- 
- 
- 
- 
Medical History Review 
X 
- 
- 
- 
- 
- 
- 
TMS Screening Checklist 
X 
- 
- 
- 
- 
- 
- 
Laboratory Data (renal and liver 
function, hematology) including 
a general chemistry 6 panel at 
baseline 
X 
- 
X 
X 
X 
- 
- 
ECG 
X 
- 
X 
X 
X 
- 
- 
Physical Exam (to include vital 
signs) 
X 
- 
X 
X 
 
 
 
Neurocognitive 
 
 
 
 
 
 
 
GOAT 
X 
- 
X 
X 
- 
X 
X 
Trail Making Test A&B 
X 
- 
X 
X 
- 
X 
X 
TBI Quality of Life 
X 
X 
X 
X 
- 
X 
X 
Ruff Neurobehavioral Inventory 
X 
X 
X 
X 
- 
X 
X 
CVLT-II  
- 
X 
X 
X 
- 
X 
X 
Brief Pain Inventory 
- 
X 
X 
X 
- 
X 
X 
Suicidality Scale 
- 
X 
X 
X 
X 
X 
X 
Beck Depression Inventory  
- 
X 
X 
X 
- 
X 
X 
British Columbia Post-
Concussion Scale  
- 
X 
X 
X 
- 
X 
X 
Neurophysiologic 
 
 
 
 
 
 
 
Eye Tracking (Pupil Activation) 
- 
X 
X 
X 
- 
X 
X 
P50/P300 (EEG) 
- 
X 
X 
X 
- 
X 
X 
SAI (TMS) 
- 
X 
X 
X 
- 
X 
X 
TMS Adverse Effects Checklist 
- 
X 
X 
X 
- 
X 
X 
Concomitant Safety 
Monitoring 
 
 
 
 
 
 
 
Drug Side Effects  
Weekly 
X 
- 
- 
Seizure Log 
Weekly 
X 
- 
- 
Medication Log (for all 
medications) 
Weekly 
- 
- 
- 
Adverse Event Monitoring  
Ongoing Monitoring 
 
 


Detailed Protocol 14 
On a weekly basis during the 12-week active treatment period, study staff will record medication 
changes that have occurred since the previous week. For inpatient subjects, study staff will conduct 
a structured chart review and speak with the treating physician/nursing staff to identify any 
medication changes. For outpatient subjects, medication monitoring will be conducted through 
weekly phone calls. In addition to recording medication changes that have occurred over the last 
week, changes in baseline symptoms, side effects from Huperzine-A, adverse events and episodes 
of seizure activity that may have occurred in the intervening week will also be recorded. 
Approximately every three months after the 12-week active treatment phase (i.e., week 24, week 
36, week 52), subjects will be monitored for the occurrence of any new serious adverse events as 
well as any unresolved/unstable AEs that may have occurred during the 12-week treatment period. 
Subjects will be instructed to call the Study Coordinator if a serious adverse event occurs between 
scheduled follow-ups. Serious adverse events will be reported immediately, in accordance with 
DSMB and IRB policies.  
 
Drug Administration and Dosing:  
 
Within 7 days of completion of the baseline assessment, subjects will receive the initial dose of 
the study drug (Huperzine A or placebo). The study drug will be prepared and stored by a third-
party vendor and will be shipped to the Spaulding pharmacy. Due to changes in Massachusetts 
State Laws compounding pharmacies are now required to prepare and label the study drug for each 
individual subject. The shelf life of the study drug has been reduced from one year to six months. 
The study drug will be couriered to SRH hopefully, within 24 hours of receiving the prescription. 
Thus, the window between randomization and first dose of study drug has been increased to seven 
days. The randomization list will be maintained by a Spaulding pharmacist with no access to 
subjects or study data. Please see Appendix I for more detailed drug administration guidelines. For 
inpatient subjects, the study drug will be administered by nursing staff. For outpatients, 
instructions will be given to the subject and/or caretaker on how to continue the dosage schedule. 
Weekly phone calls will be made to ensure compliance.  
 
Dose Titration: 
 
The starting dose of the study drug will be 100 micrograms once a day, administered in the 
morning. The starting dose will be continued for 4 days and will then be increased on a fixed 
titration schedule as follows: 100 micrograms twice a day for 4 days; 200 micrograms in the 
morning, 100 micrograms at night for 4 days; 200 micrograms twice a day for 4 days; 300 
micrograms in the morning, 200 micrograms at night for 4 days; and then 300 micrograms twice 
daily for the remainder of the 12 weeks of treatment (64 days). See the table below for a summary 
of the study drug dosages. At the completion of the 12 weeks of dosing, all subjects will terminate 
study drug. 
 
Table 2: 12-week Dosing Schedule - Titration Summary 
 
Timeframe 
Total Dose 
Amount 
4 Days 
100 micrograms 
AM: 100 Micrograms 
4 Days 
200 micrograms 
AM: 100 Micrograms; PM: 100 Micrograms 
4 Days 
300 micrograms 
AM: 200 Micrograms; PM: 100 Micrograms 


Detailed Protocol 15 
 
Drug - Dose Intolerance: 
 
In the case of a drug-dose intolerance, the dose of the study drug will be reduced by one dosage 
level. For example: from [AM: 300 mcg; PM: 300 mcg] to [AM: 300 mcg; PM: 200 mcg]. The 
subject will continue at the reduced dosage level for 7 days and then the dose will be escalated to 
the next dose level. Should the subject redevelop a drug-dose intolerance, the subject will stop the 
drug regimen. 
 
Blinding Procedure: 
 
Participants, family members, treating staff, physicians, RAs/data collectors, SRH pharmacists, 
site investigators and the statistician at the lead site will be blinded to group assignment until after 
the final analyses. The lead pharmacist who prepares the study drug kits will be blinded to group 
assignment. Johnson Compounding and Wellness Center, which is responsible for the 
randomization, will be unblinded. When Johnson Compounding and Wellness Center sends the 
medication to SRH for dispensing to the subject, a note indicating the contents, “Active” or 
“Placebo,” will be with the medication. This note will be placed in a sealed envelope for storage 
should the subject’s medication require unblinding to treat an adverse event.  
 
Guidelines and Procedures for Breaking the Blind: 
 
In rare cases, it may be necessary to break the blind to facilitate management of a serious adverse 
event (SAE). At each site, an assigned person (the PI or designee) will have access to unblinding. 
In the vast majority of cases where adverse events are noted, however, the decision about whether 
or not to continue the study drug, and what treatment to provide, if any, can be made without 
knowing whether the patient was receiving study drug or placebo. Since most SAEs might be due 
to something else even if the patient is in the active treatment group, the treating physician should 
assess multiple possible causes of the adverse event and should stop the study drug at least 
temporarily as long as there is some chance that it is responsible for the event. Just as in clinical 
practice, if an adverse event is thought to be due to a given drug, and that drug is stopped, clearance 
of the adverse event tends to support the causal connection. On the other hand, failure of the 
symptoms to resolve with stopping the drug requires the physician to search for alternative causes. 
Thus, in general, the treating physician should make a decision about whether or not an adverse 
event is likely to be connected to the study drug in a blinded fashion, and stop the drug where 
appropriate, at least temporarily. If the adverse event does not resolve, the physician may identify 
another etiology and make a decision, in the future, to restart the study drug. 
In order to meet the criteria for unblinding the following scenario should be true. In uncommon 
instances, the treating physician may feel that it is critical to know which drug the subject is 
receiving, because:  
1. The adverse event is serious; and 
2. It will be potentially harmful or costly to stop the study drug and act simultaneously on 
4 Days 
400 micrograms 
AM: 200 Micrograms; PM: 200 Micrograms 
4 Days 
500 micrograms 
AM: 300 Micrograms; PM: 200 Micrograms 
Remainder treatment  
(64 days) 
600 micrograms 
AM: 300 Micrograms; PM: 300 Micrograms 


Detailed Protocol 16 
other possible causes of the adverse event; and 
3. It will be dangerous to stop the study drug and wait for a few days to see whether the 
adverse event resolves before acting on other possible causes. 
In all cases, the Treatment Unblinding form, which prompts the PI to address the need to unblind 
to ensure unblinding is indeed necessary, should be completed prior to unblinding the participant. 
Unmasking/unblinding should be considered a serious action. The treating physician may unblind 
the participant if unblinding is considered to be essential to clinical management.  
 
The following questions should be answered on the Treatment Unblinding form by the treating 
physician whenever unblinding is being considered or has been implemented: 
 
1. What is the adverse event that leads you to want to unblind the treatment condition? 
2. What prevents you from addressing all possible causes of the adverse event 
simultaneously? 
3. What prevents you from stopping the study drug blindly and waiting a few days to evaluate 
the course of the adverse event, and then make decisions about other interventions 
accordingly?    
 
In all cases in which unblinding has occurred, the study physician treating the participant has been 
unblinded and will record and maintain these data in a confidential log so the case can be reviewed 
and the reasons for unblinding tracked. The treating physician should be reminded not to reveal 
the treatment assignment to any other staff members unless this information is essential to patient 
management, or to the patient or the patient’s family.  
 
Note: To avoid inadvertent or non-essential episodes of unblinding, the PI (or designee) should 
assure that the covering physician staff, residents, physician’s assistants and nursing staff is 
informed that stringent guidelines must be followed when starting and stopping all medications. 
Unblinding cannot occur unless the Guidelines for Unblinding have been reviewed and completed 
by the PI.  
 
A record of each subjects’ assignment from Johnson Compounding and Wellness Center, “Active” 
or “Placebo,” is placed in a sealed envelope labeled with the Study Name and ID along with the 
subject’s name and Study ID Number on the outside. This envelope is placed in a larger envelope 
labeled with the Study Name and ID in the SRH Night Pharmacy Omnicell automated dispensing 
machine. Only SRH Nursing Managers and the SRH IND Pharmacist, using the Omnicell machine 
can access the study envelope using the product identifier “unblinding key.” 
 
Follow-up Assessments: 
 
Subjects/surrogates will be contacted by the study coordinator two weeks before each scheduled 
follow-up visit to arrange an in-person visit at Spaulding. Follow-up visits will be conducted at 6, 
12, 13, 24, and 52 weeks. There will be a +/- 2-week window to complete these milestone visits 
to accommodate for scheduling issues. Actual dates for each visit will be recorded to ensure that 
the follow up visits are within the appropriate time window. In addition, we will conduct a 
telephone call to the subject at week 13 (one week after drug has stopped) as a safety assessment. 
If it is not possible to complete an in-person assessment, follow-up activities involving symptom 


Detailed Protocol 17 
reporting, adverse events and medication changes will be conducted by telephone. 
 
Inpatient-Outpatient and Acute Care Transition Procedures: 
 
As part of this study focuses on Traumatic Brain Injury in an inpatient population, there may be 
subjects who are transferred from Spaulding Rehabilitation Network (SRN) inpatient to other 
outpatient sites (either inside or outside SRN), to acute care based on clinical treatment needs, or 
home. In these cases, we will follow our subjects with an intent-to-treat procedure as outlined 
below. 
1. In all cases of discharge or transfer (prior to 12 weeks) we will attempt to continue the 
subject’s dosage schedule of the investigational drug. For all subjects, regardless of 
whether dosing continued for the full 12 weeks, we will attempt to follow up with these 
subjects for follow-up assessments. Subjects who do not return for any follow-up 
assessments will be considered lost to follow up.  
a. Upon discharge, instructions will be given to the subject and/or caretaker on how 
to continue the dosage schedule. Study staff will follow up with all monitoring 
procedures (as outlined in the study visit table) via weekly phone calls.  
2. In addition, if the subject transfers to acute care: 
a. We will attempt to continue the subject’s dosage schedule as outlined above. 
b. If this is not possible, there will be a 1 week window during which the treatment 
schedule will be deferred to accommodate for appropriate acute clinical care during 
this time; that is, study drug may be stopped for up to 1 week, and then restarted. 
This restart may only happen once per patient during the trial.  
c. If the subject returns to the study within 1 week, he/she will continue the dosage 
schedule at the last administered dose. If the subject is unable to return within 1 
week, he/she will be analyzed as a non-compliant patient. 
 
 
IX. 
DESCRIPTION OF ASSESSMENT AND OUTCOME MEASURES 
 
Outcome measures: 
 
The outcome measures, assessment schedule, and change indices we selected are designed to 
optimize capture of the primary treatment effect (CVLT-II) while considering normal variation in 
test performance as well as practice effects which may occur with repeated assessments. The 
proposed test battery consists of measures selected from the NIH NCMRR TBI study group for 
the COBRIT study [23].  
 
All outcome measures will be assessed in person, with the following schedule: baseline 
(pretreatment), 6, 12, 24, and 52 weeks post-intervention.  
 
The primary outcome measure, the California Verbal Learning Test-II (CVLT-II), is sensitive to 
attention, memory and aspects of executive functions, all of which may be altered by Huperzine 
A because of their dependence on dopaminergic and cholinergic activity. The CVLT-II will be 
administered at baseline, 6 weeks, 12 weeks, and 52 weeks. With the exception of the 24-week 
timepoint, these assessment points coincide with the neurophysiologic assessment schedule 


Detailed Protocol 18 
(described below) and will allow us to explore the correspondence between temporal changes in 
biologic and cognitive markers associated with the intervention.  
Variability in cognitive performance within and across assessments is common in both healthy and 
neurologically-impaired populations and may exceed two standard deviations in some cases. 
Practice effects are also common and generally attributable to increased familiarity with test 
materials, content, and process. To help mitigate these potential influences on test scores, Reliable 
Change Indices (RCI) [26] will be calculated at the 3 and 12 month reassessments. RCI’s are 
determined by first subtracting the mean T2 - T1 change from the difference between the two 
testings for each individual, and then comparing it to 1.64 times the standard deviation of the 
difference. A difference of this magnitude is exceeded only 10% of the time (in either the positive 
or negative direction) if there is no real change. Applying a correction to change scores is 
particularly important in samples that include patients with severe TBI, as practice effects are 
magnified at higher levels of impairment and with shorter test-retest intervals [27].  
 
All subjects will also undergo a neurophysiologic examination at the time of their baseline 
cognitive testing, and at 6 weeks, 12 weeks, 24 weeks and 52 weeks.  
  
To control for the potential confounding effects of mood, pain and subjective distress on treatment, 
we will administer self-report measures in these areas at the time of enrollment. Scores on the Beck 
Depression Inventory, modified Brief Pain Inventory and British Columbia Post-Concussion 
Scale, obtained at baseline, will be used as covariates in our primary analysis. We will also use 
time post-injury as a covariate to control for the possible influence of length of time since injury 
on treatment outcome as spontaneous recovery will be more rapid in subjects enrolled early versus 
late, and because those enrolled later will be higher functioning at baseline.  
Screening/Follow-up measures: 
• Galveston Orientation Amnesia Test (GOAT): A series of 10 questions asked to a patient 
to help evaluate posttraumatic amnesia. The test is repeated at each visit, and is scored on 
a scale of 0 to 100. A patient is determined to be out of the amnesic state when the score 
exceeds 75 on three consecutive examinations. 
 
• Trail Making Test B: Part B of this test requires the patient to alternate between numbers 
and letters (i.e., 1-A-2-B, etc.) distributed in a spatial array [28, 29]. The task alternation 
aspect of Trail Making B increases its sensitivity to aspects of executive/frontal lobe 
function that are frequently altered in patients after TBI. 
 
• Traumatic Brain Injury Quality of Life (TBI-QOL): The TBI-QOL [28] was developed as 
a comprehensive patient-reported outcomes (PRO) measurement system specifically for 
individuals with traumatic brain injury (TBI). It consists of 20 independent calibrated item 
banks and 2 uncalibrated scales that measure physical, emotional, cognitive, and social 
aspects of health-related quality of life. We will administer only the short form (6 
questions) of the TBI-QOL Learning and Memory subscale. 
 
• Ruff Neurobehavioral Inventory (RNBI): The RNBI [29] is a self-report instrument for 
assessment of a wide range of symptoms (cognitive, emotional, and physical), as well as 


Detailed Protocol 19 
quality of life and daily functioning. It was designed to assess these areas in individuals 
who have recently been affected by an injury, illness, or other stressor. We will be 
administering the 24 question Postmorbid Cognitive Domain form of the RNBI. 
Primary measure: 
• CVLT-II: The California Verbal Learning Test provides a reliable measure of memory 
encoding and retrieval, which is often affected in patients with TBI. The California Verbal 
Learning Test second edition [30, 31] measures verbal learning and memory using a 
multiple-trial list-learning task. Sixteen words from 4 semantic categories are repeated for 
each of the five learning trials for list A. A distractor list (B) of 16 words from 2 of the 
same and 2 different semantic categories is then given. Short and long free and cued recall 
measures of list A are presented. There is also a recognition paradigm. Z-scores are 
computed for total learning trials 1-5 and all recall and recognition trials.  
 
Covariates: 
 
• Beck Depression Inventory (BDI): The Beck Depression Inventory (BDI) is a 21-item test 
presented in multiple-choice format that measures the presence of and the degree of 
depression in adults [32].The BDI will be used to evaluate if mood is a confounder in this 
study.  
• The modified Brief Pain Inventory: The BPI is a short self-assessment questionnaire that 
provides information on various dimensions of pain including how pain developed, the 
types of pain a patient experiences, and time of day pain is experienced, as well as current 
ways of alleviating pain[33]. 
• The British Columbia Post-concussion Scale: The scale evaluates several clinical 
symptoms (13 categories) of concussion (i.e. headache fatigue, dizziness, sleep 
disturbance, etc) and asks individuals to endorse the level of severity [34]. Symptom 
frequency ratings over the prior two weeks are also evaluated. The scale has been normed 
to investigate the expected rate of symptomatology in the non TBI population, making it 
an attractive metric for TBI.  
• Time since injury: Time since injury will be used as a covariate to control for the possible 
influence of injury chronicity on treatment outcome. 
 
Secondary measures (neurophysiological assessments): 
In addition to the neurocognitive assessment, we will also conduct several neurophysiologic 
assessments in line with our secondary aim of investigating the neurophysiologic effects of 
Huperzine A. These assessments will provide insights into the mechanisms of Huperzine A in 
modulating mood and cognition.  
 
• P50 and P300 (via EEG) – event related potentials:  
We will measure P50 and P300 using auditory stimuli. These neurophysiological 
measurements index cortical electrical activity associated with a given stimulus and 
therefore our hypothesis is that if there is an improvement in activation of cognitive-related 


Detailed Protocol 20 
neural networks, then the signal as indexed by P50 and P300 will increase. Specifically, 
P50 represents an index of activity in the cholinergic system and has been used to 
characterize presynaptic cholinergic deficit [35] and P300 is a measure of general cognitive 
processing elicited during attention, memory, and executive tasks. A decrease in P300 
latency indicates faster processing and can serve as an index of cognitive improvement. 
 
• Short Latency Afferent Inhibition (SAI) (via TMS):  
TMS can index cortical activity as it is possible to elicit a cortical motor evoked potential. 
Before undergoing the procedure, we will administer a TMS screening checklist to the 
subject so as to assess whether the subject is eligible to receive TMS. Contraindications to 
TMS include (but are not limited to): metal in the head and/or implanted brain medical 
devices. If the patient has recently undergone a craniectomy TMS will be administered to 
the contralateral hemisphere. If it is determined that subject is not eligible for this 
procedure, he/she will continue the study without collecting the TMS data, as this measure 
is a secondary outcome. 
Responses to stimuli applied to the motor cortex will be recorded from surface 
electromyography (EMG). Silver/silver chloride electrodes will be placed over the muscle 
belly (active electrode) and joint or tendon of the muscle (reference electrode) to record 
MEPs. Conditioning stimuli will be done via single pulses of electrical stimulation applied 
to the median nerve at the wrist. The intensity of the conditioning stimulus will be adjusted 
over motor threshold for evoking a visible movement of the thenar muscles. The 
conditioning stimulus to the peripheral nerve will be applied before the magnetic test 
stimulus. Furthermore, according to the parameters used it is possible to assess the activity 
of certain neurotransmitters. In this application, we will measure SAI that can also be 
considered a measure of cholinergic activity. Our hypothesis is that cholinergic activity 
will increase in subjects randomized to Huperzine A compared to those randomized to 
placebo, and therefore a decrease in SAI will be seen in the Huperzine A arm. We will 
monitor for adverse effects using an adverse effect questionnaire designed for TMS 
stimulation. 
 
• Task-evoked pupillary response (TEPR): 
Changes in pupil diameter due a cognitive task are considered a reliable and sensitive index 
of the degree of cognitive load and resource demand to the task. We will record pupillary 
response, indexed as changes in pupil diameter, to two sets of tasks measuring cognitive 
performance: a low cognitive-load task vs. a high-cognitive load task. The first will consist 
of a set of simple stimuli presented to the patient in an organized and timely manner, which 
will then be recognized and named by the subject. The high-cognitive load task includes 
the presentation of a larger and more complex array of information for the subject to recall. 
Both sets of stimuli will be shown in a computer screen with a fixed interval between 
stimuli. Measurement of pupil diameter and reactivity will be conducted with a remote eye-
tracker device. It consists of a high-resolution 17” computer screen placed in front of the 
subject’s face that allows both stimuli display and pupil tracking through infrared diodes 
that generate reflection patterns of the user’s cornea. 
 
• Smooth pursuit eye movement (SPEM):  


Detailed Protocol 21 
Eye movements will be recorded by an infrared-based eye-tracking device consisting of a 
high-resolution computer screen that will be placed in front of the subject’s face at a 
40cm distance. Subjects will be presented with a target stimulus that will move in a 
predetermined and consistent trajectory in the fixed background screen. We will measure 
different parameters based on this recording both at baseline and treatment completion, 
including eye and target velocity, index of target prediction, eye position error and intra-
individual variability of eye error. Comparison of baseline to after-treatment 
measurements will be considered a reflection of cognitive changes. 
 
 
X. 
STATISTICAL ANALYSIS 
 
This is an exploratory phase II study with the intent of generating hypotheses for a confirmatory 
phase II/III trial, as well as in obtaining effect sizes and variances for sample size calculation for 
further confirmatory studies. The study is also designed to address the tolerability of Huperzine A 
in the moderate and severe TBI populations. 
In designing this exploratory study, we conducted a sensitivity analysis to obtain a sample size that 
would provide a reasonable effect size when comparing Huperzine A to placebo (Figure 2). This 
allows for detection of differences between the two treatments, to plan for further studies. 
Assuming a power of 80% and an alpha of 5%, we originally projected that a sample of 60 patients 
(30 patients in each group) would allow us to detect an effect size of 0.75 between the two groups 
(placebo and active). Given our prior studies in TBI and cognition, an effect size of 0.75 for the 
primary outcome measure (CVLT-II) is clinically meaningful, to compare two treatments. 
 
The authorized addition of two self-report measures to our outcome assessment battery (i.e., 
Cognitive Function subscale from the Neurological Quality of Life Questionnaire and the Ruff 
Neurobehavioral Inventory) was expected to influence our original power estimate and required 
sample size. We calculated our effect size by estimating the upper and lower limits of the change 
score that would be demonstrated by the control group on our primary outcome measure (i.e., 
California Verbal Learning Test-II). This analysis showed that, to achieve an effect size of 0.75, 
the final sample size could vary between 15 and 39 subjects per group, given a mean difference 
 
Figure 2: Effect size to total sample size ratio necessary for a powerful study 


Detailed Protocol 22 
in performance between groups of 3.59 with a standard deviation of 3.30 to 5.13. In view of the 
variability in this estimate, we proposed that we would run 10 subjects, determine the degree of 
variability in performance and then adjust the sample size accordingly.  
 
After enrolling 10 subjects, we assessed the data for an estimate of the average difference 
between groups (difference to baseline) on a ratio of 3:6. There was an estimated effect size of 
1.19 between group A (M=4.67, SD=4.51) and group B (M=0.33, SD=2.50). Using a two sample 
t-test to detect the difference between groups, a minimum sample of 26 subjects will be required 
(13 per group).  
 
The anticipated loss to follow-up rate in this study is around 15% - like our other drug studies. To 
account for this loss-to-follow-up rate, a total of 15 patients per group (30 total) will be enrolled 
in the study. The primary analysis will be an intent-to-treat analysis that will include all 
randomized participants regardless of their compliance with the study treatment or follow-up 
schedule. Study subjects should have received at least 1 dose of study drug/placebo. All hypothesis 
testing will be conducted using α = 0.05, two-sided. 
 
To analyze the data, we will compare means using t-test and modeling the data with ANOVA for 
multivariate analysis controlling for baseline values and other important covariates. If necessary, 
non-parametric approaches including Mann-Whitney and Friedman’s test for multivariate analysis 
will be used with data that are not normally distributed. We will also use Fisher’s exact test for 
comparison of categorical variables such as proportion of adverse effects. In summary, we will use 
the following statistical tests according to our primary aims: 
• Primary Aim: To determine whether Huperzine A, as compared with placebo, has a differential 
effect on learning and memory functions after moderate to severe TBI. 
For this aim we will compare means using t-test (comparing differences (Week 12 – baseline) between 
the two groups) and modeling the data with ANOVA for multivariate analysis controlling for baseline 
values and other important covariates (for secondary analysis). If necessary, non-parametric approaches 
including Mann-Whitney and Friedman’s test for multivariate analysis will be used with data that are not 
normally distributed. 
• Secondary aim 1: To determine whether administration of Huperzine A produces significant 
differences in neurophysiologic markers (as indexed by EEG event related potentials (P50 and 
P300) and TMS-indexed cortical excitability (cholinergic activity)) associated with cognition, 
relative to a placebo. 
For this aim we will also build models in which the main dependent outcome will be amplitude or latency 
measures (from P50, P300 and SAI) and the independent variables will be group, time and the interaction 
group*time. 
• Secondary aim 2: To determine whether Huperzine A reduces the prevalence of post-traumatic 
seizures after moderate and severe TBI as compared to placebo 
For this aim, we will compare proportion of seizures within the first 6 weeks between Huperzine vs. 
placebo group using a Fisher’s exact test (as we expect small numbers of expected frequencies in the 
cells). 


Detailed Protocol 23 
• Secondary aim 3: To evaluate the safety and tolerability of Huperzine A in this patient population 
as compared to placebo. Safety and tolerability will be assessed by frequency of adverse effects, 
including laboratory abnormalities. 
 
 
XI. 
RISKS AND DISCOMFORTS 
Transcranial Magnetic Stimulation: 
 
Note that we will be using single and paired-pulse TMS as a diagnostic tool. This type of TMS 
stimulation does not change cortical excitability or brain activity. There is a small chance of single 
pulse TMS inducing a mild, transient headache, or neck pain. TMS equipment produces a clicking 
sound when a current is passed through the stimulation coil. This click can result in ringing in the ear 
and temporary auditory threshold shifts if no protection is used. In order to prevent this potential 
adverse effect subjects will wear earplugs during TMS. 
 
Electroencephalography (EEG): 
 
The EEG test is performed to measure the electrical activity in the brain and to examine the 
dynamic changes. It also allows for better understanding of the effects of electrical activity 
generated in different areas of the brain. EEG only measures brain activity and does not induce 
electrical current. It is non-invasive and has been used extensively in clinical practice for diagnosis 
or neurological conditions such as epilepsy. There are no risks associated with EEG other than a 
mild discomfort caused by the tightness of the EEG net. The investigator will adjust the net to 
allow for the comfort of the subject. 
 
Eyetracking and pupil dilation measurement: 
 
The eyetracking tasks that will be performed help us to measure cognitive load capabilities of the 
subjects. Subjects will complete both task-evoked pupillary response and smooth-pursuit eye 
movements by watching a high-resolution computer screen while resting their head on a headrest. 
There are therefore no added risks associated with this device. During the procedure, participants 
may experience minor discomfort resulting from sitting still and minimizing movements during 
the experiment. Rarely, some eye dryness or watering of the eyes may result from the test. If the 
subject’s eyes become uncomfortable, the person administering the test will stop the procedure so 
the subject may rest their eyes.  
 
Huperzine-A: 
 
Potential side effects of  Huperzine A include: nausea, vomiting, diarrhea, sweating, blurred vision, 
slurred speech, loss of appetite, contraction and twitching of muscle fibers, cramping, increased 
saliva and urine, inability to control urination (incontinence), hypertension, and slowed heart rate 
(bradycardia) [36]. As with any investigational agent, there may be side effects that are unknown 
that may occur.  
 
Neuropsychological assessments/questionnaires: 
 


Detailed Protocol 24 
There are no risks involved with the neuropsychological testing batteries used in this study. If the 
subject feels uncomfortable answering any of the questions or performing any of the 
neuropsychological assessments, he/she may stop at any time. If the subject becomes bored or 
fatigued while completing the battery, he/she may be able to take a break and rest before 
completion. 
 
Expected symptoms of Traumatic Brain Injury: 
 
Based on the severity of the subject’s TBI, there are many different symptoms expected that are 
related to the injury. Thus, these symptoms can change as the patient’s injury status progresses 
from the acute to chronic phase. These expected symptoms may include medical (e.g., 
hemodynamic changes, fever, dysautonomia), cognitive (e.g., confusion, distractibility, memory 
impairment) and behavioral (e.g., agitation) problems. Please see Appendix A for the list of 
adverse events frequently seen in the TBI population. 
 
In order to determine whether side effects seen during the study are related to TBI or Huperzine 
A, we will review baseline symptoms of TBI for the patient prior to starting Huperzine A, monitor 
for exacerbation of existing symptoms and assess for the onset of new symptoms through 
systematic medical record review, medication/seizure/side effects checklists and also through 
contact with the subject and treating physician/nursing team.  
 
Data Safety Monitoring Board and stopping rules: 
 
Adverse events will be collected from the start of Huperzine A treatment through the end of study 
participation. During the 12 weeks of study drug, all adverse events will be collected. However, 
after study drug dosing is complete, we will only collect information on serious and/or 
unresolved/unstable adverse events that are clinically meaningful. All adverse events regardless of 
attribution to Huperzine A will be collected and recorded, using standard adverse event forms. All 
applicable local regulatory requirements related to the reporting of serious adverse events to 
regulatory authorities and the IRB will be followed during this study. Serious adverse events will 
be promptly reported to the IRB. The issue of placing the study on hold will be raised by the 
investigators with our local IRB if any serious adverse events occur. 
 
Importantly, a data safety monitoring board (DSMB) will be established for the study, and will be 
comprised of a physiatrist (rehabilitation physician) with expertise in traumatic brain injury 
rehabilitation, a neuropsychologist with expertise in cognitive rehabilitation, and a biostatistician 
with expertise in clinical trials. This DSMB will meet after 50% of the subjects complete the study, 
and will meet again after 20 subjects have completed the study. The DSMB will be un-blinded in 
their assessment of AEs and we will adopt the following stopping rules: 
 
• We will stop the study early if any patient dies, if any patient requires emergency surgery, 
or if any patient suffers a permanent and irreversible disability, only if the DSMB 
determines that this event was definitely or probably related to study drug.  
• We will also stop the study if there is disproportionate incidence of serious adverse events 
in the active treatment arm vs. the placebo arm. Serious adverse effects will be defined as 
any adverse event, including a significant change in lab values, that is: (i) fatal, (ii) life-


Detailed Protocol 25 
threatening, (iii) requires or prolongs inpatient hospitalization, (iv) result in persistent or 
significant disability or incapacity, v) a congenital anomaly/birth defect, or vi) an important 
medical event. Important medical events are those that may not be immediately life 
threatening, but are clearly of major clinical significance.  They may jeopardize the subject, 
and may require intervention to prevent one of the other serious outcomes noted above. For 
example, drug overdose or abuse, a seizure that did not result in in-patient hospitalization, 
or intensive treatment of bronchospasm in an emergency department would typically be 
considered serious.  
 
Because this clinical trial is largely exploratory, we will not employ a stopping rule for efficacy or 
for futility. 
 
The DSMB will review all unanticipated problems involving risk to subjects or others, serious 
adverse events and all subject deaths associated with the protocol through oversight of study 
interventions and interactions and will provide an unbiased written report of the event. At a 
minimum, the DSMB must comment on the outcomes of the event or problem and in case of a 
serious adverse event or death, comment on the relationship to participation in the study. The 
DSMB must also indicate whether it concurs with the details of the report provided by the Principal 
Investigator. Members of the DSMB may discuss the research protocol with the investigators, 
interview human subjects, and consult with others outside of the study about the research with 
respect to safety and well-being of participants. The DSMB will protect the safety and well-being 
of human subjects through its review and bring findings promptly to the Principal Investigator and 
then to the IRB/HRPO. The DSMB shall have authority to stop the protocol or remove subjects 
from the study in order to protect the well-being of the subjects enrolled, should this be necessary. 
 
XII. 
MONITORING AND QUALITY ASSURANCE 
 
Safety monitoring and subject compliance: 
 
• Medication Log: Prior to randomization, study staff will review the results of screening 
tests to identify potential drop-outs; subjects identified as being at high risk for 
noncompliance with study drug will not be randomized. For Spaulding inpatients, nursing 
staff will maintain a treatment record which will be checked weekly by study staff, and 
transcribed onto a medication log which will include the time, date, and each dosage of 
Huperzine A taken. Finally, any deviation from adherence to the medicine regime will be 
documented. Study data will be monitored for irregularities. Any irregularities in the data 
will be investigated for possible deviations in adherence. We will also track the patient’s 
other medications (not involving Huperzine) on this Log – including dosing amounts and 
times. For community-dwelling outpatients, subjects and/or caretakers will be given a 
medication diary to fill out daily. 
 
• Laboratory Data: Serum measures of renal and liver function will be obtained at baseline, 
6 weeks, 12 and 13 weeks to evaluate for eligibility to participate in the study as well as 
any potential unexpected side effects. Hematologic data will also be obtained at baseline, 
6 weeks, 12 and 13 weeks to observe for any side effects. A general chemistry panel will 
be completed at baseline. Clinical blood results obtained within 48 hours of the study time 


Detailed Protocol 26 
points will be able to be used for evaluation thereby, reducing the number of venipunctures 
for the subject. 
 
• Biomarkers: Blood samples for UCH-L1, GFAP, NF-l and Tau will be obtained at the time 
of enrollment, at 24 hours, 23 days, 30 days post injury and 90 days post injury. De-
identified samples will be shipped to Quanterix Corporation for analysis after processing 
by the SRH clinical lab.  After analysis Quanterix Corporation will return the samples to 
the Spaulding Rehabilitation Network Research Institute.   
 
 
• Study Drug Blood Levels: Blood samples will be drawn from 10% of subjects to assess 
efficacy vs. steady- state HupA concentrations and tolerability during the maintenance 
phase. Samples will be drawn pre-dose and 1 hour post dose on Day 21 or Day <21 (at the 
end of dose escalation for each patient in the 10%, whatever dose they escalate to). During 
the maintenance phase a weekly pre-dose sample will be drawn. Week 12, on the last day 
of dosing, a pre-dose and 1 hour post-dose sample will be drawn. These samples will be 
processed in the SRH clinical labs, stored at -80, and then forwarded to the Illinois Institute 
of Technology Research Institute (IITRI) in Chicago. De-identified samples will be 
shipped to IITRI and the link to the code will remain at SRH and not be provided to IITRI. 
 
• Baseline Symptom Checklist: This will be completed before study drug is started. After 
treatment is initiated, the study staff should use the baseline symptom checklist list as a 
reference to identify and code new symptoms that arise after treatment is initiated, or to 
detect an exacerbation of a previously-noted symptom. For Spaulding inpatients, these data 
will be collected from systematic medical record review and also from speaking with the 
treating medical/nursing staff on a weekly basis. For outpatient, changes in baseline 
symptoms will be monitored through weekly phone calls.  
 
• Drug Side Effects Checklist: For Spaulding inpatients, the study staff will evaluate 
potential side effects of Huperzine A using the drug side effect checklist during weekly 
systematic review of the medical record. This scale covers all known possible side effects 
of Huperzine A. In addition to speaking with medical/nursing staff, the subjects/caregivers 
may be asked to report whether they have experienced any side effects in an open-ended 
manner, and any side effects experienced by patients will be marked on a scale of 1 through 
3 to measure severity (1- Mild, 2- Moderate, 3- Severe). If any side effects are reported, 
the degree of relatedness to the Huperzine A intervention will be assessed. For outpatient, 
side effects from Huperzine-A will be monitored through weekly phone calls.  
 
• Seizure Log: Although it is not possible within the scope of this study to obtain definitive 
information regarding Huperzine A’s anticonvulsant properties, a detailed seizure log will 
be utilized to evaluate for seizure events or signs associated with seizures in Spaulding 
inpatients. This log will be maintained weekly by the study staff collecting data through 
the systematic review of the medical record and speaking to the treating medical/nursing 
staff. Study staff will review documented chart evidence of seizure including any EEG that 
is performed as part of the routine medical care. For outpatient subjects, episodes of seizure 
activity will be monitored through weekly phone calls.  


Detailed Protocol 27 
 
• Suicidality Scale – We will monitor for suicidality using the Columbia-Suicide Severity 
Rating Scale (C-SSRS) at baseline, 6 weeks and 13 weeks – as requested by the FDA 
Division of Neurology Products per our IND. 
 
• Adverse event monitoring: In addition to the above, we will continually monitor for 
changes in patient health status. The baseline symptom checklist, side effect checklist and 
seizure log will be used to help monitor for changes in symptoms or side effects in 
Spaulding inpatients, and community-dwelling outpatients. For Spaulding inpatients, we 
will also monitor the patient’s chemistry by the scheduled laboratory exams, and monitor 
compliance through the medication log. By using this multimodal approach, we will be 
able to appropriately rate events as they occur, and to accurately associate such events to 
the appropriate changes in subject status (ex. whether from TBI or Huperzine, expected 
or unexpected, etc). In addition, approximately every three months after the 12-week 
active treatment phase (i.e., week 24, week 36, week 52), subjects will be monitored for 
the occurrence of any new serious adverse events as well as any unresolved/unstable AEs 
that may have occurred during the 12-week treatment period. Subjects will be instructed 
to call the Study Coordinator if a serious adverse event occurs between scheduled follow-
ups. Serious adverse events will be reported immediately, in accordance with DSMB and 
IRB policies.  
 
Treatment Compliance: 
 
Because effects will be dependent on patients taking Huperzine, treatment compliance will be 
essential in our study. We will measure compliance by asking the subject and/or caretaker to return 
the medication bottles at the end of the titration and maintenance periods. Any remaining pills will 
be counted, returned to the pharmacy and documented in the subject’s folder, with specific forms. 
If the subject is still inpatient, we will monitor compliance through the medication administration 
system. Finally, we do not expect significant adverse effects associated with the use of Huperzine 
that would lead patients receiving this drug to become non-adherent. 
 
Dropout in the placebo group:  
 
Participants in the placebo group may disproportionately discontinue the study treatment since 
there will be little or no improvements in cognitive function. Although, this fact could bias the 
results, we expect that the placebo group will also have a small improvement (due to natural 
recovery, though smaller than in the active group) and we will conduct an analysis of the data 
using an intent-to treat population, to protect against disproportionate dropout. 
 
 
XIII. POTENTIAL BENEFITS 
 
It is possible that subjects participating in this research may benefit from the Huperzine-A given 
in the trial. Additionally, subjects may benefit from the use of TMS as the data could suggest 
plasticity changes and encourage clinicians to continue therapy or other interventions. Lastly 


Detailed Protocol 28 
subjects may benefit from the close follow-up and enhanced observation of the clinical research 
team. 
 
 
XIV. STIPEND 
 
Each subject will receive a total of $450 paid throughout their study participation. The stipend 
payment distribution is as follows: Screening $25, Baseline $25, Week 6 $50, Week 12 $100, 
Week 13 $50, Week 24 $100 and Week 52 $100. 
 
In addition, travel, parking and/or hotel costs will be reimbursed up to $200/ subject. Proof of 
payment of costs will be required for reimbursement. 
 
Plan for Future Studies: 
 
A major goal of this study is to collect data that may be useful to plan future phase II/III trials. In 
fact, this is one of the major goals of phase II studies that usually test several outcomes as there 
are no strong data to support a large trial. Therefore, with this study we expect to: 
(1) Determine parameters for sample size calculation such as effect sizes for different 
endpoints. 
(2) Based on the extensive assessments we are conducting, we will be able to determine in a 
preliminary manner the predictors associated with response. Therefore, further trials may 
optimize response. 
(3) Determine duration of treatment as we may conclude that treatment duration is excessively 
long or short. 
(4) Learn the neural mechanisms associated with Huperzine induced cognitive improvement; 
and thus, be able to optimize response. 


Detailed Protocol 29 
XV. 
REFERENCES 
 
1. 
Kraus J, N.P., The Epidemiology of Mild Head Injury. Mild Head Injury, ed. E.H. Levin 
H, Benton A. 1989: Oxford University Press. 
2. 
Faul M, X.L., Wald MM, Coronado VG. , Traumatic brain injury in the United States: 
emergency department visits, hospitalizations, and deaths., G.C.f.D.C.a.P. Atlanta, 
National Center for Injury Prevention and Control, Editor. 2010. 
3. 
Warden, Military TBI during the Iraq and Afghanistan wars. J Head Trauma Rehabil, 
2006. 21(5): p. 4. 
4. 
Zangara, A., The psychopharmacology of huperzine A: an alkaloid with cognitive 
enhancing and neuroprotective properties of interest in the treatment of Alzheimer's 
disease. Pharmacol Biochem Behav., 2003. 75(3): p. 11. 
5. 
Zhang, Y., et al., Spermidine antagonizes the inhibitory effect of huperzine A on 
[3H]dizocilpine (MK-801) binding in synaptic membrane of rat cerebral cortex. Neurosci 
Lett., 2002. 319(2): p. 3. 
6. 
Gordon, R., et al., The NMDA receptor ion channel: a site for binding of Huperzine A. J 
Appl Toxicol, 2001. 21: p. 4. 
7. 
Wang, R., H. Yan, and X. Tang, Progress in studies of huperzine A, a natural 
cholinesterase inhibitor from Chinese herbal medicine. Acta Pharmacol Sin, 2006. 27(1): 
p. 26. 
8. 
Peng, Y., et al., Effects of huperzine A on amyloid precursor protein processing and beta-
amyloid generation in human embryonic kidney 293 APP Swedish mutant cells. J 
Neurosci Res, 2006. 84(4): p. 8. 
9. 
Chu, D., et al., Pharmacokinetics of huperzine A in dogs following single intravenous and 
oral administrations. Planta Med, 2006. 72(6): p. 3. 
10. 
Garcia, G., et al., Identification and characterization of the major huperzine a metabolite 
in rat blood. J Anal Toxicol, 2004. 28(5): p. 4. 
11. 
Zhu, X. and E. Giacobini, Second generation cholinesterase inhibitors: effect of (L)-
huperzine-A on cortical biogenic amines. J Neurosci Res, 1995. 41(6): p. 7. 
12. 
Tenovuo, O., Central acetylcholinesterase inhibitors in the treatment of chronic 
traumatic brain injury-clinical experience in 111 patients. Prog Neuropsychopharmacol 
Biol Psychiatry, 2005. 29(1): p. 6. 
13. 
Zhang JH, F.J., Deng AW, A clinical study of huperzineA on mild and moderate 
traumatic brain injury in memory and cognitive impairment. Chin J Rehabil Med, 2002. 
17: p. 2. 
14. 
Wang R, Y.H., Tang XC, Progress in studies of huperzine A, a natural cholinesterase 
inhibitor from Chinese herbal medicine. Acta Pharmacol Sin 2006 27(1): p. 26. 
15. 
Xu, S.S., et al., Efficacy of tablet huperzine-A on memory, cognition, and behavior in 
Alzheimer's disease. Zhongguo Yao Li Xue Bao, 1995. 16(5): p. 391-5. 
16. 
Qian, B.C., et al., Pharmacokinetics of tablet huperzine A in six volunteers. Zhongguo 
Yao Li Xue Bao, 1995. 16(5): p. 396-8. 
17. 
Park, P., S. Schachter, and T. Yaksh, Intrathecal huperzine A increases thermal escape 
latency and decreases flinching behavior in the formalin test in rats. Neurosci Lett, 2010. 
470(1): p. 6-9. 
18. 
White, H.S., et al., First demonstration of a functional role for central nervous system 
betaine/{gamma}-aminobutyric acid transporter (mGAT2) based on synergistic 


Detailed Protocol 30 
anticonvulsant action among inhibitors of mGAT1 and mGAT2. J Pharmacol Exp Ther, 
2005. 312(2): p. 866-74. 
19. 
Schachter, S.C., Therapeutic effects of vagus nerve stimulation in epilepsy and 
implications for sudden unexpected death in epilepsy. Clin Auton Res, 2006. 16(1): p. 29-
32. 
20. 
Rafii, M.S., et al., A phase II trial of huperzine A in mild to moderate Alzheimer disease. 
Neurology, 2011. 76(16): p. 1389-94. 
21. 
Ye, J.W., et al., Improving effects of huperzine A on spatial working memory in aged 
monkeys and young adult monkeys with experimental cognitive impairment. J Pharmacol 
Exp Ther, 1999. 288(2): p. 814-9. 
22. 
Laganiere, S., et al., Acute and chronic studies with the anticholinesterase Huperzine A: 
effect on central nervous system cholinergic parameters. Neuropharmacology, 1991. 
30(7): p. 763-8. 
23. 
Villamar, M.F., et al., Noninvasive brain stimulation to modulate neuroplasticity in 
traumatic brain injury. Neuromodulation, 2012. 15(4): p. 326-38. 
24. 
Bashir, S., et al., Changes in cortical plasticity after mild traumatic brain injury. Restor 
Neurol Neurosci, 2012. 30(4): p. 277-82. 
25. 
Zhang, J., J. Fan, and A. Deng, A clinical study of huperzine A on mild and moderate 
traumatic brain injury in memory and cognitive impairment. Chinese Journal of 
Rehabilitation Medicine, 2002. 17: p. 162-164. 
26. 
Sawrie, S.M., et al., Empirical methods for assessing meaningful neuropsychological 
change following epilepsy surgery. J Int Neuropsychol Soc, 1996. 2(6): p. 556-64. 
27. 
Dikmen, S., et al., Functional status examination: a new instrument for assessing 
outcome in traumatic brain injury. J Neurotrauma, 2001. 18(2): p. 127-40. 
28. 
Tulsky, D.S., et al., TBI-QOL: Development and Calibration of Item Banks to Measure 
Patient Reported Outcomes Following Traumatic Brain Injury. J Head Trauma Rehabil, 
2015. 
29. 
Ruff, R.M., K.M. Hibbard, and Psychological Assessment Resources Inc., RNBI, Ruff 
neurobehavioral inventory. Professional manual. 2003, Lutz, FL: Psychological 
Assessment Resources. 117 p. 
30. 
Delis, D.C., et al., Recall discriminability: utility of a new CVLT-II measure in the 
differential diagnosis of dementia. J Int Neuropsychol Soc, 2005. 11(6): p. 708-15. 
31. 
Vanderploeg, R.D., G. Curtiss, and H.G. Belanger, Long-term neuropsychological 
outcomes following mild traumatic brain injury. J Int Neuropsychol Soc, 2005. 11(3): p. 
228-36. 
32. 
Beck, A.T., et al., Comparison of Beck Depression Inventories -IA and -II in psychiatric 
outpatients. J Pers Assess, 1996. 67(3): p. 588-97. 
33. 
Bryant, R.A., et al., Interaction of posttraumatic stress disorder and chronic pain 
following traumatic brain injury. J Head Trauma Rehabil, 1999. 14(6): p. 588-94. 
34. 
Iverson, G.L., M.R. Lovell, and M.W. Collins, Interpreting change on ImPACT following 
sport concussion. Clin Neuropsychol, 2003. 17(4): p. 460-7. 
35. 
Jessen, F., et al., Sensory gating deficit expressed by a disturbed suppression of the P50 
event-related potential in patients with Alzheimer's disease. Am J Psychiatry, 2001. 
158(8): p. 1319-21. 
36. 
Pepping, J., Huperzine A. Am J Health Syst Pharm, 2000. 57(6): p. 530, 533-4.