Conditions: Cervical Intraepithelial Neoplasia Grade 2/3 High-risk HPV (Any Strain)
Keywords: cervical dysplasia HPV Treatment preinvasive cervix vaginal suppository Cornelia Trimble abnormal pap CIN
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 30
Healthy volunteers: no

Brief summary:
This phase I research protocol is designed to assess immunogenicity and clinical endpoints in patients with human papillomavirus (HPV) 16 cervical intraepithelial neoplasia (CIN)2/3. The protocol tests the use of artesunate in suppository formulation applied intravaginally in patients with cervical dysplasia (CIN2/3).

The primary endpoint is to determine and evaluate the safety, tolerability, and feasibility of intravaginal administration of artesunate in health women with CIN2/3.

Detailed description:
Phase I open-label dose escalation study of intravaginal artesunate, formulated in suppositories, in adult females with biopsy-confirmed CIN2/3. Thirty (30) subjects will undergo up to a total of three cycles of intravaginal artesunate. The first cycle will be initiated on Day 0, the second at Week 4, and the third and final cycle at week 8.

Arms:
arm 1: Subjects enrolled in this cohort will receive 1 five day cycle of Artesunate suppositories prior to therapeutic resection of their lesion (if clinically indicated). arm 2: Subjects enrolled in this cohort will receive 1 five day cycle of Artesunate suppositories prior to therapeutic resection of their lesion (if clinically indicated). arm 3: Subjects enrolled in this cohort will receive 2 five day cycles of Artesunate suppositories prior to therapeutic resection of their lesion (if clinically indicated). arm 4: Subjects enrolled in this cohort will receive 3 five day cycles of Artesunate suppositories prior to therapeutic resection of their lesion (if clinically indicated).

Interventions: intervention 1: Artesunate Suppositories
Intervention descriptions: intervention 1: None

Protocol document:
Prot_SAP_000.pdf: 
A Phase I Study of intravaginally administered Artesunate in women with high grade cervical intraepithelial neoplasia (CIN2/3) 
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A Phase I Study of Intravaginally Administered Artesunate In Women with 
High Grade Cervical Intraepithelial Neoplasia (CIN2/3) 
 
 
Johns Hopkins Study Protocol Number: J1498 
NCT02354534 
 
IND Sponsor: Johns Hopkins University 
 
Principal Investigator 
Cornelia Trimble, M.D. 
Departments of Gynecology and Obstetrics, Oncology and Pathology 
Johns Hopkins University School of Medicine 
 
Lead Site Co-Investigator (Johns Hopkins Clinical Research Network) 
Kimberly Levinson, MD, MPH (GBMC) 
 
Study Coordinator – Johns Hopkins, Coordinating Center 
Mihaela Paradis 
Department of Gynecology and Obstetrics 
Johns Hopkins University School of Medicine 
 
Research Nursing 
Elizabeth Sauter - Department of Gynecology and Obstetrics, JHU School of Medicine 
Garnitha Ferguson, RN – Greater Baltimore Medical Center 
 
 
Statistician 
Chenguang Wang  
 
Research Pharmacies 
Johns Hopkins Investigational Drug Pharmacy  
GBMC Infusion Pharmacy  
 
 
 
Draft or Version Number: 5.0 
August 7, 2017 
 


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STATEMENT OF COMPLIANCE 
The study will be carried out in accordance with Good Clinical Practice (GCP) as 
required by the following regulations and guidelines: 
• United States Code of Federal Regulations (CFR) applicable to clinical studies 
(45 CFR Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
• ICH E6; 62 Federal Register 25691 (1997)  
 
All key personnel responsible for the design and conduct of this study have completed 
Human Subjects Protection Training. 


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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this trial will be conducted according to all 
stipulations of the protocol, including all statements regarding confidentiality, and 
according to local legal and regulatory requirements and applicable US federal 
regulations and ICH guidelines. 
 
Trial Site: Johns Hopkins Hospital 
 
 
 
Signed:  
Date: 
 
 
Cornelia Trimble, M.D. 
Professor of Gynecology and Obstetrics 
Professor of Oncology 
Professor of Pathology 
 
 
 
 
Trial Site: Greater Baltimore Medical Center 
 
 
 
Signed:  
Date: 
 
 
Kimberly Levinson, MD, MPH 
Assistant Professor of Gynecology and Obstetrics 
 
 
 


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TABLE OF CONTENTS 
 
Page 
1.0 Synopsis ...................................................................................................................... 7 
2.0 Background and rationale .......................................................................................... 11 
2.1 
HPV-associated malignancies of the cervix ................................................ 11 
2.2 
Noninvasive topical therapy for cervical dysplasia .....................................12 
2.3 
Rationale for study drug ...............................................................................12 
2.4 
Nonclinical safety literature review .............................................................. 13 
2.4.1 Acute (Single Dose) Toxicity ........................................................................14 
2.4.2 Sub-acute (Repeated Dose) Toxicity ..........................................................14 
2.5 
Nonclinical studies of Artesunate on HPV-infected epithelial cells ........... 16 
3.0 Clinical Safety and Tolerability ................................................................................. 25 
3.1 
Clinical Toxicology and Pharmacology ...................................................... 25 
3.2 
Dosing Rationale ......................................................................................... 29 
4.0 Overall Assessment of Risks and Potential Benefits ............................................... 29 
4.1 
Potential Risks ............................................................................................. 29 
4.2 
Known Potential Benefits ............................................................................ 30 
5.0 Rationale for clinical trial design ............................................................................... 30 
6.0 Study Objectives ........................................................................................................ 31 
7.0 Study Outcome Measures ........................................................................................ 32 
7.1 
Primary Outcome Measures ....................................................................... 32 
7.2 
Secondary Outcome Measures .................................................................. 32 
8.0 Study Design ............................................................................................................. 32 
9.0 Study Population and Enrollment ............................................................................. 33 
9.1 
Subject Inclusion Criteria ............................................................................ 33 
9.2 
Subject Exclusion Criteria ........................................................................... 33 
10.0 Study procedures ...................................................................................................... 34 
10.1 
Screening .................................................................................................... 34 
10.2 
Intervals Between Visits .............................................................................. 36 
10.3 
Description of Study Visits .......................................................................... 37 
10.4 
Examinations, Evaluations, and Procedures ............................................. 38 
10.5 
Compensation ............................................................................................. 39 
11.0 Study Drug and Administration ................................................................................. 39 
11.1 
Description of Study Medication ................................................................. 39 
11.2 
Dosage and Administration ..........................................................................41 
11.3 
Accountability Procedures for the Study Investigational Drugs ..................41 
11.4 
Assessment of Subject Compliance with Self Administration of 
Artesunate Suppositories ............................................................................ 42 
Toxicities and Adverse events ............................................................................... 43 
11.5 
Management of Toxicities ........................................................................... 44 
11.6 
Definition of a Serious Adverse Event ........................................................ 46 
11.7 
Recording and Follow-up of Adverse Events ............................................. 46 
11.8 
Definition of Dose-Limiting Toxicity ............................................................ 47 
11.9 
Early stopping rules ..................................................................................... 47 


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12.0 STATISTICAL CONSIDERATIONS ......................................................................... 48 
12.1 
Study Design/Endpoints/Accrual ................................................................ 48 
12.2 
Secondary Endpoints .................................................................................. 49 
12.3 
Other Efficacy Endpoints ............................................................................ 50 
12.4 
Additional exploratory analysis ................................................................... 50 
13.0 Subject completion/dropout ....................................................................................... 51 
13.1 
Definition of a Dropout ................................................................................. 51 
13.2 
Procedures for Handling Dropouts .............................................................. 51 
13.3 
Reasons for Dropout ................................................................................... 52 
14.0 Clinical Monitoring ..................................................................................................... 52 
15.0 Ethics and Regulatory considerations ...................................................................... 52 
15.1 
Institutional Review Board .......................................................................... 52 
15.2 
Informed Consent ........................................................................................ 52 
16.0 REFERENCES .......................................................................................................... 54 
 
 


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LIST OF ABBREVIATIONS 
 
AE 
Adverse Event 
CFR 
Code of Federal Regulations 
CIN 
Cervical Intraepithelial Neoplasia 
CRO 
Clinical Research Office 
CRC 
Clinical Research Review and Monitoring Committee 
CRF 
Case Report Form 
CTC 
Common Toxicity Criteria 
DHA 
Dihydroartemisinin 
DNA 
Deoxyribonucleic Acid 
E7 
Human Papillomavirus E7 antigen 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HSIL 
High grade squamous intraepithelial lesion 
HCG 
Human Chorionic Gonadotrophin 
HIPAA 
Health Insurance Portability and Accountability Act 
HLA 
Human Leukocyte Antigen 
HPV 
Human Papillomavirus 
IC50 
Concentration providing 50% inhibition 
IM 
Intramuscular 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
IV 
Intravenous 
JHH 
Johns Hopkins Hospital 
IND 
Investigational New Drug 
LEEP 
Loop Electrocoagulation Excision Procedure 
LSIL 
Low grade squamous intraepithelial lesion 
PCR 
Polymerase chain reaction 
SAE 
Serious Adverse Event 
TID 
Three Times A Day 
WHO 
World Health Organization 
 
 
 


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1.0 
SYNOPSIS 
 
Study drug 
Artesunate 
Title 
A Phase I study of intravaginally administered Artesunate 
In women with High Grade Cervical Intraepithelial 
Neoplasia (CIN2/3) 
Indication 
Female patients, 18 years and older, with CIN2/3 
Study 
population 
Women with biopsy-confirmed CIN2/3 with a visible lesion 
after biopsy 
Rationale 
Despite the approval of prophylactic vaccines which prevent 
infection with HPV types 16 and 18, the genotypes most 
commonly associated with squamous cancers, disease 
remains very common, even in high-resource settings, and 
is likely to remain so for decades.  Infection with a high-risk 
HPV type is necessary but not sufficient for the 
development of squamous cervical cancer (SCCx) and its 
precursor lesion, high grade cervical intraepithelial 
neoplasia (CIN2/3).  Both SCCx and CIN2/3 are associated 
with functionally obligate expression of two viral proteins, 
E6 and E7. Epithelial cells that express either or both of 
these oncoproteins also overexpress the transferrin 
receptor, and have increased levels of intracellular iron, 
compared to normal cells [1].  This observation raised the 
possibility of treating preinvasive HPV disease (i.e., 
CIN2/3) with an effective anti-malarial drug, Artesunate. 
Artesunate contains an endoperoxide bridge that reacts 
with intracellular ferrous iron to generate free radicals, 
leading to cell death [2, 3]. Artesunate, formulated as a 
suppository, is WHO-approved for first-line treatment for 
acute malaria in children who are remote from health care 
settings [4].  The toxicity profile of this formulation is well-
documented. Preclinical data suggest that Artesunate 
mediates cytotoxic effect on HPV-infected cells, while 
having no effect on normal epithelium. Thus, it is proposed 
to investigate the use of Artesunate formulated in 
suppositories applied intravaginally to treat CIN2/3. 
Evaluation 
Criteria 
 
Safety will be assessed by the absence of related dose 
limiting adverse effects.  Subjects will be monitored at each 
visit for the appearance of erythema; itching, burning, pain; 
infection; desquamation; tenesmus; bleeding; dizziness; or 
other unexpected AEs. Circulating blood cells will be 
monitored weekly for any quantitative changes. 
 


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Efficacy will be determined by the presence/absence of 
HPV, by viral load, and by regression of lesions. The 
presence of HPV will be determined by DNA tests before 
pre- and post-treatment. Histologic endpoints will be 
assessed with colposcopically-directed biopsies obtained 
before and after treatment with topical Artesunate.   
 
In this population, it is feasible to directly visualize, serially 
quantitate, and perform therapeutic resection of 
premalignant lesions.  By comparing immunologic 
parameters in the peripheral blood (systemic) compartment 
with measures obtained from the site of the lesion, pre- and 
post-drug administration, we expect to derive insights 
which will inform immune therapeutic strategies for 
incipient malignancies. 
 
IND Phase 
Phase I 
Objectives 
 
Primary Objective:  
• To determine and evaluate the safety, tolerability, and 
feasibility of intra-vaginal administration of Artesunate, 
administered via vaginal suppository, in healthy women 
with cervical intraepithelial neoplasia (CIN2/3)  
Secondary and Exploratory Objectives:  
• To measure the effect of intra-vaginal topical Artesunate 
administration on histology, based on the regression of 
CIN2/3 at study week 15, assessed as either CIN1 or no 
CIN lesion detected by colposcopy/biopsy and cytology. 
• To evaluate clearance of HPV as assessed by Hybrid 
Capture 2 DNA testing of cytologic specimens 
• To evaluate the local tissue immune response 
• To correlate measures of immune response with clinical 
response 
Study 
Population 
Women, 18 years and older, with biopsy-confirmed CIN2/3, 
and a visible lesion after biopsy. 
Inclusion/ 
Exclusion 
Criteria 
Inclusion 
• ≥ 18 years 
• Capable of informed consent 
• HPV-positive by DNA test 
• Histologically confirmed CIN2, CIN3, or CIN2/3 
• Body weight ≥ 50 kg 
• Immune competent 
 
 


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Exclusion 
• Pregnant and nursing women 
• HIV seropositive 
• Active autoimmune disease 
• Taking immunosuppressive medication  
• Evidence of concurrent adenocarcinoma in situ 
• Concurrent malignancy except for nonmelanoma skin 
lesions 
Investigational 
Treatment 
Intra-vaginal administration of Artesunate suppositories, 
one to three multi-day dosing cycles; 50-200 mg, once a day 
for  5 days per treatment cycle;  prior to scheduled LEEP or 
cone excisional procedure (standard of care for CIN2/3 
patients) 
Clinical Study 
Sites 
Johns Hopkins Hospital 
Greater Baltimore Medical Center (Johns Hopkins Clinical 
Research Network Site) 
Study Design* 
Phase I open-label dose escalation study of intravaginal 
Artesunate, formulated in suppositories, in adult females 
with biopsy-confirmed CIN2/3.  Thirty (30) subjects will 
undergo up to a total of three cycles of intravaginal 
Artesunate. The first cycle will be initiated on Day 0, the 
second at Week 2, and the third and final cycle at week 4.  
The lowest dose cohort that demonstrates most evidence of 
clinical efficacy as determined by clinical endpoints will be 
expanded.  A schematic overview of the study design is 
provided on the next page, including study arms, sample size 
and schedule of interventions. An outline of scheduled visits 
and assessments is presented below. 
Evaluation 
Criteria 
Safety: Subjects will be monitored for local and systemic 
adverse events.  
 
Efficacy: Histology, cytology, and HPV PCR at study week 15.   
Number of 
Subjects 
A total of 30 patients will be enrolled in this study 
Estimated 
Patient 
Enrollment 
Period 
12 months after the approvals of IND and JHH IRB 


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Subject 
Participation 
Duration 
41  weeks from the first dosing  
Statistical 
Analysis Plan 
Statistical analysis will be primarily descriptive due to the 
small sample size.  Treatment related safety and efficacy 
attributes will be analyzed using the Chi-square test. Any 
exploratory inferences with a p-value of ≤ 0.10 will be 
considered for further study in subsequent trials. 
 
Schematic of Study Design: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening visit
Week 0 (Cycle 1) 
Artesunate suppositories 
Week 2 (Cycle 2) * 
Artesunate suppositories 
Week 4 (Cycle 3) ** 
Artesunate suppositories + 
Colposcopy (week 6) 
Week 15 
Assess for residual disease: colposcopy, biopsy  
Some improvement observed: 
- Lesion size by colposcopy 
- CIN grade reduction (Biopsy, pap smear) 
No improvement 
Cone resection*** 
Week 28 
Assess for residual disease: colposcopy + biopsy + pap smear 
No detection of CIN2/3 lesion by biopsy + pap 
smear 
Detection of CIN2/3 lesion by biopsy + pap 
smear 
Cone resection*** 
Week 41 
Assess for relapsing disease: pap smear and if clinically indicated colposcopy + biopsy 


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There will be 4 treatment cohorts, which will be filled sequentially 
Cohort 
Number of patients 
Dose 
Treatment cycles 
I 
3 
50 mg 
1 (5 days) 
II 
9 
200 mg 
1 (5 days) 
III* 
9 
200 mg 
2 (5 days each) 
IV** 
9 
200 mg 
3 (5 days each) 
***According to the standard of care clinical practice, a postoperative visit will be done 4 weeks after the 
cone resection. 
 
2.0 
BACKGROUND AND RATIONALE 
2.1 
HPV-associated malignancies of the cervix 
Virtually all squamous cervical cancers (SCCx) are caused by a common virus, human 
papillomavirus (HPV). Exposure to HPV occurs with the onset of sexual activity. While 
most persons clear their infection without intervention, and without sequelae, a subset 
do not.  Persistent infection with a high-risk HPV type is the proximate cause of SCCs 
and their precursor intraepithelial lesions, cervical intraepithelial neoplasia (CIN2/3). 
Other factors contribute to the development of SCC, as viral integration into the host 
genome is necessary but not sufficient for the initiation and persistence of malignant 
transformation.  Currently there are no available treatments that can eradicate HPV 
infection.  Successful prevention of SCC in infected individuals is based on early 
detection of preinvasive lesions prior to the development of cancer. 
 
Despite the availability of several, inexpensive, noninvasive screening strategies to 
detect precursor lesions, the incidence of HPV disease (SCC and CIN 2/3) in women 
remains high. An effective topical intervention such as current proposal of an 
intravaginal Artesunate treatment would obviate the need for surgery and prevent 
progression to SCC. Globally, ten percent of malignancies in women are caused by HPV. 
Cervical cancer is the second leading cause of cancer death in women worldwide, and in 
the United States, cervical cancer remains the sixth most commonly diagnosed 
malignancy among women.  Over the past decade, SEER data have documented a 17% 
increase in incidence in the U.S., normalized for population growth, with a 
disproportionate increase among young women [5] . Although preventative vaccines 
that protect against infection with HPV types 16 and 18, the genotypes most commonly 
associated with squamous cancers, and have been introduced for preteen and teen girls 
and boys since 2006, their impact has not yet realized as the rates of vaccination remain 
low in the United States. Recent data indicate that the rate of preventive vaccination in 
eligible girls, in 2012, was only 33% [6].  
 
The primary strategy to decrease disease burden in HPV-infected individuals is to 
intervene when premalignant cervical disease, CIN2/3, is detected prior to the 
development of SCCs. All treatments for CIN2/3, including cryotherapy, laser 
vaporization, or cone excisions, are ablative and require repeated visits to health care 


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providers.  Because they involve tissue destruction, all current therapeutic options have 
the potential for adverse sequelae, and furthermore, are not always curative.  Among 
treated CIN2/3 in immunocompetent women, the overall risk of recurrence is less than 
ten percent when all surgical margins are clear. This risk increases to approximately 
25% in women with positive endocervical margins. Women who have undergone 
cervical conization have three times the risk of cervical stenosis as those who have not 
[7].  Several authors have documented a significantly increased risk of premature 
delivery in pregnancies subsequent to cervical conization, and subsequent neonatal low 
birth weight. [7, 8] Moreover, tissue destruction from therapeutic interventions can 
make subsequent detection and treatment of recurrent disease more difficult, as the 
healing process tends to draw the transition zone of the cervical epithelium proximally, 
into the endocervical canal [9]. While these complications are not insurmountable, an 
effective local immunotherapy such as intravaginal Artesunate suppositories is clinically 
meaningful to obviate the need for surgeries. 
 
2.2 
Noninvasive topical therapy for cervical dysplasia 
The development of a topical therapy for preinvasive CIN2/3 lesions that could be self-
administered would be a game-changing treatment option for women with preinvasive 
HPV disease.  A topical treatment option would decrease adverse sequelae related to 
ablative procedures, decrease visits to specialized health care providers, and result in 
considerably decreased health care costs.  In addition, in many cultures, HPV disease, 
particularly of the lower genital tract, carries a social stigma [10].  The development of a 
topical therapy that could potentially be self-administered, would be empowering for 
women with preinvasive lesions. A topical therapeutic also has the potential to obviate 
the need for surgery.  
 
2.3 
Rationale for study drug 
Artesunate is a semisynthetic derivative of artemisinin, a plant compound extracted 
from the leaves of sweet wormwood, Artemisia annua, an herb used in Chinese 
traditional medicine for antimalarial treatment for over two thousand years, with 
marketed formulations since the late 1980s [11].  The safety profile of Artesunate and 
related compounds has been established based on  millions of malaria patients, ranging 
from infants to adults, over decades [12].  Artesunate has been shown to be safe and 
well-tolerated when administered orally, intravenously, intramuscularly, or intra-
rectally, as a suppository [4, 5, 13-15]. Reports submitted to the FDA by both the WHO 
and by Novartis summarize many of the safety studies conducted with artemisinin and 
its derivatives, including Artesunate [16-18]. 
 
Recently, Artesunate compounds have been shown to have antitumor activity against 
several human solid tumor cell lines, including breast, colon, ovarian, prostate, renal, 
and non-small cell lung cancer [13]. Of particular relevance, artemisinin derivatives 
have also been shown to have a cytotoxic effect on HPV-immortalized epithelial cells 
[13] .  
 


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Artemisinin and its derivatives, including Artesunate, have been shown to decrease cell 
proliferation, reduce angiogenesis and trigger apoptosis in cancer cells [14, 15, 19]. 
Although the mechanisms of action are incompletely understood, in human solid 
cancers, sensitivity to Artesunate has been reported to be associated with expression of 
angiogenesis-related gene transcripts [20].  Tumor susceptibility has also been reported 
to be correlated with cell surface overexpression of transferrin receptor [21, 22] and with 
intracellular ferrous iron [1].  It has been demonstrated that artemisinin and its 
derivatives contain an endoperoxide bridge that reacts with intracellular ferrous iron to 
generate free radicals, leading to cell death [3,4].  
 
Both SCC and CIN2/3 are associated with functionally obligate expression of two viral 
proteins, E6 and E7. Epithelial cells that express either or both of these oncoproteins 
also overexpress the transferrin receptor, and have increased levels of intracellular iron, 
compared to normal cells [1].  Because cervical squamous cancers and their precursor, 
intraepithelial lesions (CIN) overexpress the transferrin receptor [21, 22], these 
observations prompted a subsequent study of the cytotoxic effect of dihydroartemisinin 
(DHA), the bioactive form of Artesunate, on papillomavirus-expressing epithelial cells 
[23] .  In vitro studies demonstrated that, while DHA had little effect on normal cervical 
epithelial cells, it had a significant cytotoxic effect on HPV-immortalized cervical cells 
[23]. In addition, formulated as a local treatment in an oral mucosal canine nonclinical 
model, DHA has been reported to inhibit papillomavirus- induced tumor formation 
[23].  
 
Together, these findings suggest that topical administration of an Artesunate compound 
may provide therapeutic benefit for intraepithelial HPV disease.  Given the unmet need 
for a local treatment for CIN2/3 lesions and that Artesunate has demonstrated a 
favorable safety profile for decades in both children and adults, a prospective, dose-
escalation, Phase 1 study of  an Artesunate suppository, administered intravaginally to 
achieve local, mucosal distribution of study drug, is proposed.   
 
2.4 
Nonclinical safety literature review 
Extensive reviews of the effects of Artesunate on the central and peripheral nervous 
system, and on cardiovascular, respiratory and gastrointestinal systems of various 
animal species (mice, rabbits, guinea pigs, dogs, and monkeys) have been compiled [18, 
24] . There are also some recent sub-chronic and chronic toxicological studies of 
Artesunate in dogs [25, 26] . These reports contain data on Artesunate toxicity when 
administered via different routes, including oral, intravenous, intramuscular and intra-
peritoneal injections at doses of up to 640 mg/kg, which is well above therapeutic levels. 
In general, minimal pathological changes were observed in animal studies at doses 
under 60 mg/kg [18, 25, 26]. The toxic effects would not therefore be considered to be of 
clinical concern in regard to human therapeutic use [18, 24] . In this clinical study, we 
propose to evaluate intravaginal suppository doses up to 200 mg (approximately ≤4 
mg/kg for subjects who weigh ≥50 kg, an inclusion criterion of this study) that are 
considerably less than doses associated with toxicities in animals (>60 mg/kg) and the 


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rectal suppository doses used in children (approximately 10 mg/kg) and adults 
(approximately 8.3 mg/kg) for malaria treatment [18]. 
 
2.4.1 Acute (Single Dose) Toxicity 
In single-dose toxicity studies of Artesunate administered intravenously or 
intramuscularly in mice, LD50 values of 520 and 475 mg/kg were determined 
respectively [18]. In another mice study, Artesunate exhibited a higher intravenous LD50 
of 699 mg/kg [18]. Rats exhibited a slightly lower intravenous LD50 at 488mg/kg [27]. 
Maximum tolerated intravenous doses for guinea pigs, rabbits, dogs and monkeys were 
240, 160, 80 and 60 mg/kg, respectively, and the minimum lethal doses for these 
species were found to be 480, 640, 480 and 160 mg/kg, respectively [18]. Toxicity 
symptoms observed included CNS effects (depression, unstable gait, tremor, 
convulsions) and respiratory suppression [18, 28] . Oral administration of Artesunate 
was better tolerated and resulted in LD50 values of approximately 1000-1300 mg/kg in 
mice, and 600-900 mg/kg in rats. In dogs, reticulocyte counts were decreased at doses 
of ≥80 mg/kg, while histopathology showed some liver damage at ≥120 mg/kg [18].  
 
2.4.2 Sub-acute (Repeated Dose) Toxicity 
In repeated-dose toxicity studies, intravenous administration of Artesunate to dogs at 
doses of 10 and 40 mg/kg for 14 days did not produce clinical symptoms or significant 
hematological, biochemical or histopathological changes [18]. Similar results were 
obtained in monkeys given daily 10 and 32 mg/kg intravenous injections for 14 days 
[18]. In rats, intravenous administration of Artesunate for 3 consecutive days at doses of 
up to 240 mg/kg induced a dose-dependent decrease in reticulocyte numbers, as well as 
decreases in erythrocytes, haematocrit and haemoglobin [18]. The decrease in 
reticulocyte numbers was generally the most sensitive measure of Artesunate effects, 
since statistical significance of the difference to the treatment controls was reached in 
females at 7.5 and in males at 15 mg/kg. At doses ≥60 mg/kg, body weight was also 
depressed [18]. Lastly, when Artesunate was administered intramuscularly to rats at a 
daily dose of 50 mg/kg for 7 days, food and water consumption declined starting from 
the first or second dose. Body weight also decreased over the treatment time. However, 
all these effects were reversible upon cessation of treatment [18].  
 
2.4.3 Subchronic Toxicity 
A 3-month study of orally administered Artesunate (6mg/kg) in dogs revealed no 
neurotoxicity [25]. Nor were any gross pathological changes observed. There was, 
however, evidence of some damage to the mitochondrial ultra-structure when studied 
using an electron microscope [25].  
 
2.4.4 Organ and system toxicities 
Artemisinins exhibit a distinctive embryotoxicity that has a very steep dose-response 
relationship [18]. In rats, treatment with Artesunate and other artemisinin derivatives 
results in large embryonic losses at modest doses . In mice, 100% resorptions were 
observed at a dose of 21.4 mg/kg, while in rats and rabbits, resorptions were observed in 


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over 90% of rats and rabbits at doses of 10.7 and 2.7 mg/kg, respectively [29]. An 
embryo-fetal no-observable-adverse-effect level (NOAEL) of 8mg/kg was recently 
established in rats [30]. Importantly, no artemisinin-induced embryotoxicity has been 
reported in humans [24]. This is remarkable because malaria treatment in much of 
tropical Africa is often dispensed on the basis of fever rather than the confirmation of 
parasitemia [31]. The parasite is widely thought to have a protective effect against 
artemisinin-induced decrease in reticulocyte count which is a marker for erythropoietic 
toxicity [31]. In any case, the use of Artesunate and other artemisinins in the first 
trimester of pregnancy is contraindicated based on observations of embryotoxicity 
between gestation days 9–11 in mice, 6–15 in rats, 7–18 rabbits, and 26-36 in monkeys 
[32].  
 
Neurotoxicity associated with artemisinins appears to be not only dependent on the 
dose, but also on the route of administration and the particular artemisinin derivative 
[24]. The vehicle used to deliver the drug is also important with the water-soluble 
Artesunate showing markedly less neurotoxicity in laboratory animals than the oil-
soluble arteether and artemether presumably because of the slower release and 
distribution of the latter compounds in blood plasma [24]. In mice, neurotoxic effects of 
Artesunate and DHA in a 28-day oral study were observed at a dose of >200 mg/kg/day 
[33]. Other effects included sedation at doses ≥200 mg/kg in mice and lower body 
temperature in rats at a dose of 450 mg/kg, and in rabbits and dogs at doses of >160 
mg/kg. In rabbits, respiratory depression was observed at doses of ≥320 mg/kg, without 
any changes in the blood gases and blood pH [18, 24] . In mice, artemether in doses of 
30, 50, 75 and 100 mg/kg, and Artesunate in doses of 30, 50 and 100 mg/kg, 
administered daily as intramuscular injections for 28 days, resulted in greater mortality 
in the high dose artemether cohort, compared to mice that received Artesunate at the 
same dose [34]. No significant effects on body weight were observed at the lowest dose. 
In the high-dose cohorts, differences in body weight were apparent between artemether 
and Artesunate [34]. Neurological observations at the high dose showed a clear 
distinction between Artesunate, which had practically no ill effect, and artemether, 
which elicited neurologic effects in 90% of treated animals. Lastly, olfactory responses, 
scored as the time needed to find food, were significantly impaired in the artemether but 
not in the Artesunate-treated groups [18, 34]. Overall, artemether appeared to have 
greater impact on neurological parameters than Artesunate, and intramuscular injection 
appeared to elicit more side effects than oral formulations [18, 24, 33, 34]  .  
 
Hematopoietic toxicities are either erythropoietic or leukopoietic. In animal 
experiments, artemisinins effects on erythropoiesis are frequently encountered [24, 31] . 
The effects are however mild and reversible in humans [31]. The data on artemisinin 
effects on leukocytes are contradictory, with some authors describing suppressed 
immune response, while others reported enhancements such as increased phagocytosis 
and interferon production [24].  
 
The generation of reactive oxygen species (ROS) and carbon-centered radicals by 
artemisinins raises the possibility of cardiotoxicity. But whereas there have been reports 


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of cardiotoxicity in dogs, no impairment of heart function has been observed in 
numerous malarial therapy trials in humans [24].  
 
Finally, at an elevated dosage of 240mg/kg/day (i.v. for 3 days), Artesunate causes renal 
failure and tubular necrosis in rats that were not infected with malaria [35]. The 
nephrotoxic effects were however reversible.  
 
2.5 
Nonclinical studies of Artesunate on HPV-infected epithelial cells 
Our research collaborators at Georgetown University have conducted several nonclinical 
studies to evaluate the cytotoxic, apoptotic and anti-tumor activity of Artesunate and its 
active metabolite dihydroartemisinin (DHA). The effects of artemisinin, Artesunate, and 
DHA were studied on a panel of cell lines including primary, HPV-immortalized, and 
cervical tumor cell lines. Both HPV-immortalized and cervical tumor cell lines are 
sensitive to DHA and Artesunate. These cells express higher levels of transferrin 
receptor and intracellular iron as compared to normal ectocervical cells. Iron is required 
for the drug-induced formation of reactive oxygen species (ROS), the activation of 
caspases, and subsequent apoptosis [23].  
 
Table 1:  List of In Vitro and In Vivo Nonclinical Pharmacology Studies of Artesunate 
and DHA 
Study 
Description 
Species/ 
Cell line 
Dose 
Method 
Primary Findings 
In vitro 
cytotoxic 
activity study 
-HPV expressing 
cell lines 
 -Normal cervical 
epithelial cells     
-Ectocervical 
epithelial cells  
- E6 or E7 
transformed 
cervical cells  
-normal cervical 
epithelial cells 
that expressed 
either E6 or E7 
5-50 µmol/L 
artemisinin, 
Artesunate 
or DHA 
Treatment of cell 
line for 3 days 
and measurement 
of viability with 
neutral red 
staining 
Artesunate and DHA 
were cytotoxic to 
cervical cancer cell 
lines and to HPV-
transformed cells, but 
not to normal cervical 
epithelium cells. 
 
DHA had a cytotoxic 
effect on all cells 
expressing E6 or E7, 
regardless of whether 
they were malignant.   
In vitro 
apoptotic 
activity study 
HPV-infected 
andnon-infected 
HeLa cells  
0, 25 or 100 
µmol/L 
DHA 
HeLa cells treated 
with 5 µmol/L 6-
carboxy-2V, 7V-
dihydrofluorescei
ndiacetate and 
DHA for 3 hours 
and then analyzed 
by flow 
cytometry. 
Cytotoxic effect on 
HPV-infected cells 
mediated by DHA is 
iron-dependent, and 
occurs via apoptosis. 
In vivo 
activity of 
DHA 
Canine  
2.22 mg 
DHA 
dissolved in 
100 µL 
Beagles 
challenged with 
purified canine 
oral 
Topical application of 
DHA inhibits the 
formation of virally 


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DMSO 
(route- 
topical) 
papillomavirus on 
the gum and 
buccal mucosa 
were 
administered 
topical DHA 
induced papilloma in 
vivo 
Note: Both DHA and Artesunate show similar in vitro activity against HPV-infected cells; however, DHA 
was selected for the in vivo study due to its favorable solubility in dimethyl sulfoxide (DMSO). HPV (Human 
papillomavirus).  
 
2.5.1 In vitro cytotoxicity assay 
The anti-tumor activity of artemisinin, Artesunate, and DHA was evaluated using a well-
characterized HPV-transformed cervical cancer HeLa cell line. HeLa cells, at a 
concentration of 5 x 104, were cultured for 3 days with each test compound at 
concentrations ranging from 5 to 50 µM (Figure 1). Viability was measured using the 
neutral red uptake assay [36].  
 
As illustrated in Figure 1, artemisinin had little effect on HeLa cells. The in vivo activity 
of artemisinin may rely on its rapid conversion into the active metabolite, DHA in the 
liver. Consistent with this hypothesis, DHA was highly effective in killing HeLa cells.  
 
Figure 1. Effects of artemisinin, Artesunate 
and DHA on normal, immortalized and 
HPV-transformed cervical epithelial cells. 
(A) HeLa cells were cultured for 3 days with 
concentrations ranging from 0-50 μM of 
artemisinin, Artesunate, or DHA. The IC50 of 
Artesunate and DHA was 5 and 7.5 μM, 
respectively. (B) Viability of normal cervical 
epithelial cells (HCX) cultured as in (A) was 
shown. (C) Ectocervical cells transduced 
either with empty retrovirus (HCX), with 
either HPV-16 E6 or E7, cervical cells 
transduced with HPV-16 E6/E7, pre-
immortalization (early p5) and post-
immortalization (late p50), and cervical 
cancer cells (HeLa, SiHa, and Caski) were 
cultured for 3 days with DHA. All 
tumorigenic and HPV-immortalized cell 
lines were sensitive to DHA, with HeLa 
showing the greatest sensitivity. In all 
culture conditions, cell viability was 
measured using the neutral red assay. Each 
data point represents the mean of two wells 
from experiments carried out in triplicate. 
 
 
 
 
 
 


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The IC50 measurements for Artesunate and DHA on HeLa cells were determined to be 5 
and 7.5 µM, respectively. Most importantly, DHA and Artesunate had little or no 
cytotoxic effect on normal cervical epithelial cells (Fig. 1B), even at 50 μM 
concentrations. As anticipated from its lack of effect on HeLa cells, artemisinin also had 
no effect on normal cervical cells. Because DHA is the active metabolite of Artesunate, it 
was used for further studies and screened for its activity against tumorigenic and HPV-
infected cervical cell lines.  
 
In the clinical setting of SCCx and its precursor lesion, CIN2/3, expression of both the 
E6 and E7 viral proteins is constitutive, and functionally required to maintain the 
transformed state [37, 38]. Cell surface expression of the transferrin receptor is 
increased in cells expressing these oncoproteins [21]. Because internalization of DHA is 
mediated in part by this receptor, we tested the effect of DHA on normal cells, on E6- 
and E7 -transformed cells, on epithelial cells that expressed either E6 or E7, but were 
not transformed, and on HPV cancer cell lines SiHa, CaSki, and HeLa.   
 
As shown in Figure 1C, DHA had a strong cytotoxic effect on all cells expressing E6 or 
E7, regardless of whether they were frankly malignant. On the other hand, DHA had no 
effect on the viability of normal cervical epithelial cells (Figure 1C). 
 
2.5.2 In vitro apoptosis assay  
Correlation of transferrin receptor level with DHA sensitivity 
It is known that the antimalarial activity of Artesunate and DHA depends on the high 
intraparasitic iron content [11]. The same panel of cells as in Figure 1C was examined for 
transferrin receptor level and cellular iron content. Cells, at a concentration of 1 x 106, 
were cultured for 24 hours and harvested for Western blot analysis (with anti-
transferrin receptor antibody, 1:1000) to determine the level of transferrin receptor. As 
shown in Figure 2A, the blot shows a clear increase in the amount of transferrin receptor 
in HPV-immortalized and tumorigenic cell lines (1.7- to 2.5-fold) than corresponding 
normal cells (HCX). The increased level of transferrin receptor in vitro correlates with 
in vivo data showing increased transferrin receptors in high-grade cervical lesions and 
cancers as measured by immunohistochemistry [21, 22, 39].  
 
Transferrin receptor overexpression correlated with total intracellular iron as measured 
by inductively coupled plasma optical emission spectrometry [23]. HeLa cells contained 
50% more iron than normal cervical epithelial cells, i.e. 1.3 versus 0.88 pg per cell. 
Whereas the absolute level of transferrin receptor and total cellular iron in HeLa cells 
were elevated, they did not correlate linearly with the dramatic increase in sensitivity to 
DHA. This discrepancy could be explained by several possibilities. For example, total 
cellular iron was measured because we have not been able to quantify the level of 
ferrous iron, the form that reacts with the DHA endoperoxide bond to generate ROS 
[23]. It is possible that there is a greater difference in the level of ferrous iron in the 
normal and HPV-expressing cells. In addition, because HPV-expressing cells are 
sensitized to apoptosis by the E6 and E7 proteins, it is conceivable that slight increases 
in iron content may amplify toxicity of DHA in HPV-infected cells.  


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Abrogation of DHA-induced cytotoxicity with an iron chelator 
To provide more direct evidence for the role of iron in DHA-induced cytotoxicity, a 
study was conducted involving an iron chelator - desferrioxamine (DFOM). HeLa cells, 
at a concentration of 2 x 104, were incubated for 6 hours with DFOM at concentrations 
ranging from 0 to 200 µM. Media containing DHA at concentrations of up to 150 µM 
and DFOM at the original concentration were subsequently added and incubated for 24 
hours before cell viability was analyzed. As shown in Fig. 2B, DHA cytotoxicity was 
reversed in a dose-dependent manner, indicating an essential role for iron. To 
circumvent potential toxicity associated with prolonged exposure of cells to 
desferrioxamine [40], these experiments were repeated with increased amounts of 
DHA, and decreasing exposure from 3 days to overnight. Under these conditions, there 
was no detectable effect of desferrioxamine on cell viability.  
 
Figure 2. Transferrin receptor levels correlate with DHA sensitivity and iron-dependent toxicity in HeLa 
cells. (A) The same panel of cells as in Fig. 1C was analyzed by Western blotting for transferrin receptor 
level. A clear increase in the amount of transferrin receptor in immortalized and tumorigenic cell lines as 
compared with normal cells (HCX) was observed. Densitometry was done on the scanned image; numbers 
below the lanes, increase in transferrin receptor levels when HCX was set at 1. Jurkat cell extract was 
included as a positive control. (B) Viability of desferrioxamine pre-treated HeLa cells exposed to DHA. 
DFOM antagonized the cytotoxic effects of DHA and increased cell survival in a dose-dependent manner 
with >85% cell survival when the cells were pretreated with 200 µM desferrioxamine. Each point 
represents the mean cell viability of three wells from experiments carried out in triplicate.  
 
DHA dose-dependent increase in presence of iron-dependent reactive 
oxygen species 
To determine whether the DHA cytotoxicity observed in HeLa cells was a function of 
generation of iron-dependent ROS, the induction of ROS in HeLa cells was measured by 
a non-fluorescent probe, 6-carboxy-2V, 7V-dihydrofluorescein-diacetate. This probe is 
oxidized by ROS and converted into a fluorescent compound, 2V, 7V-dichlorofluorescein 
to provide a measurable indicator for the level of ROS. HeLa cells, at a concentration of 
1 x 106, were incubated for 1 hour in 5 µM 6-carboxy-2V, 7V-dihydrofluoresceindiacetate 
in Dulbecco’s modified eagle medium (DMEM). Subsequently, the probe was removed 
by rinsing in phosphate buffered saline. Cells were incubated with either fresh DMEM, 
DMEM + DHA (25 or 100 µM) or DMEM containing 150 µM DFOM and DHA (25 or 


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100 µM) for 3 hours. Cells were then analyzed by fluorescence-activated cell sorting 
(FACS) analysis. 
 
As shown in the left panel of Figure 3A, The DHA-treated HeLa cells demonstrated a 
dose-dependent increase in fluorescence (2-fold and 4.2-fold at 25 and 100 µM doses, 
respectively), indicating an increase in production of ROS in response to DHA 
treatment. This increase in ROS was abrogated when the cells were pretreated with 
DFOM as shown in the right panel of Figure 3A. Altogether, these data underscore the 
essential role for intracellular iron in mediating the cytotoxic effects of DHA, via 
generation of ROS.  
 
DHA-induced apoptosis 
We further hypothesized that the cytotoxicity mediated by formation of ROS could be 
due to the induction of apoptosis. This is based on the observation that DHA-treated 
cervical cancer cells ceased proliferating, rounded up, and detached from the plate, 
suggesting an apoptotic process.  
 


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Figure 3. DHA-induced apoptosis is caused by iron-dependent ROS formation. (A) HeLa cells were 
treated with a fluorescent probe for ROS, and simultaneously with 0 µM (red line), 25 µM (blue line), or 
100 µM (purple line) DHA for 3 hours (left). The black lines represent untreated cells in both panels. 
HeLa cells cultured with DHA demonstrated a dose-dependent increase in fluorescence (2-fold and 4.2-
fold at 25 and 100 µM doses, respectively), indicating an increase in production of ROS in response to 
DHA treatment. The production of ROS was markedly inhibited by pretreating the cells with the iron 
chelator DFOM (right), indicating that the formation of ROS in response to DHA treatment is iron-
dependent. (B) Control cervical cells (HCX) and HeLa cells were treated at 0, 10, 25, 50 µM DHA for 3 
days, and stained for apoptosis with FITC-conjugated Annexin V and propidium iodide. HCX showed only 
a minor increase (from 2.7% to 4.4%; top) in stained cells while HeLa cells showed a significant increase 
in Annexin V and propidium iodide (from 0.1% to 64.8%; bottom).  
 
To determine the mode of DHA-induced cell death, HeLa cells and control normal 
cervical epithelial cells were stained with fluorescent-conjugated Annexin V for early 
apoptotic detection, and with fluorescent-conjugated propidium iodide for late 
apoptotic events. The frequency of stained cells was quantified by flow cytometry.  


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As shown in Figure 3 (top), control normal cervical epithelial cells (HCX) showed only a 
marginal increase of either Annexin V-stained or propidium iodide-stained cells 
following treatment with increasing concentrations of DHA for 3 days (increase from 
2.7% to 4.4% at the highest DHA concentration of 50 µM). In contrast, DHA-treated 
cervical cancer cell lines stained with Annexin V and rapidly became propidium iodide 
positive, indicating irreversible loss of membrane integrity. After 3 days in 50 µM DHA, 
approximately 65% of the cervical cancer cells were stained positively with propidium 
iodide, with an additional 9% staining positive for Annexin V. 
  
2.5.3 In vivo activity of DHA in a canine oral mucosa model  
To determine whether in vitro activities of DHA shown in previous sections were 
applicable in vivo, a canine oral papillomavirus (COPV) model was used. This animal 
model emulates the effects of HPV on the mucosal sites in human [41] and has been 
previously used for efficacy evaluation of papillomavirus vaccines. In this model, 100% 
of challenged animals become infected and develop tumors. Canine oral papillomas 
have an incubation period of 4–8 weeks following inoculation, and typically undergo 
spontaneous regression in the ensuing 4-8 weeks [42].  
 
The maxillary buccal mucosae of six 10-week-old, female beagles were abraded with a 
sterile wire brush. Wart homogenate containing 500 ng (based on L1 concentration) of 
oncogenic COPV was then applied to the excoriated mucosa with a cotton swab. After 24 
hours, the infected areas of three beagles were treated topically with 2.22 mg DHA 
dissolved in 100 µL dimethyl sulfoxide (DMSO) (78 mM). The infected areas of the 
other three beagles were treated with 100 µL DMSO as the placebo control. Treatment 
was applied once daily, five times a week, for 6 weeks. 
 
Figure 4A depicts a schematic of viral challenge, drug treatment, tumor appearance, and 
tumor regression. In all control animals, tumors appeared approximately 3 weeks after 
the COPV challenge. Figure 4B is a photograph of virus-infected dogs treated with 
DMSO (left) or DHA (right) at 6 weeks post-challenge. The tumor shown in the DMSO-
treated dog is representative of the size of tumors observed in all tumor-positive dogs 
and was taken at the pinnacle of tumor growth. The DHA-treated dog is free of tumor. 
In addition, the oral mucosa treated with DHA shows no evidence of ulceration or 
inflammation, consistent with the lack of DHA cytotoxicity for normal epithelial cells in 
vitro. All tumors were regressed by 8 weeks following initial viral challenge. Study 
animals were observed for another week after regression to ensure that no additional 
tumors were formed. Peripheral blood was obtained 1, 3, and 5 weeks after the onset of 
tumor formation.   
 
Figure 5A summarizes the results of tumor formation in the six study dogs. All virus-
infected dogs treated with DMSO developed tumors at 3 to 4 weeks post-challenge. In 
contrast, DHA treatment abolished tumor formation in two of three infected animals. 
The lesions that developed in the DHA-treated animal (dog 5) underwent regression 2 
weeks earlier than those in the animals treated with DMSO alone, although the mass 
was of comparable size to those in control animals.  


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Figure 4. Topical therapeutic effect of DHA in a preclinical papillomavirus model. (A) a schematic 
representation of the experimental protocol. Treatment was initiated 24 hours after the COPV challenge 
and stopped when all tumors had regressed. For this COPV model, tumors appeared approximately 3 
weeks after viral challenge and all tumors had regressed 8 weeks post-challenge.  (B) Representative 
images of dogs with and without tumor formation.  
 
Although the study sample size was small, precluding statistical analysis, it should be 
emphasized that the COPV model resulted in a 100% tumor formation rate during 
decades of working with this model by the group. Thus, the absence of tumors in 2 of 3 
treated animals is biologically significant, suggesting that DHA is effective in the 
prevention of tumor formation. 
 
Visual observations of the oral mucosa approximately 3 weeks post-challenge identified 
a similar mucosal ‘‘roughness’’ in all of the challenged dogs, including the DHA-treated 
animals, indicative of early papilloma formation. We postulated that DHA might be 
inhibiting tumor formation rather than virus infection.  
 
To determine whether the DHA-treated dogs had a subclinical viral infection, serum 
samples obtained at the end of the trial were used to detect and quantify antibody 
responses to the L1 viral capsid protein. Serum samples were analyzed for 
papillomavirus antibody using an ELISA immunoassay. The results are shown in Figure 
5B. Despite the finding that only one DHA-treated dog developed a tumor, all DHA-
treated animals developed antibody titers against the L1 protein, suggesting that the 
tumor-free animals indeed had been infected with virus and that the virus had 
replicated and synthesized significant amounts of L1 protein. These data suggest that 
DHA inhibits tumor formation rather than viral replication. As anticipated, all of the 


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control animals also developed antibodies to L1, which is normally observed during the 
natural regression of these tumors.  
 
Figure 5. Topically applied DHA inhibits papilloma formation by COPV. (A) Tabulation of tumor 
formation in dogs. All 3 control animals and 1 DHA-treated animal developed tumors that reached a 
similar size to those shown in Figure 4B, left. The tumor in the DHA-treated dog regressed 2 weeks sooner 
than the tumors in DMSO-treated dogs. (B) ELISA results from dog serum collected during the last week 
of the study. All dogs were positive for antibodies against L1, indicating an initial infection in all dogs. 
Dogs 4 and 6 without the presence of tumors showed antibodies against L1, suggesting DHA can inhibit 
tumor formation without inhibiting the initial infection or replication of the virus. 
 
In summary, and taking into account the large body of clinical experience garnered from 
the use of systemically administered Artesunate for the treatment of acute malaria, these 
data provide rationale for the use of a topical formulation of Artesunate in the treatment 
of incipient, intraepithelial neoplasia caused by HPV.  
 


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3.0 
CLINICAL SAFETY AND TOLERABILITY 
Commercially available Artesunate has been used for the treatment of acute malaria for 
decades using systemic dosing that is at least 5-fold higher than the suppository dosing 
proposed in this clinical trial. Rectal suppository formulations are approved for use as a 
second-line treatment for acute malarial infections in children, and used off-label in 
adults, who cannot tolerate oral administration and are distant from a hospital setting 
[4]. For severe malaria during pregnancy, there is less certainty about the safety of 
Artesunate during the first trimester, but Artesunate is recommended as first-line 
therapy during the second and third trimesters [43]. Two reports submitted to the FDA 
by the WHO and Novartis summarize many of the safety studies conducted with 
artemisinin and its derivatives, including Artesunate [4, 17, 18, 29].  
 
For young children of less than 5 years of age, rectal Artesunate suppository 
administered at a single dose of 10 mg/kg is the only WHO recommended safe pre-
referral treatment for severe malaria. Rectal suppository dosing has also been tested in 
adults, including regimens of 500 mg/day for 5 consecutive days [44]. No local or 
serious systemic side effects have been reported at these doses. The initial starting 
intravaginal suppository doses proposed, 50 mg (≤1 mg/kg in individuals who weigh 
≥50 kg, a subject inclusion criterion for this study) and the highest proposed dose, 200 
mg (≤4 mg/kg) are approximately 10 and 2.5-fold lower than the approved pediatric 
rectal suppository dose.   
 
3.1 
Clinical Toxicology and Pharmacology 
Clinical effects reported in subjects treated with artemisinin compounds have included 
mild gastrointestinal disturbances, dizziness, and tinnitus, albeit with weak evidence of 
association [45]. There have also been reports of transient reticulocytopenia, 
neutropenia and elevated liver enzyme values, though none has been considered to be of 
clinical significance [45]. Electrocardiographic abnormalities such as bradycardia and 
prolongation of the QT interval, have been reported but again, no impairment of heart 
function has been observed in numerous malarial therapy trials in humans [24, 45] . 
The only potentially serious adverse effects that have been convincingly associated with 
administration of artemisinin compounds are the type 1 hypersensitivity reactions in 
approximately 1 in 3000 patients [46]. In nonclinical studies in mice, CNS effects have 
been observed; however, such effects have not been observed in humans. Because the 
dosage evaluated in mice (100 mg/kg, i.m., artemotil and artemether) is at least 6 fold 
higher than therapeutic doses in humans, these toxicities are likely of little clinical 
relevance (see nonclinical toxicology section 2.4 above).  
 
With regard to strong nonclinical evidence of artemisinin compounds-induced 
embryotoxicity, the tight relationship to a specific period of the gestational cycle has 
informed the contraindication of their usage during the first trimester of pregnancy in 
humans [29, 32] . Importantly, in malaria treatment, there is no evidence for adverse 
effects on the fetus following exposure during the second and third trimesters, when 
these drugs are recommended depending on the safety profile of the partner drug in the 
artemisinin-based combination treatment [45]. This recommendation can be 


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sidestepped in cases of severe malaria where artemisinin derivatives are clearly superior 
in terms of life-saving efficacy [45]. Prospective tracking of over 1000 pregnancies did 
not reveal any significant adverse effects following artemisinins’ use during the 2nd and 
3rd trimesters [47, 48].  
 
Finally, artemisinins are generally well tolerated and human studies with the various 
drugs used in combinatorial therapy against malaria have not revealed major clinical 
concerns regarding untoward drug interactions. However, because such interactions are 
peculiar to the compounds themselves, each potential drug interaction has to be 
considered on its own [18, 49].  
 
In this clinical protocol, intra-vaginal Artesunate suppositories will be tested. Direct 
application to the mucosal surface at the doses we propose is unlikely to result in 
significant systemic absorption. While there are no published pharmacologic data for 
intra-vaginal administration of Artesunate suppositories, clinical pharmacology of 
Artesunate and its metabolite DHA following rectal, IV, IM, and oral administration are 
well documented in the anti-malaria literature [4, 18, 24, 28, 50-52]. Years of 
pharmacokinetic data already performed with Artesunate informed the choice of intra-
vaginal dosing [49-51, 53, 54].  A review of clinical toxicology and pharmacology 
assessments of Artesunate and its metabolite DHA is summarized in Table 1.  
 
Artesunate is rapidly absorbed, with peak plasma levels occurring 1.5 h, 2 h and 0.5 h 
after oral, rectal and intramuscular administration, respectively [3, 55-58].  It is almost 
entirely converted to its active metabolite DHA [59].  Elimination of Artesunate is very 
rapid, and its activity is determined by DHA elimination, which has a half-life of 
approximately 45 min [11, 52].  
 
Table 2: Clinical Toxicity and Pharmacology of Artesunate 
Route of 
Administration 
Dosing Regime 
Toxicity Summary 
Oral 
Administration of 
Artenimol-R 
(DHA) in patients 
with squamous cell 
carcinoma stage 3 
or 4 [60]  
Treatment Phase: 7 
days @ 100mg/day, 
and if no NCI CTCAE 
grade 3 or 4, then 
additional 21 days @   
200 mg/day 
 
Follow Up Phase: Four 
patients with relapse 
received 28 days of 
DHA at 200 mg/day 
Treatment Phase: Zero incidence of 
grade 3 or 4 AEs; five patients with 
grade 1 or 2 AEs described as 
transient ‘flu-like syndrome’, 
headache, and abdominal pain 
Rectal 
Artesunate – data 
from 1167 patients 
in 15 clinical trials 
A single high dose 
of rectal Artesunate 
(10mg/kg) was 5X 
more likely to 
Total incidence of AEs considered 
by clinicians to be possibly drug-
related was approx. between 2.7% 
and 9.0% of all rectally 


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Route of 
Administration 
Dosing Regime 
Toxicity Summary 
of rectal 
artemisinin 
derivative 
(Artesunate, 
artemisinin and 
artemether) 
therapy [61]  
 
achieve >90% 
parasite reductions 
at 24 hours than 
were multiple lower 
doses of rectal 
Artesunate, or a 
single lower dose 
administration of 
rectal artemether. 
artemisinin-treated patients, 
compared with 22% of quinine-
treated patients. Majority of the 
possibly drug-related adverse 
events involved either the 
gastrointestinal system or were 
generalized and non-specific in 
nature and were not severe. 
IV Administration 
of Artesunate in US 
Phase I safety trial 
[51]  
Dose escalation of 
single dose of 0.5, 1, 2, 
4, or 8 mg/kg over 2 
min  
No dose-limiting toxicity  
IV Administration 
of Artesunate in a 
Phase 1b Study 
[50]  
3 days @ 2, 4, or 8 
mg/kg over a 2-min 
infusion 
• No subject drop-outs for 
adverse events or other 
treatment-related issues.  
• Dose-related decrease in 
reticulocyte count noted that 
peaked 4 days after dosing and 
returned to normal by study 
day 7 in most cases, and day 10 
in all patients.  
• No other clinically significant 
laboratory abnormalities 
detected.  
• No deleterious hemodynamic 
or ECG effects  
• A transient, reversible 
sensation of altered or unusual 
taste, lasted 30 minutes in all 
cases at higher doses of 
Artesunate 
IM Administration 
of Artesunate in 
Children with 
Severe Malaria [54]  
IM 2.4 mg/kg at 0, 12, 
and 24 h in children 
from 6 months to 11 
years old 
No direct report of treatment-specific 
complications 
Comprehensive 
review of clinical 
pharmacology 
following 
administration of 
Artesunate by the 
IV Route 
• High initial plasma concentration subsequently declined 
rapidly with a half-life of less than 15 min.  Clearance and 
volume estimates averaged 2 - 3 L/kg/hr and 0.1 - 0.3 
L/kg, respectively.  
• The metabolite DHA concentrations peaked within 25 
minutes post-dose, and a half-life of 30 - 60 minutes. 


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Route of 
Administration 
Dosing Regime 
Toxicity Summary 
IV, IM, oral or 
rectal routes [52]  
DHA clearance and volume averaged between 0.5 - 1.5 
L/kg/hr and 0.5 - 1.0 L/kg, respectively 
 
IM Route 
• Lower peaks, longer half-life values, and higher volumes 
of distribution for Artesunate 
• Delayed peaks for DHA; other parameters were generally 
similar due to the high bioavailability, assessed by 
exposure to DHA, associated with IM Artesunate 
administration (> 86%). 
Oral Route 
• High bioavailability of DHA (> 80%).   
• Artesunate Cmax within 1 hour, and a half-life of 20 - 45 
minutes 
• DHA Cmax within 2 hours and a half-life of 0.5 - 1.5 hours.  
• AUC values reported for Artesunate often substantially 
lower than those reported for DHA following oral 
administration 
Rectal Route 
∗ Similar PK results as oral administration, with the 
exceptions of delayed Cmax and longer half-life 
 
Based on clinical safety studies of Artesunate in the U.S. [62], tests on daily IV boluses 
of up to 8 mg/kg of Artesunate did not result in dose-limiting toxicities [55].  At 8 
mg/kg, a transient, reduced reticulocyte count had been observed, but resolved  without 
intervention over a timeframe of 4-7 days [24].  Drug concentrations did not accumulate 
in subjects receiving multiple daily IV treatments, corroborating with known rapid 
elimination rates for artemisinin derivatives [50].  A significantly lower maximum 
plasma concentration after intra-vaginal administration is expected, compared to IV 
administration. This prediction is based on the pharmacokinetic studies of IM 
Artesunate [52], in which the rate of tissue absorption slows drug distribution; on 
average, one thirtieth and one fifth of Cmax observed by the IV route for Artesunate and 
DHA, respectively. The differences between Artesunate and its principal metabolite are 
due to a combination of differences in solubility, and to rapid conversion of Artesunate 
to DHA [52].  Despite the prediction of low systemic concentration of drug, rapid 
elimination following intra-vaginal administration would be expected, as seen with IM 
administration [52].  
    


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3.2 
Dosing Rationale 
Although no serious adverse events are anticipated with the doses we propose, the first 
cohort of subjects will be a run-in of three patients receiving 50mg suppositories.  If no 
mucosal or systemic adverse events are reported, we will proceed with 200mg dosing.  
 
A five-day dosing regimen with a single dose of 200 mg per 24-hour period is selected 
based on the clinical toxicology and pharmacology database from anti-malarial and 
cancer trials as well as the performance data presented on the inhibition of HPV infected 
cells in vitro and in vivo. We anticipate that intra-vaginal administration of 200 mg 
Artesunate suppositories to the cervix will provide a local drug concentration well above 
IC50 of HPV-infected cells.  Based on our experimental experience in nonclinical studies, 
we plan to assess more than one 5-day treatment cycle to explore potential dosing 
regimens suitable for subsequent phases of clinical studies.   
 
The doses selected for this study are 50 mg and 200 mg. A dose level of 200 mg is 
equivalent to ≤ 4mg/kg based on a minimum body weight of 50 kg as a subject inclusion 
criterion.  The proposed dose level should be extremely safe as it is well within WHO 
recommended pre-referral dose for parenteral Artesunate treatment of severe malaria in 
young children; that is a single dose of 10mg/kg body weight.  The proposed dosing 
regimen also resembles WHO recommended multi-day dosing regimen for adults and 
children with severe malaria upon hospitalization; that is 2.4mg/kg IV or IM TID on day 
1 (or 7.2 mg/kg) and continued daily dosing at 2.4mg/kg for a total of 7 days [43].  Due 
to the rapid rate of elimination in vivo, negligible systemic accumulation of Artesunate 
or DHA metabolite is expected over multi-day dosing, especially in the case of local 
administration. 
 
In summary, local intravaginal administration of Artesunate as a suppository at the 
highest dose in this study, 200 mg or approximately ≤ 4 mg/kg is not expected to cause 
any systemic toxicity. Although no serious adverse events are anticipated with the 
proposed doses, dose escalation will only occur if no mucosal or systemic adverse events 
are reported in the first cohort of three subjects who receive the lowest dose. Subjects 
will be monitored for any unexpected adverse events locally and systemically.   
 
4.0 
OVERALL ASSESSMENT OF RISKS AND POTENTIAL BENEFITS 
4.1 
Potential Risks 
The major risks for the study subjects are associated with surgical excision procedures 
that are the standard of care for patients with CIN2/3.  Other potential risks posed by 
this clinical protocol would include local tissue irritation due to the presence of the 
investigational drug, and venipuncture for peripheral blood collection.   
Intra-vaginal administration of Artesunate suppositories has not been approved for 
general use by the Food and Drug Administration (FDA). An Investigational New Drug 
(IND) application covering the proposed was submitted to the FDA.  Approval to 
conduct this clinical trial was obtained from the FDA before clinical enrollment began.  
The IND governing this protocol is IND 124299.  In parallel, the clinical trial protocol 


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was reviewed and approved by the Johns Hopkins Hospital Institutional Review Board, 
and the Johns Hopkins Cancer Center Clinical Research Committee.    
 
4.2 
Known Potential Benefits 
This study is designed to assess safety, tolerability, and feasibility of using Artesunate 
suppositories in women with CIN2/3. Treated subjects whose CIN lesions regress 
during the study period would avoid the need for surgical LEEP or cone excisional 
biopsy procedure.   
 
In summary, the potential to develop a non-invasive therapy for this disease is worth the 
minimal risk with this study.  
 
5.0 
RATIONALE FOR CLINICAL TRIAL DESIGN 
This trial is a prospective, open, dose-escalation study of topical Artesunate, formulated 
as a suppository, administered intravaginally, prior to standard therapeutic resection of 
the squamocolumnar junction at study week 15. An initial dose cohort of 3 subjects will 
receive intravaginal Artesunate for one 5-day treatment cycle. Based on clinical 
experience with Artesunate, we expect that absorption in the cervical mucosa, which is 
stratified squamous epithelium, will be less than what is observed with rectal 
administration, which is a single layer of glandular epithelium.  Because cervical stroma 
is much less vascularized than colorectal stroma, we also expect significantly lower 
systemic levels of Artesunate in subjects receiving intravaginal dosing. The goal of 
topical administration is to achieve local, mucosal distribution of study drug, as opposed 
to systemic levels.  
 
If we do not observe untoward side effects, including mucosal excoriation, in the first 
dose cohort, we will proceed with dose-escalation. The subsequent cohort will undergo 
two cycles of treatment, spaced at 2-week intervals.  The final cohort will undergo a total 
of three cycles of treatment, spaced at 2-week intervals.  All subjects will undergo an 
interval colposcopic exam at study week 6. At week 15, if there is still a colposcopically 
detectable lesion, subjects will undergo standard therapeutic resection.  If no lesion is 
obvious, a biopsy will be taken at the site of the original lesion. The cohort receiving the 
fewest cycles of Artesunate, and showing therapeutic effect will be expanded (see 
Section 12 for details).  At each vaginal exam, cervical swabs will be obtained and 
cryopreserved.  Peripheral blood will be obtained by venipuncture at baseline, and at 
study weeks 5, 15, and 28, 41, and the postop visit, and will be cryopreserved for 
assessment of biomarkers. 
 
This clinical cohort, healthy subjects with colposcopically-directed, biopsy-confirmed 
CIN2/3, was selected for safety reasons. The study protocol window (15 weeks), is within 
the standard of care.  In pregnant women who have a cervical HSIL, the standard of care 
is to follow colposcopically until postpartum [7]. We and others have reported that the 
probability that CIN2/3 will progress in this timeframe is essentially zero [63]. In fact, 


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we and others have also reported that in this timeframe, approximately 20-25% of 
CIN2/3 lesions associated with HPV16 will undergo regression [63].  Lesions that are 
associated with HPV genotypes other than HPV16 are even more likely to regress in this 
timeframe [64].  Moreover, in this population, it is feasible to visualize directly, 
quantitate serially, and perform therapeutic resection of premalignant lesions.  Based on 
safety and tolerability data in pediatric populations receiving intrarectal suppositories at 
a dose that is approximately five-fold compared to the regimen we propose in this 
clinical protocol, we do not expect dose-limiting toxicities with the dosage and regimen 
proposed in this proof-of-principle study. The Trimble group has a decade of experience 
in the design and execution of prospective clinical protocols specific for this patient 
cohort, testing immunotherapeutic interventions prior to standard therapeutic resection 
of any residual lesions. [65, 66]. The parallel design of this trial will allow comparison of 
tissue-based endpoints in this cohort. 
Despite the experience with active immunotherapy in other tumor models, it is still not 
clear what measures of immune response are relevant to lesion regression.  In 
particular, it does not appear that classic measures of immune response, measured in 
the circulation, correlate at all with clinical response.  Because the biology of HPV 
disease is extensively characterized; because there are known, quantifiable risk factors 
for persistence and progression of disease; because we have ready access to an 
immunocompetent population of patients at elevated risk for carcinoma development; 
and because we can directly visualize and subsequently therapeutically resect and 
analyze affected tissue; we are presented with an opportunity to rapidly evaluate 
therapeutic regimens, characterize relevant immune responses, and provide direction 
for future interventions, both in this disease and in other tumor models.  The relatively 
long precursor phase in HPV disease also provides many opportunities for intervention, 
including, optimally, prophylaxis against the development of cancer. 
A larger goal of these clinical trials is to correlate measures of immune response with 
known clinical parameters of disease. The larger implications of this effort lie in the 
identification of appropriate and relevant measures of the mucosal immune 
microenvironment. The identification of intermediate biomarkers in the tissue not only 
serves to further our understanding of the immune response and our efforts to 
manipulate it, but also has practical implications in the setting of monitoring patients at 
risk for disease recurrence or progression.  In the case of high grade cervical 
intraepithelial neoplasia, lesion size and viral load are both quantifiable risk factors for 
persistent and progressive HPV disease.  Therefore in these trials we will quantitate 
both outcome measures sequentially and correlate them with local tissue and peripheral 
measures of immune response. 
 
6.0 
STUDY OBJECTIVES 
 
This trial is designed to test the hypothesis that intra-vaginal application of Artesunate 
is well-tolerated in women with CIN2/3.   
 


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The primary objective is to evaluate the safety and tolerability of the intra-vaginal 
administration of Artesunate in study subjects.   
 
The secondary objective is to explore clinical activity, such as clearance of HPV disease, 
corresponding to intra-vaginal administration of Artesunate.  
 
7.0 
STUDY OUTCOME MEASURES 
7.1 
Primary Outcome Measures 
Safety will be assessed by the findings of adverse events, including dose related limiting 
AEs. Subjects will be monitored for the appearance of erythema, itching, burning, pain, 
infection, desquamation, tenesmus, bleeding, dizziness, and any unexpected AEs.  
7.2 
Secondary Outcome Measures 
Indicators of drug efficacy (see below) will be drawn from any evidence demonstrating a 
significant difference (by simple chi-square analysis; p<0.1) between treated subjects 
and the baseline data in the Johns Hopkins Colposcopy database 
 
• Extent of lesion clearance (by lesion number, size, and/or area of coverage) 
• Presence/absence of HPV DNA 
• Regression of CIN stage by histopathology (comparison of initial biopsy to 
LEEP/cone excision-procured tissue). 
 
Histologic regression will be judged as none (persistent CIN2/3 in week 15 resection              
specimen) or total (no CIN2/3 in resection specimen).   
 
Virologic HPV viral load.  Longitudinally obtained cervical swabs will be used to 
quantitate HPV viral load as a continuous variable.  
 
Tissue sections from the diagnostic biopsy obtained as part of the screening process, and 
from the resection specimen obtained post-treatment will be assessed for expression of 
CD8 (effector T cells), CD71 (transferrin receptor), and cleaved caspase 3 (apoptotic cell 
death). 
 
8.0 
STUDY DESIGN 
Prior to enrollment, patients referred for evaluation of an HSIL pap smear will undergo 
a full colposcopic evaluation, with biopsy confirmation.  Clinical, histopathologic, 
virologic, and immunologic parameters will be assessed longitudinally in each study 
participant, as outlined below.  The trial is a dose-escalation design, with a run-in cohort 
to assess the likelihood of mucosal irritation. 
These serial assessments will directly parallel those obtained in the currently open 
CIN2/3 cohort study (JHMI JCCI# 99-08-27-04).  Those patients who have measurable 
disease after biopsy will be eligible for study participation. 


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Cervical tissue specimens obtained as routine diagnostic and therapeutic specimens will 
be analyzed to characterize local tissue immune response.  Diagnostic biopsies obtained 
at initial colposcopy will be used to confirm the diagnosis of CIN2/3.  The initial 
diagnostic biopsy specimens will also be characterized in terms of type and quantitation 
of inflammatory cell infiltrates.  Immunocytochemical techniques will be used to 
determine presence and numbers of macrophages, dendritic cells, T cells, B cells, and 
NK cells.  We will also use standard staining techniques to detect cleaved caspase 3. As 
tissue availability permits, the specimens will also be analyzed for other immune cells.  
We will be comparing tissue profiles with those obtained on circulating PBLs in our 
study population.  In the post- suppository administration, therapeutic LEEP/cone 
biopsy specimens, markers of local tissue immune response will be similarly 
characterized and quantitated.  Dr. Trimble will review all of the histologic slides. 
 
9.0 
STUDY POPULATION AND ENROLLMENT 
Patients with high-grade cervical intraepithelial lesions (CIN2/3) will participate in this 
study.  At Johns Hopkins, patients will be recruited from the Johns Hopkins Colposcopy 
Service, and from referring physicians.  Annually, the Colposcopy Service treats over 
800 patients, approximately 15% of whom have high-grade lesions.  In terms of 
feasibility, over the past 12 years we have recruited nearly 500 patients for screening to 
our cohort and vaccine studies, which consists of the same study population as the one 
described for this interventional trial.    Of the HSIL patients approached for study 
entry, 80% have consented for screening.  Of the patients who consented for screening, 
33% were African American.  This percentage is representative of our colposcopy 
population.  It is anticipated that as therapeutic options become available through the 
upcoming Phase I trial, that referral volume will continue to increase. 
 
9.1 
Subject Inclusion Criteria 
∗ Patients with high-grade cervical intraepithelial lesions (CIN2/3) 
confirmed by colposcopy and biopsy. 
∗ Patients who have measurable disease after diagnostic biopsy. 
∗ Patients who weigh ≥50 kg 
∗ Patients who are HPV-positive by DNA test 
∗ Patients who are age 18 or older. 
∗ Patients who are able to give informed consent. 
∗ Patients who are immunocompetent. 
∗ Patients who are not pregnant; committed to using adequate 
contraception through week 15 if of childbearing age. Condom use will be 
encouraged. 
∗ Patients who have the ability to collaborate planned follow-up 
 
9.2 
Subject Exclusion Criteria 
∗ Patients with cytologic evidence of glandular dysplasia  
∗ Patients with cytologic evidence of adenocarcinoma in situ 


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∗ Patients who are taking immunosuppressive medication 
∗ Patients with concurrent malignancy except for nonmelanoma skin lesions 
∗ Patients who have an active autoimmune disease or history of 
autoimmune disease requiring medical treatment with systemic 
immunosuppressants, including: inflammatory bowel disease, systemic 
vasculitis, scleroderma, psoriasis, multiple sclerosis, hemolytic anemic, or 
immune thrombocytopenia, rheumatoid arthritis, SLE, and Sjogren’s 
syndrome, sarcoidosis. Asthma or COPD that does not require systemic 
corticosteroids or routine use of inhaled steroids is acceptable 
 
10.0 STUDY PROCEDURES 
10.1 Screening  
All subjects will receive medical care that is routine to clinical practice.  
 
Drs. Trimble (JHU) and Levinson (GBMC) and the study nurses or coordinators will 
recruit patients at the time they are seen for their initial clinical evaluation, or when they 
return to discuss their colposcopy results.  All eligible patients will be asked if they 
would like to be screened for participation in this clinical trial.  Drs. Trimble and 
Levinson and the study nurses or coordinators will be responsible for recruiting study 
participants and for obtaining written informed consent.  They will allow as much time 
as necessary to review the consent forms with subjects, and to allow them to ask 
questions.  Participants will receive a copy of the consent form at the time of their 
appointment, and consented subjects will undergo the procedures listed in Table 3 
below.  
 
Once patients sign consent, will be assigned a study ID, and their demographic and 
clinical information will be entered in the Cervical Dysplasia Research Program 
database.  This information is also entered in the institutional database CRMS.  To 
maintain confidentiality, the study-specific ID will be used to label all specimens 
collected and banked during the study.  Only the clinical team and program manager 
will have access to PHI.  All other members of the research team will only have access to 
study-specific IDs. 
 
A schema of the data flow is outlined in Figure 1 below.   
 
 
 


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Figure 1: Data Flow Diagram 
 


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Table 3: Outline of Evaluation Procedures 
 
 
-3wks 
Wk 0 
Wk
2 1,2 
Wk 
42 
Wk 
6 
Wk 
15 
Postop ** 
Wk 
28 
Wk 
41 
Informed consent 
X 
 
 
 
 
 
 
 
 
Artesunate dosing 
 
X 
X 
X 
 
 
 
 
 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Cervix check 
 
 
X 
X 
X 
X 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
History of disease 
X 
 
 
 
 
 
 
 
 
Demographic data 
X 
 
 
 
 
 
 
 
 
Diary cards (give to pt) 
 
X 
X 
X 
 
X 
 
 
 
Diary cards (collect from 
pt) 
 
 
X 
X 
 
X 
 
 
 
Adverse events 
 
X 
X 
X 
X 
X 
X 
X 
X 
Inclusion/exclusion 
criteria 
X 
 
 
 
 
 
 
 
 
Colposcopy 
X 
 
 
 
X 
X 
 
X 
(X)* 
Pap smear 
X 
 
 
 
X 
X 
 
X 
X 
HPV genotyping 
X 
 
 
 
X 
X 
 
X 
X 
Cervical swabs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
LEEP/Cone** 
 
 
 
 
 
(x) 
 
(x) 
 
HLA testing *** 
X 
 
 
 
 
 
 
 
 
PBL for banking**** 
X 
(X) 
 
 
X 
X 
X 
X 
X 
Urine HCG 
X 
X 
X 
X 
 
X 
 
 
 
Optional pheresis****** 
 
 
 
 
 
 
 
 
 
 
1. Treatment Group III 
2. Treatment Group IV 
∗       Colposcopy only if clinically-required at wk 41 
** 
if residual disease is present (the procedure can happen any time after week 
15, based on the physician’s clinical judgment). The Postoperative visit will 
take place 4 +/- 1 weeks after the LEEP/Cone procedure. 
*** 
HLA can be done at any time during the study, not an eligibility criterion 
**** 
First blood draw can be done at either the screening visit or Wk 0, prior to 
dosing 
***** pheresis is optional and can happen any time after week 15 
 
10.2 Intervals Between Visits 
The intervals between screening and first administration may be 3 ± 2 weeks, if 
necessary.  A subject is considered enrolled when she receives the first Artesunate 
suppository at the scheduled Week 0 visit.  After the first administration of Artesunate, 
the time window for each subject to complete 4 remaining daily doses of Artesunate 
suppositories may be between 4 days and 2 weeks (for example, to minimize potential 
wash-off of the treatment during a menstrual period).  There will be a window of ± 1 
week at weeks 2, 4, 6, 15, and post-operative visit, and ± 2 weeks at the week 28, and 41 
visits. 


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10.3 Description of Study Visits 
All clinically relevant data collected during the study procedures must be recorded first 
in the patient file.  These data will then be transcribed into the CRF.  All visits will be 
outpatient visits. 
 
At the initial screening visit, in addition to undergoing a standard of care colposcopic 
evaluation, we will obtain cervical brush specimens for HPV typing, and peripheral 
blood specimens to determine HLA phenotype, HIV status, and immunologic studies. 
Subjects with biopsy-confirmed CIN2/3 will be enrolled into treatment cohorts as 
depicted in Table 4 below. 
 
Table 4 – Treatment Cohorts 
Cohort 
Number of patients 
Dose 
Treatment cycles 
I 
3 
50 mg 
1 (5 days) 
II 
9 
200 mg 
1 (5 days) 
III 
9 
200 mg 
2 (5 days each) 
IV 
9 
200 mg 
3 (5 days each) 
 
In cohort I, at the first study visit, the subjects will be given a total of five suppositories 
and applicators.  The first suppository will be inserted in clinic, by the patient.  Patients 
will be given diary cards, and will return 1-2 weeks after completing the regimen, for 
visual assessment of the cervicovaginal mucosa. 
 
In cohorts II-IV, the first study visit will be the same as the first study visit for the run-in 
cohort. Subjects will return to clinic 1-2 weeks later for visual assessment of the 
cervicovaginal mucosa. At study week 2, patients enrolled in cohorts III and IV will 
undergo a visual assessment of their cervicovaginal mucosa and will be given an 
additional 5 suppositories and applicators.  Patients enrolled in cohort III will return to 
clinic 1-2 weeks later for visual assessment of the cervicovaginal mucosa.  At study week 
4, subjects enrolled in cohort IV will undergo a visual assessment of their cervicovaginal 
mucosa, and will also be given five suppositories and applicators. At study week 6, all 
subjects will undergo an interval colposcopic assessment.  If their lesions do not 
appear to be more severe than at study entry, they will return at study week 15. At study 
weeks 15 and 28, all subjects will undergo colposcopic evaluation.  If there is any 
residual colposcopically abnormal tissue, it will be removed using standard excisional 
procedures. Peripheral blood and cervical brush specimens will be obtained at study 
entry, at week 5, at week 15, and at the follow-up visits: postoperative visit, and at weeks  
28 and 41. 
 


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10.4 Examinations, Evaluations, and Procedures 
Vital signs 
Supine blood pressure and heart rate will be measured both prior to each procedure and 
drug administration.  Additional investigation may be added at the discretion of the 
investigator. 
Demographic data/medical history 
The patient’s age, weight, height, and ethnicity will be ascertained at the screening visit.  
Information will be collected on the patient’s past significant medical history including 
tobacco exposure, gynecologic and obstetric history including contraceptive use, total 
and current number of partners, and current medical status.   
 
History of HPV disease 
The following information will be noted at the screening visit: 
• month/year of detection of CIN lesions 
• Details of previous therapies 
 
Colposcopic examination 
Patients will undergo serial colposcopic examinations; at the screening visit, and at 
weeks 6, 15, 28.   Exfoliated cell samples will be obtained using Digene swabs at the time 
of colposcopy, for HPV typing.  Specimens for HPV genotyping will be processed by 
Quest Diagnostics, Nichols Institute.  Specimens for quantitation of HPV viral load 
should be directed to the GYN biorepository, where they will be processed and 
cryopreserved for batch analysis.  Swabs of cervical secretions will be banked in the 
tissue bank.  Cervical lymphocyte samples will be processed directly. 
 
LEEP/Cone Excisional Biopsy Procedure 
After week 15, if there is residual colposcopic disease, the affected area will be excised 
using standard LEEP or cold knife cone technique.  The resection specimens will be 
taken fresh to surgical pathology.  Frozen section will be performed to confirm the 
presence or absence of CIN2/3.  The frozen section block will be banked at –70°C, and a 
sliver of tissue immediately facing the frozen section will be reserved for isolation of 
immune cells. This will allow unhampered routine diagnostic ability as well as preserve 
tissue sections frozen for characterization of inflammatory cell infiltrates.  Routinely 
processed formalin-fixed, paraffin-embedded sections will also be analyzed for the 
characterization and quantitation of tissue infiltrates.   
 
Peripheral blood samples 
Blood will be drawn at various visits as noted in the protocol for analysis of various 
parameters and markers.  The volume of blood will vary depending on the tests to be 
performed.   
 
HLA typing:  one 10 ml yellow top tube  


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Immunology samples:  approximately 50 mls of blood drawn in green top 
(heparinized) tubes to the tissue core.    
These specimens will be separated by standard Ficoll centrifugation, and the serum and 
PBLs aliquoted and banked at –700C. 
 
Histologic response 
We will evaluate pathologic changes in diagnostic biopsy and post-suppository   
therapeutic resection material, on standard hematoxylin and eosin slides. 
 
Urine sample 
A urine pregnancy test for HCG will be performed in patients of childbearing potential 
prior to each suppository administration. 
 
HIV Testing 
HIV testing will be performed using standard JHH procedures including the JHH 
consent forms. 
 
Immune Response 
We will assess peripheral blood specimens for immune responses to HPV antigens, 
using standard ELISA and ELISPOT assays. We will also perform quantitative digital 
image analyses of the tissue specimens obtained at diagnosis, and at week 15.   
 
10.5 Compensation 
Study subjects will receive a total of $200 if they participate in the entire study. A check 
will be mailed to their home within a month after each visit. Study subjects will receive 
$20 for visits at weeks -3, 0, 2, 4, 5, 28, 41 and the postop visit and $40 for the visit at 
week 15. 
 
Upon completion of the postoperative visit, patients will have routine follow-up, 
including examinations at three-month intervals for the first year according to the 
standard of care.  
 
11.0 STUDY DRUG AND ADMINISTRATION 
11.1 
Description of Study Medication 
 
Study Medication: Suppository Formulation of Artesunate 
 


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Drug Substance 
Artesunate is a derivative of artemisinin which is the active principle of the Chinese 
medicinal herb Artemisia annua.  
 
Nomenclature  
 
Artesunate, Artesunic acid 
 
Chemical Name 
[3R, 5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-
trimethyldecahydro-3,12-epoxypyrano[4,3-j]-1,2-
benzodioxepin-10-ol, hydrogen succinate; 
 
CAS Reg. No. 
88495-63-0 
 
General Properties 
Molecular Formula  
C19H28O8 
Molecular Weight   
384.4 g/mol 
Physical Description  
A fine, white crystalline powder 
Solubility  
Very slightly in water; very soluble in dichloromethane; 
freely soluble in ethanol (~ 750 g/l) and acetone 
Melting Point 
 
132-135ºC 
Optical Rotation  
R; [α] D 20 °C = +2.5° to +3.5° in a 10mg/mL in 
dichloromethane 
pH  
 
  
 
3.5-4.5 in an aqueous suspension containing 10 mg/g 
 
Suppository Formulation 
Artesunate suppositories will be produced under controlled environmental conditions as 
prescribed in USP Chapter 795.  Artesunate will be added and dissolved into a molten 
fatty acid base.  The mixture will then be transferred into disposable pre-formed 
suppository molds to cool into solid suppositories.  
 
Clinical trial supplies will be produced by Buderer Drug Co. in Perrysburg, Ohio.  
Buderer maintains compliance with USP Chapter 795 (Pharmaceutical Compounding – 
Nonsterile Preparations) and USP Chapter 797 (Pharmaceutical Compounding – Sterile 
Preparations) which establish practice standards and outline the responsibility of the 
compounder, selection and appropriate sources of ingredients, quality control, and 
considerations regarding the stability of compounded preparations. Two additional USP 
informational chapters are in effect, including, USP Chapter 1075 – Good Compounding 


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Practices, and USP Chapter 1160 – Pharmaceutical Calculations in Prescription 
Compounding. 
 
Packaging and Labeling 
Suppositories for dispensing will be packaged in standard prescription use plastic or 
cardboard containers. Proper labeling will be applied per state regulations. 
 
Product Storage and Stability 
The suppositories may be stored at controlled room temperature.  Suppositories for 
dispensing will be packaged in plastic or cardboard container, and kept in their plastic 
mold until administration.  
USP 795 designates a beyond use date for this preparation not longer than 6 months.  
Potency testing will be obtained to validate the preparation process.   
11.2 Dosage and Administration  
At each dosing visit, after a negative pregnancy test is confirmed, the study physician or 
nurse will provide five daily vaginal suppositories.  During that visit, the subjects will be 
trained in the vaginal insertion of Artesunate suppositories followed by a tampon, and 
instructed to administer all daily doses at home at bedtime.  
 
The first 3 study subjects (healthy volunteers with CIN2/3) will receive five (5) daily 
suppositories that contain 50 mg Artesunate. The 50 mg dose (or ≤ 1mg/kg for subjects 
weighing 50kg at minimum) is considerably lower than that used when the anal 
suppository is intended to deliver a systemic dose of drug for malaria – the 50 mg 
suppository is recommended by WHO for pediatric care.   If no local irritation or 
systemic toxicities observed in the first 3 patients at 50mg, the dosage will escalate to 5 
daily doses of 200 mg per day for the remainder of study enrollments (see treatment 
cohort table).   
 
11.3 Accountability Procedures for the Study Investigational Drugs 
Clinical investigators will issue a prescription for each enrolled patient per USP 1168 
and State and Federal Law.  Buderer Drug Co. will schedule, prepare, package, label and 
ship clinical trial materials to participating facilities.   
The study drug will be kept in the institutional Investigational Drug Pharmacy.  Upon 
receipt of drug orders, and documentation of negative pregnancy test, the pharmacist 
will dispense enough suppositories for a full cycle (5 days).   
 


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11.4 Assessment of Subject Compliance with Self Administration of 
Artesunate Suppositories 
Eligible patients will have clinic visits scheduled at weeks 0, 2, and 4 for a urine 
pregnancy test and dosing per their treatment cohort.  Once a negative pregnancy test is 
obtained, the patient will receive 5 doses of Artesunate for the respective treatment 
cycle.  They will receive instructions on how to apply the vaginal suppositories a tampon 
for medication barrier at bedtime. Patients will be instructed to keep the tampon for the 
remainder of the night. Before the patient leaves the clinic, the study team will confirm 
contact information where they can be reached the following week. Patients will 
document any adverse events, and will undergo a cervix check in clinic when they return 
for the following Artesunate dosing visit.   


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Toxicities and Adverse events 
 
Table 5: Solicited General Signs and Symptoms  
Adverse 
Event 
Short 
Name 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Allergic 
reaction/ 
hyper-
sensitivity 
(including 
drug fever) 
Allergic 
reaction 
Transient 
flushing 
or rash;  
drug fever 
<380C 
(<100.40F
) 
Rash; 
flushing; 
urticaria; 
dyspnea; 
drug fever 
>380C 
(>100.40F) 
Symptomatic 
bronchospasm, 
with or without 
urticaria;  
parenteral 
mediation(s) 
indicated; 
allergy-related 
edema/angioede
ma; hypotension 
Anaphylaxi
s 
Death 
Fever (in 
the absence 
of 
neutropenia
, where 
neutropenia 
is defined 
as ANC 
<1.0 x 
109/L) 
Fever 
38.0-
39.00C 
(100.4-
102.20F) 
 
>39.0-
40.00C 
(102.3-
104.00F) 
 
>40.00C 
(104.00F) <24 
hours 
 
>40.00C 
(104.00F) 
>24 hours  
Death 
 
Cytokine 
release 
syndrome/a
cute 
infusion 
reaction 
Cytokine 
release 
syndrom
e 
Mild 
reaction; 
infusion 
interrupti
on not 
indicated; 
interventi
on not 
indicated 
 
Requires 
therapy or 
infusion 
interruption 
but responds 
promptly to 
symptomatic 
treatment 
(e.g., 
antihistamin
es, NSAIDS, 
narcotics, IV 
fluids); 
prophylactic 
medications 
indicated for 
<24hrs 
 
Prolonged (i.e., 
not rapidly 
responsive to 
symptomatic 
medication 
and/or brief 
interruption of 
infusion); 
recurrence of 
symptoms 
following initial 
improvement; 
hospitalization 
indicated for 
other clinical 
sequelae (e.g. 
renal 
impairment, 
pulmonary 
infiltrates) 
 
Life-
threatening
; pressor or 
ventilatory 
support 
indicated 
 
Death 


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Nervous 
system 
disorder 
Head
aches 
Mild 
pain  
Moderate 
pain, 
limiting 
instumental 
ADL 
Severe pain, 
limiting self 
care, ADL 
_ 
- 
Nervous 
system 
disorder 
Dizzines
s 
Mild 
unsteadin
ess or 
sensation 
of 
movemen
t 
Moderate 
unsteadines
s or 
sensation of 
movement, 
limiting 
instrumenta
l ADL 
Severe 
unsteadiness 
or sensation 
of 
movement; 
limiting self 
care ADL 
_ 
- 
Ear and 
labyrinth 
disorder 
Tinnitus 
Mild 
symptom
sintervent
ion not 
indicated 
Moderate 
symptoms, 
limiting 
onstrument
al ADL 
Severe 
symptoms, 
limiting self 
care ADL 
- 
- 
 
Patients will be monitored throughout the study for both known potential risks and for 
unexpected risks.  During office visits, patients will be monitored specifically for any 
signs of: 
• Symptoms of itching, burning, pain, warmth, or tenesmus 
• Signs of skin breakdown including erythema, blistering, desquamation, 
ulceration or bleeding 
• Neurologic symptoms 
• Unexpected adverse events 
 
Subjects will be given diary cards to record local and general signs and symptoms most 
likely to occur between office visits.  The diary cards will be collected, signs and 
symptoms verified, and inserted into the CRF at each subsequent visit.   
Subjects who become pregnant during study will be required to discontinue the use of 
suppositories.  Subjects removed from the study will be replaced in the treatment group. 
Although not considered an adverse event, pregnancy will be reported in the same way.  
Whenever possible, a pregnancy should be followed to term, and the status of mother 
and child reported.  The Pregnancy Notification form will be sent by sites to the study PI  
(Dr. Trimble) and to the sponsor (Frantz Viral Therapeutics)  within 48 hours of 
discovery.  Whenever possible, the Follow-up Pregnancy Outcome Form will be filled 
after delivery.   
 
 
11.5 
Management of Toxicities 
All local reactions will be considered causally related to administration of the 
suppositories.  Causality of all other adverse events will be assessed individually.  We 
will follow up subjects with non-serious adverse events until symptoms end.  Those with 


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related serious adverse events will be followed until resolved, until no further 
improvement is expected, until a non-study antitumor therapy is initiated, or for 30 
days after last study treatment, whichever comes first (with the exception of neuropathy, 
which must be followed until resolution to ≤ grade 1 or until stable for 60 days).  
 
Outcome will be assessed as 
       1=Recovered 
2=Recovered with sequelae 
3=Ongoing at subject study conclusion 
4=Died 
5=Unknown 
 
Any serious study events that are unexpected and related to the study drug or procedure 
occurring during the period starting from day of administration of the first dose of 
Artesunate suppository to each subject and ending at the last study visit for that subject 
(i.e., scheduled LEEP or cone excisional biopsy procedure), will be reported by the 
principal investigator. 
 
Subjects who become pregnant during study will be required to discontinue drug.  
Subjects removed from the study prior to week 15 will be replaced in the dose cohort.  
Although not considered an adverse event, pregnancy will be reported in the same way.  
Whenever possible, a pregnancy should be followed to term, and the status of mother 
and child reported.  The Pregnancy Notification form will be sent by sites to the study 
sponsor (Dr. Trimble) within 48 hours of discovery.  Whenever possible, the Follow-up 
Pregnancy Outcome Form will be filled after delivery.   
 
Significant local inflammation will be treated symptomatically with cold packs and oral 
analgesics.  Local infection or ulceration will be treated with routine wound care and 
antibiotics if necessary. 
 
Subjects will be given diary cards to record local and general signs and symptoms most 
likely to occur at home.  The diary cards will be collected, signs and symptoms verified, 
and inserted into the CRF at each subsequent visit.  Subjects will be monitored at 
regular intervals to ensure that all adverse events are recognized, documented, and 
treated.  Patients will be educated to contact medical staff should they feel that an 
adverse event has occurred at a time when they are not scheduled for follow-up 
monitoring and will have access to medical staff to follow up on any such concerns. 
 
All unexpected and related SAEs will be reported immediately to Dr. Trimble, who will 
remove any at-risk patient from the study. Any subject who shows evidence of a drug-
related SAE (grade III or IV) will be removed from the study and the SAE will be 
reported to the FDA and the JHMIRB within 7 working days of finding out. 
 
Risks to privacy of individuals, confidentiality of data:  There is a potential risk of loss of 
confidentiality. To ensure that all information and specimens collection remain 


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confidential and anonymous, all study data will be de-identified.  Only the research 
team will have access to study participant private data.  All study specimens are de-
identified and cannot be linked to the subjects. Specimens will be barcoded before 
storage so that patient identifiers will not be available to the laboratories performing the 
assays proposed in this application. 
 
To ensure that all study data remains confidential, all study data will be maintained on a 
password-protected database. Patient identifying information will not be associated 
with the study research data. Only the research investigators will be able to access a list 
connecting patient identifiers with the study ID, and this can only be provided upon 
special request from the study database managers.  
 
The rate at which invasive cancer develops from CIN is slow and can take years and 
perhaps decades [49]. The fifteen-week treatment study window precedes the standard 
LEEP or cone excisional biopsy procedure given to the targeted study population, and 
therefore will not extend the normal time clinicians allow between disease detection and 
therapy and as such, participation in this study will not affect optimal patient treatment. 
  
11.6 Definition of a Serious Adverse Event 
A serious adverse event or reaction is any untoward medical occurrence that at any 
dose: 
• 
results in death 
• 
is life-threatening (an event in which the subject is at risk of death at the 
time of the event;  it does not refer to an event which hypothetically might 
have caused death if it were more serious) 
• 
requires inpatient hospitalization or prolongation of existing 
hospitalization 
• 
results in persistent or significant disability or incapacity 
• 
is a birth defect/congenital anomaly 
 
11.7 
Recording and Follow-up of Adverse Events 
The severity of each adverse event must be assessed using the NCI Common 
Terminology Criteria for Adverse Events, v4.0 (CTCAE 4.0). 
 
All adverse events that occur from the time of study drug administration through 30 
days after the final treatment will be recorded. Duration, severity, treatments 
administered and outcome for each adverse event will be recorded on the CRF for the 
following visit. Drug related adverse events will be followed until resolved, until no 
further improvement is expected, until a non-study antitumor therapy is initiated, or for 
30 days after last study treatment, whichever comes first (with the exception of 
neuropathy, which must be followed until resolution to ≤ grade 1 or until stable for 60 
days).  
 


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Adverse events beginning 30 days after the last treatment that the investigator considers 
to be related to study treatment will be reported to the appropriate contact person at any 
time such events occur. 
Conditions that were present at the study start and that worsen   during the study 
should be reported as beginning on the date the event worsened, not the date it began 
pre-study. Event text may include the word “worsened” or “exacerbated.” 
Conditions which were recorded as “intermittent” at the study start which occur during 
the study must be reported if they are more frequent or of greater severity. 
 
AE Reporting 
Expected AEs will be reported to the institutional IRB and reviewed at the time of the 
annual review. Any SAEs or multiple occurrences of an AE determined to be an 
unanticipated problem will be reported to Johns Hopkins Institutional Review Board, 
the Johns Hopkins Cancer Center Clinical Research Committee, and the Johns Hopkins 
Institutional Biosafety Committee. SAE’s will be reported to all regulatory agencies 
within 7 working days. Unanticipated or unexpected AE is defined as any AE not listed 
in available sources including the labeling, the Investigator’s Brochure, or this protocol. 
 
11.8 Definition of Dose-Limiting Toxicity 
A dose-limiting toxicity is defined as any grade 3 toxicity in any organ system, or grade 2 
or higher allergic reaction/hypersensitivity reaction (allergy/immunology), as 
delineated in CTCv4.0. 
 
11.9 Early stopping rules 
Because this is a pilot study evaluating tolerability of a new intra-vaginal treatment, the 
stopping rule for safety is the presence of Grade 3 toxicity in any organ system, or grade 
2 or higher allergic/hypersensitivity reaction as delineated in CTCAEv4.0.  In the event 
of two identical unexpected related treatment-related Grade 3 toxicities, accrual will be 
suspended pending further review. 
 
Subjects who become pregnant during study will be required to discontinue drug.  
Although not considered an adverse event, pregnancy will be reported in the same way.  
Whenever possible, a pregnancy should be followed to term, and the status of mother 
and child reported.  The Pregnancy Notification form will be sent by sites to the study 
sponsor (Dr. Trimble) within 48 hours of discovery.  Whenever possible, the Follow-up 
Pregnancy Outcome Form will be filled after delivery.   
 
Finally, if there is evidence of disease progression at the interval visit regardless whether 
the progression may or may not be associated with Artesunate, patients will undergo 
resection at that point.   
 


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12.0 STATISTICAL CONSIDERATIONS   
 
12.1  Study Design/Endpoints/Accrual 
The primary statistical outcome of the trial is safety. The treatment groups will be 
filled sequentially in a non-blinded manner. Study dropouts will be replaced. 
The dose escalation algorithm is described as follows (see Table 4 for dose levels to 
be tested): 
1. A run-in cohort of three patients will be treated with one 5-day cycle of 
50mg vaginal suppositories.  If no patient has a DLT observed by the cervix 
check visit after the last suppository, the trial will proceed to the next level. 
2. A cohort of 9 patients will be enrolled and treated with one 5-day cycle of 
200mg vaginal suppositories. If no patient has a DLT observed by the cervix 
check after the last suppository, the dose level will be considered tolerable 
and the trial will proceed to the next level.  
3. If exactly one of the 9 patients has a DLT by Week 1-2 after the last 
suppository, three more patients will be enrolled and treated at the current 
dose level. If none of the three patients has a DLT by the cervix check after 
the last suppository, the current dose level will be considered safe and dose 
will be escalated.  
4. If two or more of the 9 or 12 patients have a DLT observed by Week 1-2 
cervix check, dose escalation will be stopped. The current dose level will be 
considered unsafe. 
 
5.  A cohort of 9 patients will be enrolled and treated with 2 cycles of 200 mg 
vaginal suppositories, at 2-week intervals.  If no patient has a DLT observed in 
Cohorts II and III, then the dose level will be considered safe, and the dose will be 
escalated. 
 
6.  A cohort of 9 patients will be enrolled and treated with 3 cycles of 200 mg 
vaginal suppositories, at 2-week intervals. If no patient has a DLT observed by 
the cervix check after the last suppository, then the dose level will be considered 
safe.  
With the dose escalation algorithm, there will be less than 9% probability to 
escalate to the next higher dose level if the DLT rate for the current dose 
level is 30%. 


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The maximum sample size for dose escalation is 39. Based on our experience in 
investigator-initiated therapeutic trials in this patient population at this 
institution, our anticipated accrual rate is 20-25 patients per year.   
At the end of dose escalation phase, one dose will be selected for the expansion 
phase from dose levels II-IV that are considered safe. In general, the lowest dose 
level with significant within subject decreases in viral load (from baseline at Week 
15) and lesion clearance to CIN1 or less will be selected (see Section 12.2 for 
details). If none of the safe doses achieve significance for decrease in viral load and 
lesion clearance, a decision will be made based on the totality of safety and efficacy 
profiles of the dose levels considered safe.  
A cohort of 12 patients will be enrolled and treated at the selected dose level at the 
expansion phase to further evaluate the safety and efficacy of Artesunate. 
 
12.2  Secondary Endpoints 
Secondary endpoints include change in histology (CIN2/3 or no CIN2/3) at week 
15 and changes in cervical HPV viral load.  These endpoints are chosen because in 
other trials they have been shown to be quantifiable and reproducible.  They do 
reflect the biology of HPV infection and cervical intraepithelial disease, and 
furthermore have been shown to be modulated in response to intervention in other 
trials.[67]  The histological outcome will be determined by comparing the initial 
diagnostic biopsy used for protocol entry with the exit cone resection.  HPV viral 
load will be ascertained from longitudinally obtained cervical cell samples, using 
quantitative PCR.[68]  This data will be used to estimate the regression rate within 
the fifteen-week window in the exploratory analysis. 
Secondary endpoints will also include change in lesion size by colposcopy, and 
characterization of peripheral and local tissue response to the suppository.  These 
measures will be correlated with clinical response, and with preclinical 
experimental data in the exploratory analysis.  In every case possible, changes in 
lesion size by colposcopy will be ascertained by analyzing serially obtained digital 
colposcopic images.  Measures of immune response will be ascertained on serially 
obtained peripheral blood specimens, and on flash frozen tissue from the 
therapeutic resection specimens. 
 
To test that HPV viral load is different during and after treatment as compared to 
before treatment, paired t-tests and signed rank tests will be used.  Log 
transformation (or other appropriate transform) will be performed to symmetrize 
the distributions and achieve normality.  For each of these variables, a paired t-test 
will be performed.  Due to the small sample size and potential non-normality of 
the data, the non-parametric signed rank test will also be used.  The paired t-test 
tests whether or not the mean HPV viral loads at baseline and follow-up are 


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equivalent, whereas the Wilcoxon matched-pairs signed-ranks test tests whether 
the distributions of HPV viral loads is the same at baseline and follow-up.   
 
12.3  Other Efficacy Endpoints 
In every case possible, change in lesion size from week 0 to week 15 will be 
measured using serial digital colpographs.  Changes in HPV16 E7-specific T-cell 
response (CD8) will be compared at week 0 to week 15.   Differences will be 
calculated between baseline and follow-up and the appropriate transform 
performed.  Point estimates, confidence intervals, and hypothesis testing (i.e. 
paired t-test and signed rank test) will be done.  
 
12.4  Additional exploratory analysis 
In addition to hypothesis testing, we will characterize the change in HPV viral load 
over the course of the trial using longitudinal data modeling.  For each individual, 
we will have data on HPV viral load at weeks -2 (Screening visit), 9-10, and 15.   A 
random effects model will be used to model the distribution of HPV viral load over 
time: 
hpv
t
t
it
i
it
=
+
+
+
β
β
β
ε
0
1
2
2
 
where hpvit is individual i’s HPV viral load at week t.  Correlations between 
observations within an individual are accounted for by the random intercept (i.e. 
random effect) term.  A quadratic model is used to account for non-linearity in the 
change of HPV viral load.  From the fitted model, we can estimate the effect of the 
vaccine treatment on viral load.  The plot below is based on simulated data and 
shows the type of result we hypothesize:  a downward trend in HPV viral load that 
flattens as time increases.  Based on the random effects model shown above, we 
can estimate the average response (denoted by the thick line) and better 
understand the association between the vaccine and response and specifically 
when changes in viral load tend to occur.  
 
 
HPV viral load
Week on Study


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Lesion size will also be described using the random effects linear longitudinal 
model described above to look for time trends. 
The secondary efficacy endpoints will be compared with analogous data obtained 
in a currently open cohort study, “Evaluation of Tissue and Peripheral Blood 
Immune Response in Women with HPV16+ Cervical Intraepithelial Lesions” 
(Johns Hopkins IRB# NA_00037578).  This observational cohort protocol has 
been open since 1999, and currently includes over 250 subjects.  These subjects 
have the same eligibility criteria as the criteria for this trial, and are followed 
prospectively for the same timeframe, before undergoing standard therapeutic 
resection at week 15.  To date, no CIN2/3 lesions have progressed to invasive 
disease in this timeframe, which is well within the standard of care. We will 
compare changes in HPV viral load and lesion size from baseline at Week 15, rates 
of histologic regression, and change in viral load over the 15 week window, in both 
cohorts.  
 
We will correlate immune response with clinical response by looking at the 
association with disease regression.  A logistic regression analysis will be 
performed with regression as the outcome and baseline and change between 
baseline and follow-up HPV viral load as predictors, where HPV viral load and the 
change have been transformed for normality.  Using these two predictors, we can 
assess how change in viral load is associated with clinical response, adjusting for 
baseline level.   Other predictors of interest will also be evaluated, including 
measures of E7-specific immune response.  To explore the association between 
continuous outcomes of interest and time, the random effects model described 
above will be used. 
The frequency and severity of all toxicities will be tabulated from CRFs and 
summarized for review in a descriptive fashion.  The initial review of toxicity will 
be completed after the first treatment group has completed the intervention 
portion of the study (through week 5).   
 
13.0 SUBJECT COMPLETION/DROPOUT 
13.1 Definition of a Dropout 
A dropout is defined as any subject who did not come back for at least week 15 visit 
foreseen in the protocol.  A subject who returns for the concluding visit foreseen in the 
protocol is considered to have completed the study participation. Dropouts will be 
replaced to reach the target enrollment. 
 
13.2 Procedures for Handling Dropouts 
The investigator will make a written attempt to contact those subjects who do not return 
for scheduled visits or follow-up.  Information gathered should be described on the 
Study Conclusion page of the CRF. 
 


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13.3 Reasons for Dropout 
It should be specified on the Study Conclusion page of the case report form which of the 
following possible reasons were responsible for dropout of the subject from the study: 
• serious adverse event 
• non-serious adverse event 
• Protocol violation (specify) 
• Consent withdrawal, not due to an adverse event 
• Migration from the study area 
• Lost to follow-up 
• Other (specify) 
 
14.0 CLINICAL MONITORING 
This trial will be monitored by the Sidney Kimmel Cancer Center data managers. Data is 
also reviewed on a bi-weekly basis within the Division of Immunology Clinical Trials 
Working Group meetings. The principal investigator, study nurse, and data manager 
participate in these weekly meetings. The JHU and GBMC teams will have weekly 
conference calls to discuss study progress, and coordinate specimen collection and study 
activities. The PI and Co-investigator will meet at least monthly.  Johns Hopkins 
Comprehensive Cancer Center (SKCCC) monitoring will occur on a regular basis with 
the frequency dependent on the subject accrual and the rate of study’s progress. This is a 
DSMP Level II study under the SKCCC Data Safety Monitoring Plan (12/6/12).  Data 
Monitoring of this protocol will occur on a regular basis with the frequency dependent 
on the rate of subject accrual and the progress of the study. The protocol will be 
monitored internally at SKCCC by the Principal Investigator and externally by the 
SKCCC CRO in accordance with SKCCC guidelines. Trial monitoring and reporting will 
be done through the Safety Monitoring Committee (SMC) at SKCCC.   (See Appendix A) 
 
15.0 ETHICS AND REGULATORY CONSIDERATIONS 
15.1 
Institutional Review Board 
This protocol and an eIRB application and any other required documents will be 
submitted to the Johns Hopkins IRB and CRO, and their written unconditional approval 
will be in the possession of the investigator before commencement of the study protocol.  
No deviations from, or changes to, the protocol will be initiated without prior written 
IRB approval.  The IRB/CRO will be informed of all protocol violations, informed 
consent changes, or revisions of other documents originally submitted for review.  The 
IRB/CRO will also be informed of serious adverse events occurring during the study, all 
subsequent protocol modifications, or new information that may affect adversely the 
safety of the subjects or the conduct of the study.  Annual updates will be submitted. 
 
15.2 Informed Consent 
Information should be given in both oral and written form whenever possible.  Subjects 
will be given ample opportunity to ask about details of the study.  Subjects will be 


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informed about the expected risks and benefits, and procedures of the study protocol.  
They will be informed of alternative procedures.  The informed consent form will be 
approved by the IRB/CRO.  Written consent will be obtained before screening for study 
enrollment. 
 
 
 
 


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