A Phase II Single-arm Intervention Trial of Nelfinavir in Patients With Grade 2/3 or 3 Cervical Intraepithelial Neoplasia

2.0 BACKGROUND AND RATIONALE 2.1 Cervical Cancer Etiology and Targets for Intervention
Cervical cancer is one of the most common causes of cancer-related deaths in women
worldwide. Implementation of widespread screening has created drastic improvements in the
incidence of cervical cancer as well as significant decreases in mortality over the last 30
years. It is estimated that in 2011, there will be 12,710 women in the US diagnosed with
cervical cancer and ultimately 4,290 women will die from the disease. Cervical cancer is
unique in that there exists a well-defined pre-malignant phase, referred to as cervical
dysplasia, which can be suspected on cytological examination (pap smear) of exfoliated
cervical cells and confirmed on histological examination of cervical biopsy material. The
pre-cancerous changes represent a spectrum of histological abnormalities or cervical
intraepithelial neoplasia (CIN) ranging from CIN 1 (mild dysplasia) to CIN2 (moderate
dysplasia) to CIN3 (severe dysplasia/ carcinoma in situ). The timeline of progression for
CIN 3 to invasive cancer in untreated women averages between 8.1 and 12.6 years.The surgical
treatment of cervical pre-cancerous is therapeutically efficacious. However, it is an
invasive procedure which has been associated with fertility and obstetrical complications in
reproductive aged women. An attractive alternative to an excisional or ablative procedure
would be to utilize an agent which could be applied topically or taken by mouth to foster
regression of cervical dysplasia. The use of natural, synthetic, or biologic chemical agents
to reverse, suppress, or prevent carcinogenic progression is called chemoprevention.

A prerequisite for cervical cancer is persistent infection by a high-risk human
papillomavirus allowing viral gene products sufficient time with which to induce
carcinogenesis through a generally well understood natural history. Cancer-associated HPV
types encode three oncogenes, E5, E6 and E7, and the E6 and E7 proteins have a significant
role in malignant transformation. E6 and E7 stimulate cell proliferation by interfering with
the function of regulatory proteins in cells, including the tumor suppressors p53 and pRB.
Co-expression of both E6 and E7 leads to indefinite proliferation and immortalization of
keratinocytes and induces premalignant neoplasms. HPV-E5, a transmembrane protein, can
activate PI3K-Akt signalling. The up-regulation of PI3K-Akt survival signaling protects
HPV-16 E5-expressing cells from apoptosis induced by ultraviolet irradiation. The major
transforming capability of HPV is dependent on E6 and E7. However, E5 is necessary for full
activation of E7, and the induction of PI3K-Akt-dependent apoptotic inhibition by E5
contributes to E7-mediated oncogenesis.

The work done to evaluate PI3K activity as a potential marker of CIN progression further
supports the idea that this pathway is also up-regulated in pre-cancerous lesions. The
catalytic subunit alpha of PI3K is expressed in non-invasive cervical lesions and has shown
potential as a carcinogenesis-related marker for early intraepithelial lesion of the uterine
cervix in cytology samples. Among other potential markers, PIK3CA showed a superior
specificity to distinguish CIN3 from other groups. Yao et al 2008 showed that positive rates
of PI3K and Akt were significantly lower in normal cervical epithelium and cervical
intraepithelial neoplasia than in cervical carcinoma (0.0% and 42.9% vs. 69.7%, P<0.01;
10.0% and 52.4% vs. 75.0%,P<0.01). Specifically, PI3K/Akt activation increased with grade of
dysplasia with invasive carcinoma having the highest activity. They also showed that
expression of PI3K protein positively correlated to the expression of Akt protein(r=0.425,
P<0.01).

2.2 Nelfinavir (NFV) Background Nelfinavir (NFV) induces cell cycle arrest, endoplasmic
reticulum stress, autophagy, and apoptosis is cancer cells. As a protease inhibitor used in
patients with HIV, NFV predominately targets proteases 1 and 2, but it is also known that
part of the efficacy is due to selective inhibition of the proteosome. The pathogenecity of
high risk HPV is dependent on expression of viral E6 proteins which inappropriately activate
the 26S proteosome to degrade p53 and other cellular proteins that are detrimental to viral
replication; specific HIV protease inhibitors can block the ability of HPV16 E6 to degrade
p53 and selectively kill E6-dependent cervical carcinoma cells. Furthermore, HIV protease
inhibitors have been shown to inhibit the PI3K/Akt signaling pathway with NFV being the most
potent. NFV's mechanism of action involves decreasing Akt phosphorylation. In addition, at
concentrations (micromolar) achievable in the serum of patients taking usual dosing of the
drug (1250mg PO BID), Akt phosphorylation is inhibited. At our institution, we have shown
that HPV-infected cervical cancer cells lines treated with NFV underwent apoptosis and
decreased cell proliferation in a dose dependent manner. Furthermore, we have shown through
a recent retrospective study that HIV-infected women with cervical dysplasia who were
treated with protease inhibitors (PI), including NFV were more likely to regress than those
not on a PI-containing regimen.

Because PI3K/Akt is up-regulated in CIN2/3 and NFV has been shown to inhibit this pathway in
HPV-infected cervical cancer cell lines, we aim to further evaluate the effectiveness of NFV
as an intervention for CIN2/3 and correlate these findings with phospho-Akt levels in
cervical tissue. Several intervention trials have been conducted in women with CIN using
retinoids, ß-carotene, difluoromethylornithine, and indole-3- carbinol and over 300 women
with CIN 3 were followed in these trials for one to 15 months while none of them developed
invasive cervical cancer. Therefore, this proposed intervention trial of 24 weeks (6 months)
duration should allow a safe evaluation of NFV as a potential agent for a future randomized
Phase III trial.

Development of a new drug for cancer treatment is expensive and time consuming.
Repositioning of a drug which is already FDA approved for another indication allows for more
rapid evaluation and potential implementation of a novel cancer therapy. NFV has been
identified to have anti-cancer effects in vitro, and compared to other protease inhibitors
it is more potent in causing cell death. NFV has over 10 years of safety and pharmacokinetic
data in HIV-infected individuals. Standard dosing for the treatment of HIV is 1,250 mg PO
BID, and this dose has demonstrated plasma concentrations in vivo which correlate with
necessary concentrations of successful in vitro studies. Furthermore, evaluation of the
standard dose offers the availability of commercially produced tablets, the known and
acceptable side effect profile, and the ease of twice daily dosing. For these reasons NFV is
an excellent candidate for repositioning as an intervention for cervical dysplasia, which
can be quickly evaluated in this phase II clinical trial.

Inclusion Criteria:

- Patients must have a Cytology report with a High Grade Squamous Intra Epithelial
Lesion "HSIL" or have histologically proven CIN 2/3 or CIN 3 diagnosed by cervical
biopsy between 2 and 8 weeks prior to enrollment. For a patient to be eligible, the
Cytology report must state High Grade Squamous Intra Epithelial Lesion "HSIL" or the
pathology report must clearly state "CIN 2/3" or "CIN 3" or must state
"moderate-severe dysplasia", "moderate severe dyskaryosis," "severe dysplasia," or
"severe dyskaryosis." Patients with a diagnosis of CIN 2 alone or moderate dysplasia
or dyskaryosis alone are not eligible for this study.

- Patients must be at least 18 years of age.

- Patients must have a satisfactory (readable, good quality) colposcopic evaluation at
least 14 days after diagnostic biopsy.

- Patients must have colposcopically visible cervical lesion at entry consistent with
biopsy.

- Patients must have a negative urine pregnancy test within 14 days of starting the
NFV. Women of childbearing potential must practice an acceptable form of
contraception (e.g. intrauterine device, contraceptive pills, diaphragm, condoms).

- Patients must have a GOG Performance Status of 0, 1, or 2.

- Patients must be good candidates for delayed treatment of their CIN, i.e. they must
be reliable to return for follow-up and provide a combination of at least three phone
numbers or addresses for contact.

- Patients must have adequate*:

- CBC/Platelets: Hemoglobin (HgB) greater than 10.0g/dl; white blood cell (WBC) count
greater than 3000/mcl; Platelet count greater than 125,000/mcl.

- Renal function: Creatinine less than or equal to 1.5 x Upper Limit Normal (ULN).

- Hepatic function: Total bilirubin less than or equal to 1.5 x ULN excluding Gilbert's
disease; aspartate aminotransferase (AST) and alanine aminotransferase (ALT) < 2.0 x
ULN.

- Glycemic control: fasting glucose < 126; random glucose <200; hemoglobin A1C ≤ 7.0%

*These lab results must be evaluated prior to patient enrollment.

- Patients must have signed an approved informed consent and authorization permitting
release of personal health information.

- Patients must have a negative HIV test within 14 days of starting the NFV.

- Patients with allergies or known sensitivity to Nelfinavir will be excluded or
withdrawn after treatment, if deemed a safety concern by the principal Investigator.

Exclusion Criteria:

Ineligible Patients

- Patients who are pregnant or breast-feeding.

- Patients with cytologic or biopsy evidence of endocervical dysplasia or invasive
cancer.

- Patients with undiagnosed abnormal vaginal bleeding.

- Patients with a known immunocompromised condition or a positive HIV test. Patients
with a prior history of cervical cancer.

- Patients with a chronic or acute renal, or hepatic disorder, a significant bleeding
disorder, or any other condition which in the Investigator's opinion might preclude
study participation for the duration of the trial.

- Patients taking concurrent medication that is metabolized by the CYP3A4 isoenzyme.

- Patients taking the following concurrent medications: astemizole, cisapride,
salmeterol, alfuzosin, terfinadine, amiodarone, midazolam, quinadine, ergot
derivatives, pimozide, rifampin, triazolam, warfarin, azithromycin, carbamezpine,
cyclosporine, didanosine, fluticasone propionate, phenobarbital, phenytoin,
trazadone, sirolimus, tacrolimus , and St. John's wart.

- Patients who are unwilling, or unable, to practice an acceptable form of
contraception (e.g. intrauterine device, contraceptive pills, diaphragm, condoms).
For those women who choose to use oral contraceptive pills, they will be encouraged
to use a second form of contraception, such as condoms, because of the potential for
altered serum levels of oral contraceptives.

- Patients with uncontrolled diabetes; as defined by hemoglobin A1C ≥ 7.1%