Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus(EBV) related malignancy and is an endemic disease in Southeast Asian countries. EBV had been identified as a therapeutic target in some EBV related cancer such as lymphoma and NPC. In cancer cell, EBV was in latent phase and expressed 8-11 genes for maintaining EBV proliferation. After switching to lytic phase, almost all the EBV encoding genes were expressed including thymidine kinase (TK) and some highly immunogenetic genes. These latent-lytic phase swifter included DNA methyltransferase inhibitors, various histone deacetylase (HDAC) inhibitors, radiotherapy and chemotherapy. Recently, combined chemotherapy and viral lytic therapy, cytolytic viral activation therapy (CVAT) had been shown some promising result in pilot study of NPC. In our patient derived xenograft (PDX) animal model drug sensitivity screening, gemcitabine (GEM) was shown to be the most effective drug. Furthermore, CVAT with GEM + Valproic acid (VPA) + ganciclovir (GCV) maintaining chemotherapy may benefit but reduce chemotherapy related side effect and prolonging treatment response duration. The following phase I clinical trial will be proposed to test the optimal combination of these drugs.

1. Number of patients: total 18 patients are needed
2. Inclusion criteria:(1) used as 2nd line regimen in recurrence/metastasis NPC patients with tissue proved of World Health Organization (WHO) type II or type III.(2) Performance status: eastern cooperative oncology group performance status (ECOG PS) ≤2.
3. Chemotherapy regimen: Gemcitabine (GEM, TTY) + Valproic acid (VPA, generic medicine) for viral activation + Valganciclovir (VGC, Roche) for antiviral medication
4. This treatment cycle of 28 days was repeated maximum 6 times. (Q4wks/cycle, max: 6 cycles)
5. Dosage:

(1) GEM: 600, 800, 1000, 1250 mg/m^2, D1 & D8, intravenously. (2) VPA 12.5 mg/kg/day D1~14, per os. (3) VGC (2-3) x 450 mg/day D9~15, per os. 6. Objectives:

1. primary: to find the best combination of these 3 drugs in recurrent/metastatic NPC patients.
2. second: to evaluate the response and disease control rate in this pilot study.

Key words: NPC, cytolytic viral activation therapy, gemcitabine, valproic acid, ganciclovir.

1. Introduction to investigational treatment(s) and other study treatment(s)

   1.1 Overview of gemcitabine Gemcitabine (29,29-difluoro 29-deoxycytidine, dFdC) which developed from cytosine arabinoside (Ara-C) is an nucleoside analog used as chemotherapy. It was intended as an antiviral drug in initial, but preclinical testing showed that it killed leukemia cells and a spectrum of solid tumor in vitro. In cell, gemcitabine undergoes complex intracellular conversion to the active forms of nucleotides gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) that will influence DNA synthesis. dFdCTP competes with deoxycytidine triphosphate (dCTP) and dFdCDP is a potent inhibitor of ribonucleoside reductase (1). Gemcitabine is administered by the intravenous route with dose ranges from 0.6-1.2 g/m^2 of body surface area (2), and has been used in a broad spectrum cancer management including lung, breast, pancrease, and bladder (3).

   The toxic profile of gemcitabine was major in hematopoietic system especially neutropenia and thrombocytopenia (4). In NPC treatment, gemcitabine had been the focus of several reports, with interesting OR rates in the range of 23-48% and median progression-free survival (PFS) of between 3.6 and 5.1 months (5). In combination chemotherapy with cisplatin, the OR rate 42.7 to 73% had been reported and median PFS were 5.6 to 10.6 months (6). In combined with oxaliplatin, the OR rate was 56.1 % and median PFS was 9 months (6). In combined with vinorelbine, OR rate 36 to 37.7% had been reported and median PFS were 5.2 to 5.6 months. In multiple drugs combination with carboplatin, paclitaxel, 5-Fluoro-Uracil/LeucoVorin, high OR rate with 86% had been reported but with median PFS of 8 months (6). Still these trials were done in small series and lacking randomized large scale phase III trial.

   1.2 Valproic acid (VPA) Valproic acid (VPA), a branched short-chain fatty acid, is widely used in clinical as an antiepileptic drug and a mood stabilizer, primarily in the treatment of epilepsy, bipolar disorder, and prevention of migraine headaches (7). The antiepileptic properties of VPA have been attributed to inhibition of Gamma Amino Butyrate (GABA) trans aminobutyrate and of ion channels.

   VPA was recently classified among the Histone Deacetylase (HDAC) inhibitors, acting directly at the level of gene transcription by inhibiting histone deacetylation and making transcription sites more accessible. Chromatin is formed of DNA packaged in nucleosome structures. The condensed form of chromatin (heterochromatin) is inactive in terms of transcription whereas the decondensed form (euchromatin) corresponds to an active form. The histone acetylation leads to relaxation of the nucleosome structure, releasing the DNA and allowing transcription. Inhibition of histone deacetylases (HDACs) promotes decondensed chromatin formation, thereby promoting the expression of genes (7).

   Valproic acid (VPA), as a HDAC inhibitor, can specifically target at class I a, I b, and II a HDACs (8). VPA also down regulates expression of proteins essential for chromatin maintenance: Structural Maintenance of chromatin (SMC), DNA methyl transferase-1 (DNMT1), and heterochromatin protein-1 (HP1) (9). VPA had been shown to induce histone 3 methylation which would increase transcriptional activity (8). VPA had been shown some anticancer effect, major through its HDAC inhibitor, in single agent or combined with other anticancer medication including myeloid and lymphoid malignancies, breast cancer, prostate cancer, and NPC (10-13) Long-term VPA treatment may cause central nervous system (CNS) dysfunction, liver toxicity, and coagulopathy including thrombocytopenia and platelet dysfunction (7).

   1.3 Ganciclovir (GCV) and Valganciclovir (VGC) GCV was an antiviral agent had been used in treatment or prophylaxis of cytomegalovirus infection in solid organ transplantation recipients or bone marrow transplantation (14). GCV is a synthetic analogue of 2'-deoxy-guanosine and can be phosphorylated to ganciclovir triphosphate, a competitive inhibitor of deoxyguanosine triphosphate (dGTP) incorporation into DNA and preferentially inhibits viral DNA polymerases more than cellular DNA polymerases, by viral and cell kinase. In addition, ganciclovir triphosphate serves as a poor substrate for chain elongation, thereby disrupting viral DNA synthesis by a second route (15).

   VGC, a valyl ester prodrug of GCV, had a high oral bioavailability of about 60% with similar efficacy of GCV in management of cytomegalovirus infection (16). The most common side effect of IV GCV is fever and leukopenia (16).

   1.4 Combination of GEM, VPA and GCV in NPC treatment In EBV-related malignancy, antiviral drugs exhibit no direct effect on cancer cell except when used combined with epigenetically active agents (17, 18). Recently, Wildeman et al, had shown some efficacy by combining chemotherapy of GEM with VPA and GCV in control of locally advanced/metastatic NPC patients (13). In this article, both GEM and VPA could shift EBV from latent phase into lytic phase and had synergetic effect when combined used. Further adding GCV in this regimen could suppress virion formation. The combination therapy had been tested in three locally advanced/metastatic NPC patients showing promising results with tumor regressing/stable in image and plasma EBV DNA load monitoring with few side effects Similar manageable side effects of these three drugs combined treatment were also proved by Stoker et al (19). These results encouraged us to develop a more practical regimen in this trial.

2. Test products, dosage, and mode of administration:

2.1. Chemotherapy regimen: Gemcitabine (Gemmis injection，200 mg) (GEM, TTY) + Valproic acid (Depakine gastro-resistant tablet，200 mg) (VPA, Sanofi) for viral activation + Valganciclovir (Valcyte film-coated tablets，450 mg) (VGC, Roche) for antiviral medication

2.2. Dosage GEM: 600~1250 mg/m^2, D1 & D8, intravenously. VPA: 12.5 mg/kg/day D1~14, per os. VGC: (2~3) x 450mg/day D9~15, per os.

1. This treatment cycle of 28 days will be repeated maximum 6 times. (Q4wks/cycle, max: 6 cycles)
2. Four dosage of GEM combined with fixed dosage of VPA and VGC will be tested
3. The rationale of seven days treatment duration of VGC come from (A) 7 days treatment duration of valacyclovir in herpes zoster in immunocompetent patients (20) (B) overlap side effect of myelosuppression between GEM and VGC(13).
4. Efficient dose intensity chemotherapy (gemcitabine) is essential in this three combined drugs regimen (21) and this trial will be started with dose level 0 (Gemcitabine 800 mg/m^2)

2.3. Concomitant treatment

2.3.1 Permitted: The related treatment for relieve symptoms caused from tumor.

2.3.2 Prohibited:

1. Radiation therapy, operation, and other chemotherapy for eradicating tumor
2. Valganciclovir concomitant with Imipenem-cilastatin could result in convulsion; with zidovudine could result in neutropenia; with Probenecid would increase toxicity of ganciclovir
3. Co-administration of valproate with amitryptyline/nortryptyline, and warfarin need to be adjusted if necessary
4. Co-administration of valganciclovir with didanosine, Mycophenolate mofetil, and bone marrow suppression drugs (e.g., dapsone, pentamidine, flucytosine, vincristine, vinblastine, adriamycin, amphotericin B, nucleoside analogues, hydroxyurea) would need to be monitored more seriously because of their toxicity might increase
5. All other drugs prohibited co-administration with valproic acid absolutely

3. Duration of treatment: This treatment cycle of 28 days will be repeated maximum 6 times. (Q4wks/cycle, max: 6 cycles) A standard 3 + 3 phase I dose escalation study design was used (22). A minimum of three evaluable patients were to be treated at each dose level. According to Worst Toxicity CTCAE v4.03 Grade and FDA indication of gemcitabine, the dose limiting toxicity (DLT) of this trial was determined in the first treatment cycle.

In the absence of DLT, patients were enrolled in the next dose level. If 1 of three patients had a DLT, the cohort was expanded to include six patients. If ≥2 patients experienced DLT, maximum tolerated dose (MTD) was exceeded and further enrollment at that dose level was stopped. MTD was defined as the highest dose level at which ≤1 of 6 patients experienced a DLT. Only DLT that occurred during the first treatment cycle were used to determine the MTD.

4. Patient examination and re-evaluation: Laboratory data of complete blood count (CBC)/differential count (DC), creatinine, alanine aminotransferase (ALT) will be routine checked every weeks during the first 3 treatment cycles. During all treatment courses, if grade 4 neutropenia or grade 4 thrombocytopenia attacked, the laboratory will be checked every 3 days until recovered to pre-treatment baseline. Around 70% responsive and stable cases could be enrolled in the 4th treatment cycle will be routine checked the laboratory data before gemcitabine administration. Plasma EBV DNA copies number will be monitored before each cycle treatment. Systemic re-evaluation will be performed after every three cycles treatment. Re-evaluation including physical examination, image studies including CT/MRI for head and neck area, chest x-ray, abdominal echo, and Gallium whole body tumor scan, and blood exam including complete CBC count, biochemical profile, and plasma EBV DNA copies number.

Inclusion Criteria:

1. Have the ability to understand and willingness to sign a written informed consent document

2. Identified as recurrent nasopharyngeal carcinoma or distant metastases of male or female subjects who had failed in 1st line therapy including radiotherapy, not suitable for radiotherapy or unwilling to receive radiotherapy, 1st line chemotherapy excluding gemcitabine, and no curative treatment options

3. Biopsy confirmed belong to World Health Organization classification of nasopharyngeal carcinoma type II or type III

4. Men and women between aged 20 to 80 years of age; female patients with childbearing potential will routinely consult obstetric doctor for contraception and need to have contraception at least 6 months after finished this trial

5. Adequate internal organs including liver, kidney and bone marrow function

   • white blood cell count of >3,000/µL; platelet count of ≥100,000/µL; absolute neutrophil count >1,500/µL
   • total bilirubin <2.0 mg/dL, aspartate aminotransferase (AST), alanine transaminase(ALT) <2.5x upper limit of normal range (ULN)
   • serum creatinine <2.0 mg/dL

6. The daily performance status ECOG ≤ 2 points

Exclusion Criteria:

1. Pregnancy or breast-feeding women, and plans within six months of pregnancy

2. Contraindication to Gemmis injection, Depakine gastro-resistant tablet, and Valcyte film-coated tablets, including:

   • Allergy to Gemmis injection, Depakine gastro-resistant tablet, Valcyte film-coated tablets, and other similar drugs
   • Patients with hepatic B or C, patients with human immunodeficiency virus, or viral related disease receiving anti-viral treatment
   • Acute or chronic hepatitis not related to NPC with liver metastasis
   • Using drugs which ineligible combination with valproic acid, including mefloquine, St.-John's-Wort, lamotrigine, Topiramate, quetiapine, cyclosporin, hepatic enzyme-inducing drugs (e.g., phenytoin, carbamazepine phenobarbital, primidone, rifampin), enzyme inhibitors (e.g., felbamate), aspirin, cimetidine, erythromycin, carbapenem (e.g., Panipenem, Aztreonam, Imipenem, Meropenem), diazepam, ethosuximide, lamotrigine, phenytoin, nimodipine

3. With insomnia, anxiety or spiritual concerns, or are receiving mental illness treatment

4. Has been diagnosed with a second cancer, except to basal cell carcinoma

5. Patients unsuitable to this trial, including:

   • Patients with significant disease
   • PI evaluated with high risk group patients
   • Patients not recovered from previous anti-cancer treatment
   • Recent major surgery

6. Patients with creatinine clearance rate <40 ml/min