**Neoadjuvant Chemotherapy Using Platinum-Based Regimens for Stage Ib2-II Squamous Cell Carcinoma and Non-Squamous Cell Carcinoma of the Cervix**

Tadahiro Shoji et al.\* *Department of Obstetrics and Gynecology, Iwate Medical University School of Medicine Japan* 

#### **1. Introduction**

The methods used for treating stage Ib2-IIb cervical cancers, with a bulky mass, differ between Japan and Western countries. In Western countries, concurrent chemoradiation (CCRT) has been recommended as a standard therapy for such tumors based on the results of multiple large-scale randomized trials and meta-analyses (Morris et al., 1999; Rose et al., 1999; Whitney et al., 1999; Pearcey et al., 2002; Eifel et al. 2004; Green et al. 2001; Lukka et al., 2002). In Japan, Korea, Italy and some other countries, the neoadjuvant chemotherapy (NAC) approach has been extensively introduced to clinical practice (Sugiyama et al., 1999). NAC is considered to be clinically significant in 2 respects: it is expected to improve the radicality and safety of surgery by reducing tumor size; and it is expected to exert systemic effects, i.e., effects on lymph node occult micrometastases, etc. A disadvantage of NAC is delayed initiation of the primary treatment, suggesting the necessity of completing NAC as an auxiliary therapy within a short period of time. Therefore, we may find that NAC is valuable if it can exert efficacy rapidly with high platinum dose intensity (DI), assuring that subsequent primary surgical therapy can be performed as soon as possible. At our facility, a platinum-based regimen has been used for NAC in patients with cervical cancer. Herein, we review the efficacy and safety data on NAC for squamous cell carcinoma of the uterine cervix. We previously reported our interim data and now present the results of an ongoing pilot study on the efficacy and safety of NAC for non-squamous cell carcinoma of the uterine cervix.

#### **2. Subjects and methods**

#### **2.1 Subjects**

We studied 43 patients with locally advanced cancer of the uterine cervix (clinical stage Ib2 to IIb) who gave informed consent to participate in this study between January 2002 and

<sup>\*</sup>Eriko Takatori, Hideo Omi, Masahiro Kagabu, Tastuya Honda, Yuichi Morohara, Seisuke Kumagai, Fumiharu Miura, Satoshi Takeuchi, Akira Yoshizaki and Toru Sugiyama

*Department of Obstetrics and Gynecology, Iwate Medical University School of Medicine, Japan* 

Neoadjuvant Chemotherapy Using Platinum-Based Regimens

Fig. 1. Treatment protocol of NAC for cervical cancer

**2.4.2 NAC for non-squamous cell carcinoma** 

treatment were administered to each patient (Fig.1) .

**2.4.2.1 Criteria for starting the next course of treatment** 

**2.4.1.3 Dose reduction criteria** 

vomiting.

75,000/mm3.

75,000/mm3, 3) serum creatinine 1.5 mg/dl.

for Stage Ib2-II Squamous Cell Carcinoma and Non-Squamous Cell Carcinoma of the Cervix 81

postponed by 2 weeks at a maximum: 1) neutrophil count 1,500/mm3, 2) platelet count

In cases exhibiting the following signs of toxicity during the first course of treatment, the CPT-11 and CDDP doses for the second course were reduced from 70 mg/m2 to 60 mg/m2: Grade 4 neutropenia lasting 7 days or more; febrile neutropenia lasting 4 days or more; Grade 4 thrombocytopenia; Grade 3 thrombocytopenia accompanied by bleeding; and Grade 3 or more severe non-hematological signs of toxicity other than nausea and

One course of treatment was 21 days, with a PTX dose of 175 mg/m2 or DTX dose of 70 mg/m2 on Day 1 and intravenous CBDCA AUC 6 on Day 1. As a rule, 2 courses of

In cases in which hematological data within 2 days before the planned start of the second course of treatment did not satisfy the following criteria, starting the second course was postponed by 2 weeks at a maximum: 1) neutrophil count 1,000/mm3, 2) platelet count

September 2010. All 43 were scheduled to undergo a radical hysterectomy, including 23 with squamous cell carcinoma and 20 with non-squamous cell carcinoma.

#### **2.2 Inclusion criteria**

The following set of inclusion criteria was employed for selection of study subjects. (1) Histologically verified squamous cell carcinoma or non-squamous cell carcinoma of the uterine cervix; (2) locally advanced stage Ib2 to IIb; (3) age: 20 years upward and less than 70 years; (4) Eastern Cooperative Oncology Group (ECOG) performance status (PS): 0-2; (5) initially treated case; (6) the presence of an MRI-measurable bulky mass in the uterine cervix; (7) hematologic and blood biochemical findings meeting the following criteria [WBC count 4,000/mm3 ; neutrophil count 2,000/mm3 ; platelet count 100,000/mm3 ; hemoglobin 10.0 g/dl ; AST and ALT levels 2 times the upper limit of normal reference range at study site ; serum total bilirubin level 1.5 mg/dl ; serum creatinine 1.5 mg/dl ; and creatinine clearance 60 ml/min]; (8)life expectancy 6 months; and (9) written informed consent personally given by the subject.

#### **2.3 Exclusion criteria**

Exclusion criteria were prescribed as follows. (1) Patients with overt infection; (2) patients with a serious complication(s) (e.g., cardiac disease, poorly controlled diabetes mellitus, malignant hypertension, bleeding tendency); (3) patients with active multiple cancer; (4) patients with interstitial pneumonia or pulmonary fibrosis; (5) patients with effusions; (6) patients with a history of unstable angina or myocardial infarction within 6 months after registration, or with a concurrent serious arrhythmia requiring treatment; (7) patients in whom treatment with cisplatin (CDDP), irinotecan (CPT-11), paclitaxel (PTX), docetaxel (DTX) and carboplatin (CBDCA) is contraindicated; (8) patients with (watery) diarrhea; (9) patients with intestinal paralysis or ileus; (10) pregnant women, nursing mothers or women wishing to become pregnant; (11) patients with a history of serious drug hypersensitivity or drug allergy; and (12) patients who were inadequate for safe conduct of this study as judged by the attending physician.

#### **2.4 Administration method and criteria for modification**

#### **2.4.1 NAC for squamous cell carcinoma**

One course of NAC consisted of 21 days, with a CDDP dose of 70 mg/m2 on Day 1 and intravenous CPT-11 doses of 70 mg/m2 on Days 1 and 8. As a rule, 2 courses of NAC were administered to each patient (Fig.1).

#### **2.4.1.1 Criteria for skipping CPT-11**

In cases in which hematological data within 2 days before Day 8 did not satisfy the following criteria, CPT-11 was skipped on Day 8: 1) neutrophil count 1,000/mm3, 2) platelet count 75,000/mm3.

#### **2.4.1.2 Criteria for starting the next course of NAC**

In cases in which hematological data within 2 days before the planned start of the next course of treatment did not satisfy the following criteria, starting the second course was postponed by 2 weeks at a maximum: 1) neutrophil count 1,500/mm3, 2) platelet count 75,000/mm3, 3) serum creatinine 1.5 mg/dl.

Fig. 1. Treatment protocol of NAC for cervical cancer

#### **2.4.1.3 Dose reduction criteria**

80 Squamous Cell Carcinoma

September 2010. All 43 were scheduled to undergo a radical hysterectomy, including 23

The following set of inclusion criteria was employed for selection of study subjects. (1) Histologically verified squamous cell carcinoma or non-squamous cell carcinoma of the uterine cervix; (2) locally advanced stage Ib2 to IIb; (3) age: 20 years upward and less than 70 years; (4) Eastern Cooperative Oncology Group (ECOG) performance status (PS): 0-2; (5) initially treated case; (6) the presence of an MRI-measurable bulky mass in the uterine cervix; (7) hematologic and blood biochemical findings meeting the following criteria [WBC count 4,000/mm3 ; neutrophil count 2,000/mm3 ; platelet count 100,000/mm3 ; hemoglobin 10.0 g/dl ; AST and ALT levels 2 times the upper limit of normal reference range at study site ; serum total bilirubin level 1.5 mg/dl ; serum creatinine 1.5 mg/dl ; and creatinine clearance 60 ml/min]; (8)life expectancy 6 months; and (9) written

Exclusion criteria were prescribed as follows. (1) Patients with overt infection; (2) patients with a serious complication(s) (e.g., cardiac disease, poorly controlled diabetes mellitus, malignant hypertension, bleeding tendency); (3) patients with active multiple cancer; (4) patients with interstitial pneumonia or pulmonary fibrosis; (5) patients with effusions; (6) patients with a history of unstable angina or myocardial infarction within 6 months after registration, or with a concurrent serious arrhythmia requiring treatment; (7) patients in whom treatment with cisplatin (CDDP), irinotecan (CPT-11), paclitaxel (PTX), docetaxel (DTX) and carboplatin (CBDCA) is contraindicated; (8) patients with (watery) diarrhea; (9) patients with intestinal paralysis or ileus; (10) pregnant women, nursing mothers or women wishing to become pregnant; (11) patients with a history of serious drug hypersensitivity or drug allergy; and (12) patients who were inadequate for safe conduct of this study as judged

One course of NAC consisted of 21 days, with a CDDP dose of 70 mg/m2 on Day 1 and intravenous CPT-11 doses of 70 mg/m2 on Days 1 and 8. As a rule, 2 courses of NAC were

In cases in which hematological data within 2 days before Day 8 did not satisfy the following criteria, CPT-11 was skipped on Day 8: 1) neutrophil count 1,000/mm3, 2)

In cases in which hematological data within 2 days before the planned start of the next course of treatment did not satisfy the following criteria, starting the second course was

with squamous cell carcinoma and 20 with non-squamous cell carcinoma.

informed consent personally given by the subject.

**2.4 Administration method and criteria for modification** 

**2.4.1 NAC for squamous cell carcinoma** 

**2.4.1.2 Criteria for starting the next course of NAC** 

administered to each patient (Fig.1). **2.4.1.1 Criteria for skipping CPT-11** 

platelet count 75,000/mm3.

**2.2 Inclusion criteria** 

**2.3 Exclusion criteria** 

by the attending physician.

In cases exhibiting the following signs of toxicity during the first course of treatment, the CPT-11 and CDDP doses for the second course were reduced from 70 mg/m2 to 60 mg/m2: Grade 4 neutropenia lasting 7 days or more; febrile neutropenia lasting 4 days or more; Grade 4 thrombocytopenia; Grade 3 thrombocytopenia accompanied by bleeding; and Grade 3 or more severe non-hematological signs of toxicity other than nausea and vomiting.

### **2.4.2 NAC for non-squamous cell carcinoma**

One course of treatment was 21 days, with a PTX dose of 175 mg/m2 or DTX dose of 70 mg/m2 on Day 1 and intravenous CBDCA AUC 6 on Day 1. As a rule, 2 courses of treatment were administered to each patient (Fig.1) .

#### **2.4.2.1 Criteria for starting the next course of treatment**

In cases in which hematological data within 2 days before the planned start of the second course of treatment did not satisfy the following criteria, starting the second course was postponed by 2 weeks at a maximum: 1) neutrophil count 1,000/mm3, 2) platelet count 75,000/mm3.

Neoadjuvant Chemotherapy Using Platinum-Based Regimens

ligament, or evident invasion of the vasculature.

**3.1. Results of NAC for squamous cell carcinoma** 

and IIb in 16 (69.6%). All patients received 2 courses of NAC (Table 1).

**2.7 Primary treatment** 

**2.8 Postoperative therapy** 

**3.1.1 Background variables**

Table 1. Patient characteristics

**3.1.2 Anti-tumor response** 

**3. Results** 

for Stage Ib2-II Squamous Cell Carcinoma and Non-Squamous Cell Carcinoma of the Cervix 83

Patients with stage Ib2-IIb carcinoma underwent a radical hysterectomy unless the response of the tumor to preoperative treatment was progressive disease (PD) and the tumor was up-

Postoperative radiotherapy or chemotherapy was undertaken additionally in patients with positive vaginal stump, positive lymphadenopathy, positive invasion of the cardinal

The median age of the 23 patients was 40 (range: 25-63) years. PS was 0 in 20 cases (87.0%) and 1 in 3 (13.0%). The clinical stage of the tumor was Ib5 in 5 cases (21.7%), IIa in 2 (8.7%),

The response of the tumor to treatment was assessed in all cases. Five (21.7%) showed a complete response (CR), 15 (65.2%) a partial response (PR), 2 (8.7%) stable disease (SD), and 1 (4.3%) PD. Thus, the response rate was 87.0% (Table 2). Among the cases rated as showing

staged. In cases in which surgery was not possible, concurrent CCRT was adopted.

### **2.4.2.2 CBDCA dose reduction criteria**

In cases exhibiting the following signs of toxicity during the first course of treatment, the CBDCA dose for the second course was reduced from AUC 6 to 5. If signs of toxicity remained after this dose reduction, that for the third course of treatment was reduced from AUC 5 to 4: Grade 4 thrombocytopenia; and Grade 3 thrombocytopenia accompanied by bleeding.

#### **2.4.2.3 PTX dose reduction criteria**

In cases exhibiting signs of Grade 2 or more severe peripheral nerve toxicity during the first course, the PTX dose for the second course was reduced from 175 mg/m2 to 135 mg/m2. If Grade 2 or more severe peripheral nerve toxicity remained after dose reduction, the PTX dose for the third course was reduced from 135 mg/m2 to 110 mg/m2.

#### **2.4.2.4 DTX dose reduction criteria**

In cases exhibiting the following signs of toxicity during the first course, the DTX dose for the second course was reduced from 70 mg/m2 to 60 mg/m2. If signs of toxicity remained after this dose reduction, the DTX dose for the third course was reduced from 60 mg/m2 to 50 mg/m2: Grade 4 neutropenia lasting 7 days or more; and febrile neutropenia lasting 4 days or more.

### **2.5 Supportive therapy**

A granulocyte-colony stimulating factor (G-CSF) preparation was administered in patients developing Grade 4 neutropenia during the first course of NAC. Administration of the G-CSF preparation was permitted for prophylactic purposes during the second and subsequent courses of NAC in cases exhibiting Grade 4 neutropenia during the first course. Anti-emetics were additionally used for prophylactic purposes.

#### **2.6 Observations and tests**

The primary endpoint was anti-tumor response. Secondary endpoints were adverse events, surgery completion rate, progression-free survival period, and overall survival period. Hematological tests and urinalysis were carried out before the start of treatment and once weekly, as a rule, after starting treatment. Electrocardiograms and chest X-rays were obtained before the start and at the end of treatment.

## **2.6.1 Evaluation of anti-tumor response**

Anti-tumor response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) by comparing the baseline findings (before the start of treatment) on magnetic resonance imaging (MRI) with the MRI findings at the end of treatment courses. Efficacy evaluation adopted the best rating, without incorporating the response period.

#### **2.6.2 Evaluation of adverse events**

Adverse events were evaluated employing the National Cancer Institute Common Toxicity Criteria (NCI-CTCAE) version 3.0.

#### **2.7 Primary treatment**

82 Squamous Cell Carcinoma

In cases exhibiting the following signs of toxicity during the first course of treatment, the CBDCA dose for the second course was reduced from AUC 6 to 5. If signs of toxicity remained after this dose reduction, that for the third course of treatment was reduced from AUC 5 to 4: Grade 4 thrombocytopenia; and Grade 3 thrombocytopenia accompanied by

In cases exhibiting signs of Grade 2 or more severe peripheral nerve toxicity during the first course, the PTX dose for the second course was reduced from 175 mg/m2 to 135 mg/m2. If Grade 2 or more severe peripheral nerve toxicity remained after dose reduction, the PTX

In cases exhibiting the following signs of toxicity during the first course, the DTX dose for the second course was reduced from 70 mg/m2 to 60 mg/m2. If signs of toxicity remained after this dose reduction, the DTX dose for the third course was reduced from 60 mg/m2 to 50 mg/m2: Grade 4 neutropenia lasting 7 days or more; and febrile neutropenia lasting 4

A granulocyte-colony stimulating factor (G-CSF) preparation was administered in patients developing Grade 4 neutropenia during the first course of NAC. Administration of the G-CSF preparation was permitted for prophylactic purposes during the second and subsequent courses of NAC in cases exhibiting Grade 4 neutropenia during the first course.

The primary endpoint was anti-tumor response. Secondary endpoints were adverse events, surgery completion rate, progression-free survival period, and overall survival period. Hematological tests and urinalysis were carried out before the start of treatment and once weekly, as a rule, after starting treatment. Electrocardiograms and chest X-rays were

Anti-tumor response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) by comparing the baseline findings (before the start of treatment) on magnetic resonance imaging (MRI) with the MRI findings at the end of treatment courses. Efficacy

Adverse events were evaluated employing the National Cancer Institute Common Toxicity

evaluation adopted the best rating, without incorporating the response period.

dose for the third course was reduced from 135 mg/m2 to 110 mg/m2.

Anti-emetics were additionally used for prophylactic purposes.

obtained before the start and at the end of treatment.

**2.6.1 Evaluation of anti-tumor response** 

**2.6.2 Evaluation of adverse events** 

Criteria (NCI-CTCAE) version 3.0.

**2.4.2.2 CBDCA dose reduction criteria** 

**2.4.2.3 PTX dose reduction criteria** 

**2.4.2.4 DTX dose reduction criteria** 

bleeding.

days or more.

**2.5 Supportive therapy** 

**2.6 Observations and tests** 

Patients with stage Ib2-IIb carcinoma underwent a radical hysterectomy unless the response of the tumor to preoperative treatment was progressive disease (PD) and the tumor was upstaged. In cases in which surgery was not possible, concurrent CCRT was adopted.

#### **2.8 Postoperative therapy**

Postoperative radiotherapy or chemotherapy was undertaken additionally in patients with positive vaginal stump, positive lymphadenopathy, positive invasion of the cardinal ligament, or evident invasion of the vasculature.

## **3. Results**

#### **3.1. Results of NAC for squamous cell carcinoma**

#### **3.1.1 Background variables**

The median age of the 23 patients was 40 (range: 25-63) years. PS was 0 in 20 cases (87.0%) and 1 in 3 (13.0%). The clinical stage of the tumor was Ib5 in 5 cases (21.7%), IIa in 2 (8.7%), and IIb in 16 (69.6%). All patients received 2 courses of NAC (Table 1).


Table 1. Patient characteristics

#### **3.1.2 Anti-tumor response**

The response of the tumor to treatment was assessed in all cases. Five (21.7%) showed a complete response (CR), 15 (65.2%) a partial response (PR), 2 (8.7%) stable disease (SD), and 1 (4.3%) PD. Thus, the response rate was 87.0% (Table 2). Among the cases rated as showing

Neoadjuvant Chemotherapy Using Platinum-Based Regimens

Table 3. Toxicity of CPT-11+CDDP therapy (n=23)

16 (80.0%), and 3 courses to 3 (15.0%) (Table 1).

1 (4.3%), with the response rate being 75.0% (Table 2).

**3.2.1 Background variables** 

**3.2.2 Anti-tumor response** 

**3.2.3 Adverse events** 

neurotoxicity (Table 4).

**3.2 Results of NAC for non-squamous cell carcinoma** 

for Stage Ib2-II Squamous Cell Carcinoma and Non-Squamous Cell Carcinoma of the Cervix 85

The median age of the 20 patients was 51 (range: 30-63) years. PS was 0 in 15 cases (75.0%) and 1 in 5 (15.0%). The clinical stage was Ib2 in 5 cases (25.0%) and IIb in 15 (75.0%). The histological type was mucinous adenocarcinoma in 9 cases (45.0%), endometrioid adenocarcinoma in 3 (15.0%), clear cell adenocarcinoma in 1 (5.0%), and adenosquamous carcinoma in 7 (35.0%). One course of NAC was administered to 1 case (5.0%), 2 courses to

The response was rated as CR in 4 cases (20.0%), PR in 11 (55.0%), SD in 5 (10.0%), and PD in

Grade 3 or more severe leukopenia and neutropenia were seen in 10 (50.0%) and 19 (95.0%) cases, respectively. Grade 3 febrile neutropenia was noted in 2 cases (10.0%). The G-CSF preparation was used for 13 (65.0%) of the 20 cases; it was administered during 19 (45.2%) of the 42 cycles in total. The mean duration of G-CSF preparation treatment during each course was 3.0 days. None of the cases showed Grade 3 or more severe anemia or thrombocytopenia. The only sign of Grade 3 or more severe non-hematological toxicity was nausea, seen in one case (5.0%). None of the cases had signs of Grade 2 or more severe


CR or PR, none showed tumor growth between the end of the first course and the end of the second course of treatment.

Table 2. Response and clinical outcome

## **3.1.3 Adverse events**

Grade 3 or more severe leukopenia and neutropenia were seen in 6 cases (26.1%) and 14 cases (60.9%), respectively. Grade 3 febrile neutropenia was seen in 1 case (4.3%). The G-CSF preparation was used in 11 (55.0%) of the 23 cases; during 17 (42.5%) of the 46 treatment cycles in total. The mean duration of G-CSF treatment during each course was 3.1 days. Grade 3 or more severe anemia was noted in 3 cases (15.0%), including one patient with Grade 1 anemia requiring blood transfusion. None of the patients developed Grade 3 or more severe thrombocytopenia. Signs of Grade 3 or more severe non-hematological toxicity included nausea in 2 cases (8.7%) and vomiting in 1 (4.3%) (Table 3). No treatmentassociated deaths occurred. Chemotherapy was completed as scheduled in 21 (91.3%) of the 23 cases. In the remaining 2 cases, the CPT-11 dose on Day 2 of the second course was skipped. In these 2 cases, the dose was skipped at the discretion of the attending physician because of persistent Grade 3 nausea. There were 2 cases (8.7%) in which the start of the second course was postponed because the neutrophil count criterion was not satisfied. In both cases, the second course was started within 7 days. In one case (4.3%) showing febrile neutropenia lasting at least 4 days, the CDDP and CPT-11 doses for the second course were reduced from 70 mg/m2 to 60 mg/m2.

#### **3.1.4 Surgery completion rate and survival period**

The completion rate of radical hysterectomy after NAC was 100%. The median follow-up period was 35 months (range: 8-93 months). The median progression-free survival period was 30 months (8-93 months). The median overall survival period was 34 months (8-93 months) (Table 2).


Table 3. Toxicity of CPT-11+CDDP therapy (n=23)

#### **3.2 Results of NAC for non-squamous cell carcinoma**

#### **3.2.1 Background variables**

84 Squamous Cell Carcinoma

CR or PR, none showed tumor growth between the end of the first course and the end of the

Grade 3 or more severe leukopenia and neutropenia were seen in 6 cases (26.1%) and 14 cases (60.9%), respectively. Grade 3 febrile neutropenia was seen in 1 case (4.3%). The G-CSF preparation was used in 11 (55.0%) of the 23 cases; during 17 (42.5%) of the 46 treatment cycles in total. The mean duration of G-CSF treatment during each course was 3.1 days. Grade 3 or more severe anemia was noted in 3 cases (15.0%), including one patient with Grade 1 anemia requiring blood transfusion. None of the patients developed Grade 3 or more severe thrombocytopenia. Signs of Grade 3 or more severe non-hematological toxicity included nausea in 2 cases (8.7%) and vomiting in 1 (4.3%) (Table 3). No treatmentassociated deaths occurred. Chemotherapy was completed as scheduled in 21 (91.3%) of the 23 cases. In the remaining 2 cases, the CPT-11 dose on Day 2 of the second course was skipped. In these 2 cases, the dose was skipped at the discretion of the attending physician because of persistent Grade 3 nausea. There were 2 cases (8.7%) in which the start of the second course was postponed because the neutrophil count criterion was not satisfied. In both cases, the second course was started within 7 days. In one case (4.3%) showing febrile neutropenia lasting at least 4 days, the CDDP and CPT-11 doses for the second course were

The completion rate of radical hysterectomy after NAC was 100%. The median follow-up period was 35 months (range: 8-93 months). The median progression-free survival period was 30 months (8-93 months). The median overall survival period was 34 months (8-93

second course of treatment.

Table 2. Response and clinical outcome

reduced from 70 mg/m2 to 60 mg/m2.

months) (Table 2).

**3.1.4 Surgery completion rate and survival period** 

**3.1.3 Adverse events** 

The median age of the 20 patients was 51 (range: 30-63) years. PS was 0 in 15 cases (75.0%) and 1 in 5 (15.0%). The clinical stage was Ib2 in 5 cases (25.0%) and IIb in 15 (75.0%). The histological type was mucinous adenocarcinoma in 9 cases (45.0%), endometrioid adenocarcinoma in 3 (15.0%), clear cell adenocarcinoma in 1 (5.0%), and adenosquamous carcinoma in 7 (35.0%). One course of NAC was administered to 1 case (5.0%), 2 courses to 16 (80.0%), and 3 courses to 3 (15.0%) (Table 1).

#### **3.2.2 Anti-tumor response**

The response was rated as CR in 4 cases (20.0%), PR in 11 (55.0%), SD in 5 (10.0%), and PD in 1 (4.3%), with the response rate being 75.0% (Table 2).

#### **3.2.3 Adverse events**

Grade 3 or more severe leukopenia and neutropenia were seen in 10 (50.0%) and 19 (95.0%) cases, respectively. Grade 3 febrile neutropenia was noted in 2 cases (10.0%). The G-CSF preparation was used for 13 (65.0%) of the 20 cases; it was administered during 19 (45.2%) of the 42 cycles in total. The mean duration of G-CSF preparation treatment during each course was 3.0 days. None of the cases showed Grade 3 or more severe anemia or thrombocytopenia. The only sign of Grade 3 or more severe non-hematological toxicity was nausea, seen in one case (5.0%). None of the cases had signs of Grade 2 or more severe neurotoxicity (Table 4).

Neoadjuvant Chemotherapy Using Platinum-Based Regimens

the time interval from NAC to surgery.

further follow-up is needed.

for Stage Ib2-II Squamous Cell Carcinoma and Non-Squamous Cell Carcinoma of the Cervix 87

dose was set at 70 mg/m2 for both CDDP and CPT-11, and the therapy was administered for 2 cycles at an interval of 3 weeks, with CDDP administered on Day 1 and CPT-11 on Days 1 and 8. In this way, the DI of CDDP was raised to 23.3 mg/m2/week, and this schedule was expected to reduce the need to skip treatments. Thus, it seems valuable to be able to reduce

In an analysis of adverse events, Grade 3 or more severe neutropenia developed in 14 (60.9%) of the 23 cases, but subsided in response to short-term treatment with a G-CSF preparation. Severe diarrhea, specific to CPT-11, was not seen in any case when this agent was administered at a dose of 70 mg/m2, suggesting that the quality of life (QOL) of patients was maintained during this therapy. The first course of treatment was administered as scheduled in all cases. The start of the second course was delayed, by no more than 7 days, in 3 cases. Furthermore, the CPT-11 dose on Day 8 was skipped in 2 cases. Dose reduction during the second course was necessary in only 2 cases, suggesting that this regimen does not increase the toxicity of these drugs as compared to the dosing regimen with 4-week intervals. Furthermore, the response rate (87.6%) and the surgery completion rate (100%) were satisfactory. Regarding the outcomes of patients treated with this regimen,

Non-squamous cell carcinoma of the uterine cervix has been steadily rising in Japan, currently accounting for approximately 10% to 15% of all cervical cancer cases. Lymph node metastasis is more frequent in cases with invasive non-squamous cell carcinoma than in those with invasive squamous cell carcinoma (Aoki et al., 2002) and sensitivities to radiotherapy and chemotherapy are considered to be lower with non-squamous cell carcinoma (Landoni et al., 1997). Thus, squamous and non-squamous cell carcinomas must be analyzed separately. It is advisable to try new therapeutic strategies for non-squamous cell carcinoma, but the number of published studies involving cases with this type of cervical cancer is small, and the number of cases analyzed is also small. Thus, no high-level evidence has been obtained for this type of cervical cancer. The response rates of adenocarcinoma are reportedly 20% (Thigpen et al., 1986), 15% (Sutton et al., 1993), 14% (Look et al., 1997), and 12% (Rose et al., 2003) to uncombined therapies with CDDP, ifosmide, 5-FU, and oral etoposide, respectively, indicating that the response rates of adenocarcinoma to these therapies tend to be lower than those of squamous cell carcinoma. However, according to the report by Curtin et al, the response rate of adenocarcinoma was as high as 31% even when PTX was used independently (Curtin et al., 2001). DTX has also been attracting considerable interest. Nagao et al evaluated the efficacy of combined chemotherapy using DTX + CBDCA (DTX 60 mg/m2 on Day 1, CBDCA AUC 6 on day 1 and then every 21 days) in 17 patients with advanced/recurrent cervical cancer, including 6 with adenocarcinoma and 1 with adenosquamous carcinoma, reporting that a PR was obtained in 6 of the 7 cases with adenocarcinoma (including the one with adenosquamous carcinoma) and that the response rate was thus 86% (Nagao et al., 2005). Following these findings, we conducted a pilot study involving standard regimens of PTX/CBDCA and

DTX/CBDCA conventionally used for the treatment of ovarian cancer.

In the analysis of adverse events, Grade 3 or more severe neutropenia developed in 19 (95.0%) of the 20 cases, but subsided in response to short-term treatment with a G-CSF preparation (mean dosing period: 3.0 days/course). During the first course of DTX/CBDCA

In 3 cases (15.0%), the start of the second course of treatment was postponed because the neutrophil count criterion was not satisfied. In all 3 of these cases, the second course was started within 7 days. Both cases (10.0%) with Grade 3 febrile neutropenia for 4 days or more had received DTX/CBDCA therapy prior to the development of neutropenia. In these 2 cases, DTX (from 70 mg/m2 to 60 mg/m2) and CBDCA (from AUC 6 to 5) doses were reduced for the second course of treatment.

#### **3.2.4 Surgery completion rate and survival period**

A radical hysterectomy after NAC was completed in 15 of the 20 cases, i.e., the surgery completion rate was 75.0%. The median follow-up period was 20 months (6-70 months). The median progression-free survival period was 10.5 months (3-70 months) and the median overall survival period was 20 months (6-70 months) (Table2).


Table 4. Toxicity of TC or DC therapy (n=20)

## **4. Discussion**

A meta-analysis of the results of NAC for squamous cell carcinoma of the uterine cervix ruled out the effectiveness of radiotherapy applied as the primary treatment but suggested the effectiveness of surgery employed as primary therapy. This analysis suggested the effectiveness of NAC, if: one cycle of treatment lasted no more than 14 days; and the DI of CDDP exceeded 25 mg/m2/week (Neoadjuvant Chemotherapy for Cervical Cancer Metaanalysis Collaboration., 2003). Sugiyama et al reported a CDDP/CPT-11 therapy schedule involving CPT-11 doses on Days 1, 8, and 15 (one course = 28 days) (Sugiyama et al., 1999). We evaluated the efficacy and safety of CDDP/CPT-11 therapy, reportedly an effective NAC regimen, using modified doses and administration schedules. In our study, a single

In 3 cases (15.0%), the start of the second course of treatment was postponed because the neutrophil count criterion was not satisfied. In all 3 of these cases, the second course was started within 7 days. Both cases (10.0%) with Grade 3 febrile neutropenia for 4 days or more had received DTX/CBDCA therapy prior to the development of neutropenia. In these 2 cases, DTX (from 70 mg/m2 to 60 mg/m2) and CBDCA (from AUC 6 to 5) doses were

A radical hysterectomy after NAC was completed in 15 of the 20 cases, i.e., the surgery completion rate was 75.0%. The median follow-up period was 20 months (6-70 months). The median progression-free survival period was 10.5 months (3-70 months) and the median

A meta-analysis of the results of NAC for squamous cell carcinoma of the uterine cervix ruled out the effectiveness of radiotherapy applied as the primary treatment but suggested the effectiveness of surgery employed as primary therapy. This analysis suggested the effectiveness of NAC, if: one cycle of treatment lasted no more than 14 days; and the DI of CDDP exceeded 25 mg/m2/week (Neoadjuvant Chemotherapy for Cervical Cancer Metaanalysis Collaboration., 2003). Sugiyama et al reported a CDDP/CPT-11 therapy schedule involving CPT-11 doses on Days 1, 8, and 15 (one course = 28 days) (Sugiyama et al., 1999). We evaluated the efficacy and safety of CDDP/CPT-11 therapy, reportedly an effective NAC regimen, using modified doses and administration schedules. In our study, a single

reduced for the second course of treatment.

Table 4. Toxicity of TC or DC therapy (n=20)

**4. Discussion** 

**3.2.4 Surgery completion rate and survival period** 

overall survival period was 20 months (6-70 months) (Table2).

dose was set at 70 mg/m2 for both CDDP and CPT-11, and the therapy was administered for 2 cycles at an interval of 3 weeks, with CDDP administered on Day 1 and CPT-11 on Days 1 and 8. In this way, the DI of CDDP was raised to 23.3 mg/m2/week, and this schedule was expected to reduce the need to skip treatments. Thus, it seems valuable to be able to reduce the time interval from NAC to surgery.

In an analysis of adverse events, Grade 3 or more severe neutropenia developed in 14 (60.9%) of the 23 cases, but subsided in response to short-term treatment with a G-CSF preparation. Severe diarrhea, specific to CPT-11, was not seen in any case when this agent was administered at a dose of 70 mg/m2, suggesting that the quality of life (QOL) of patients was maintained during this therapy. The first course of treatment was administered as scheduled in all cases. The start of the second course was delayed, by no more than 7 days, in 3 cases. Furthermore, the CPT-11 dose on Day 8 was skipped in 2 cases. Dose reduction during the second course was necessary in only 2 cases, suggesting that this regimen does not increase the toxicity of these drugs as compared to the dosing regimen with 4-week intervals. Furthermore, the response rate (87.6%) and the surgery completion rate (100%) were satisfactory. Regarding the outcomes of patients treated with this regimen, further follow-up is needed.

Non-squamous cell carcinoma of the uterine cervix has been steadily rising in Japan, currently accounting for approximately 10% to 15% of all cervical cancer cases. Lymph node metastasis is more frequent in cases with invasive non-squamous cell carcinoma than in those with invasive squamous cell carcinoma (Aoki et al., 2002) and sensitivities to radiotherapy and chemotherapy are considered to be lower with non-squamous cell carcinoma (Landoni et al., 1997). Thus, squamous and non-squamous cell carcinomas must be analyzed separately. It is advisable to try new therapeutic strategies for non-squamous cell carcinoma, but the number of published studies involving cases with this type of cervical cancer is small, and the number of cases analyzed is also small. Thus, no high-level evidence has been obtained for this type of cervical cancer. The response rates of adenocarcinoma are reportedly 20% (Thigpen et al., 1986), 15% (Sutton et al., 1993), 14% (Look et al., 1997), and 12% (Rose et al., 2003) to uncombined therapies with CDDP, ifosmide, 5-FU, and oral etoposide, respectively, indicating that the response rates of adenocarcinoma to these therapies tend to be lower than those of squamous cell carcinoma. However, according to the report by Curtin et al, the response rate of adenocarcinoma was as high as 31% even when PTX was used independently (Curtin et al., 2001). DTX has also been attracting considerable interest. Nagao et al evaluated the efficacy of combined chemotherapy using DTX + CBDCA (DTX 60 mg/m2 on Day 1, CBDCA AUC 6 on day 1 and then every 21 days) in 17 patients with advanced/recurrent cervical cancer, including 6 with adenocarcinoma and 1 with adenosquamous carcinoma, reporting that a PR was obtained in 6 of the 7 cases with adenocarcinoma (including the one with adenosquamous carcinoma) and that the response rate was thus 86% (Nagao et al., 2005). Following these findings, we conducted a pilot study involving standard regimens of PTX/CBDCA and DTX/CBDCA conventionally used for the treatment of ovarian cancer.

In the analysis of adverse events, Grade 3 or more severe neutropenia developed in 19 (95.0%) of the 20 cases, but subsided in response to short-term treatment with a G-CSF preparation (mean dosing period: 3.0 days/course). During the first course of DTX/CBDCA

Neoadjuvant Chemotherapy Using Platinum-Based Regimens

issue.

NAC are clearly needed.

patients with non-squamous cell carcinoma is awaited.

**5. Conclusions** 

**6. References** 

0090-8258

for Stage Ib2-II Squamous Cell Carcinoma and Non-Squamous Cell Carcinoma of the Cervix 89

radiotherapy (7-year survival rate: 41%) as compared to surgery + radiotherapy (41%) (Sardi et al., 1997). Serur et al retrospectively compared the outcomes of treating stage Ib cases between a NAC + surgery group and a surgery alone group, demonstrating a higher 5-year survival rate in the NAC + surgery group although the difference was not statistically significant (80% vs 69%) (Serur et al., 1997). Tierney et al reported the results of a metaanalysis, stating that there was no prognostic improvement (Neoadjuvant Chemotherapy for Cervical Cancer Metaanalysis Colloaboration, 2003). Thus, there is no consensus on this

The JCOG0102 was a representative randomized study of NAC conducted in Japan, designed as an RCT comparing the outcomes of treatment for stage Ib2-IIb cases with bulky tumors between radical hysterectomy (+RT) and NAC + radical hysterectomy (+RT). The JCOG0102 used bleomycin/vincristine/mitomycinC/cisplatin (BOMP) as the NAC regimen. In that study, the response rate to BOMP therapy was low as 61%, and the interim results did not endorse the usefulness of this therapy, forcing the study to be discontinued prematurely (Katsumata et al., 2006). The JGOG1065 was a phase II clinical study on NAC + radical hysterectomy, using nedaplatin and CPT-11 for NAC, carried out in 66 patients with stage Ib2-IIb cervical cancer with a bulky tumor. In that study, the response rate was 75.8% and the 2-year recurrence-free survival period was 73.8% (Shoji et al., 2010). This therapy is expected to reduce nephrotoxicity and adverse events such as nausea and vomiting and appears to be a useful regimen for patients with renal dysfunction and elderly patients from the viewpoint of QOL. However, the response rate to this therapy has not exceeded that to CDDP + CPT-11. At present, there is no plan to launch a phase III clinical study on NAC (NDP/CPT-11) + radical hysterectomy vs. CCRT. There is no evidence supporting the view that NAC improves the outcomes of patients with cervical cancer, and NAC has not been recommended in any set of guidelines. Further studies on the indications for and efficacy of

Irinotecan/cisplatin therapy for squamous carcinoma of the uterine cervix and PTX/ CBDCA and DTX/CBDCA therapies for non-squamous cell carcinoma of the uterine cervix showed high anti-tumor efficacy, and the adverse reactions to these therapies could be dealt with satisfactorily, thus allowing safe treatment. In cases with squamous cell carcinoma, outcomes are expected to be improved by NAC, but further evaluation of the outcomes of

Aoki, Y.; Sato, T. & Watanabe, M. et al. (2002). Neoadjuvant chemotherapy using low-dose

consecutive intraarterial infusion of cisplatin combined with 5FU for locally advanced cervical adenocarcinoma. *Gynecologic Oncology*, 81, pp. 496-499, ISSN

therapy, Grade 3 febrile neutropenia developed in 2 cases. In these 2 cases, the dose was reduced during the next course of treatment (DTX, from 70 mg/m2 to 60 mg/m2; CBDCA, from AUC 6 to 5). All signs of peripheral neuropathy specific to taxanes, observed during this study, were Grade 1 or less severe, allowing continuation of treatment while preserving the QOL of individual patients. No serious adverse events occurred, and the response rate was 75%, but the completion rate of surgery (radical hysterectomy) was 75%. Thus, the outcomes of treatment in this study were not satisfactory. Possible reasons are: rapid progression of non-squamous cell carcinoma, frequent invasion of tissues/organs surrounding the uterus, and frequent lymph node metastasis.

Numerous reports on phase II studies of NAC for cervical cancer have been published, demonstrating effectiveness in 70%-80% of all cases. Table 5 shows the results of the present study in comparison to those of previous reports (Sugiyama et al., 1999; Hwang et al., 2001; Dueñas-Gonzalez et al., 2001; D'Agostino et al., 2002; Di Vagno et al., 2003; Dueñas-Gonzalez et al., 2003; Umesaki et al., 2004; Shoji et al., 2010; Shoji et al., 2010) and this study.


Table 5. Phase II study of NAC for cervical cancer

Most of the reports shown pertain to evaluation of both squamous cell carcinoma and nonsquamous cell carcinoma. There is an urgent need to conduct clinical studies on each histological type of cervical cancer and to establish new methods of treatment specific to each type. Only a limited number of reports have demonstrated a high response rate to correlate with a better outcome. Thus, randomized controlled trials (RCT) designed to assess improvement of long-term outcomes are essential. As an RCT evaluating outcomes after NAC, Sardi et al reported a study involving comparisons among 4 groups (NAC + surgery + radiotherapy, surgery + radiotherapy, uncombined radiotherapy, NAC + radiotherapy). They found that the survival rate improved significantly with NAC + surgery + radiotherapy (7-year survival rate: 41%) as compared to surgery + radiotherapy (41%) (Sardi et al., 1997). Serur et al retrospectively compared the outcomes of treating stage Ib cases between a NAC + surgery group and a surgery alone group, demonstrating a higher 5-year survival rate in the NAC + surgery group although the difference was not statistically significant (80% vs 69%) (Serur et al., 1997). Tierney et al reported the results of a metaanalysis, stating that there was no prognostic improvement (Neoadjuvant Chemotherapy for Cervical Cancer Metaanalysis Colloaboration, 2003). Thus, there is no consensus on this issue.

The JCOG0102 was a representative randomized study of NAC conducted in Japan, designed as an RCT comparing the outcomes of treatment for stage Ib2-IIb cases with bulky tumors between radical hysterectomy (+RT) and NAC + radical hysterectomy (+RT). The JCOG0102 used bleomycin/vincristine/mitomycinC/cisplatin (BOMP) as the NAC regimen. In that study, the response rate to BOMP therapy was low as 61%, and the interim results did not endorse the usefulness of this therapy, forcing the study to be discontinued prematurely (Katsumata et al., 2006). The JGOG1065 was a phase II clinical study on NAC + radical hysterectomy, using nedaplatin and CPT-11 for NAC, carried out in 66 patients with stage Ib2-IIb cervical cancer with a bulky tumor. In that study, the response rate was 75.8% and the 2-year recurrence-free survival period was 73.8% (Shoji et al., 2010). This therapy is expected to reduce nephrotoxicity and adverse events such as nausea and vomiting and appears to be a useful regimen for patients with renal dysfunction and elderly patients from the viewpoint of QOL. However, the response rate to this therapy has not exceeded that to CDDP + CPT-11. At present, there is no plan to launch a phase III clinical study on NAC (NDP/CPT-11) + radical hysterectomy vs. CCRT. There is no evidence supporting the view that NAC improves the outcomes of patients with cervical cancer, and NAC has not been recommended in any set of guidelines. Further studies on the indications for and efficacy of NAC are clearly needed.

## **5. Conclusions**

88 Squamous Cell Carcinoma

therapy, Grade 3 febrile neutropenia developed in 2 cases. In these 2 cases, the dose was reduced during the next course of treatment (DTX, from 70 mg/m2 to 60 mg/m2; CBDCA, from AUC 6 to 5). All signs of peripheral neuropathy specific to taxanes, observed during this study, were Grade 1 or less severe, allowing continuation of treatment while preserving the QOL of individual patients. No serious adverse events occurred, and the response rate was 75%, but the completion rate of surgery (radical hysterectomy) was 75%. Thus, the outcomes of treatment in this study were not satisfactory. Possible reasons are: rapid progression of non-squamous cell carcinoma, frequent invasion of tissues/organs

Numerous reports on phase II studies of NAC for cervical cancer have been published, demonstrating effectiveness in 70%-80% of all cases. Table 5 shows the results of the present study in comparison to those of previous reports (Sugiyama et al., 1999; Hwang et al., 2001; Dueñas-Gonzalez et al., 2001; D'Agostino et al., 2002; Di Vagno et al., 2003; Dueñas-Gonzalez et al., 2003; Umesaki et al., 2004; Shoji et al., 2010; Shoji et al., 2010) and this study.

Most of the reports shown pertain to evaluation of both squamous cell carcinoma and nonsquamous cell carcinoma. There is an urgent need to conduct clinical studies on each histological type of cervical cancer and to establish new methods of treatment specific to each type. Only a limited number of reports have demonstrated a high response rate to correlate with a better outcome. Thus, randomized controlled trials (RCT) designed to assess improvement of long-term outcomes are essential. As an RCT evaluating outcomes after NAC, Sardi et al reported a study involving comparisons among 4 groups (NAC + surgery + radiotherapy, surgery + radiotherapy, uncombined radiotherapy, NAC + radiotherapy). They found that the survival rate improved significantly with NAC + surgery +

surrounding the uterus, and frequent lymph node metastasis.

Table 5. Phase II study of NAC for cervical cancer

Irinotecan/cisplatin therapy for squamous carcinoma of the uterine cervix and PTX/ CBDCA and DTX/CBDCA therapies for non-squamous cell carcinoma of the uterine cervix showed high anti-tumor efficacy, and the adverse reactions to these therapies could be dealt with satisfactorily, thus allowing safe treatment. In cases with squamous cell carcinoma, outcomes are expected to be improved by NAC, but further evaluation of the outcomes of patients with non-squamous cell carcinoma is awaited.

## **6. References**

Aoki, Y.; Sato, T. & Watanabe, M. et al. (2002). Neoadjuvant chemotherapy using low-dose consecutive intraarterial infusion of cisplatin combined with 5FU for locally advanced cervical adenocarcinoma. *Gynecologic Oncology*, 81, pp. 496-499, ISSN 0090-8258

Neoadjuvant Chemotherapy Using Platinum-Based Regimens

*Oncology,* 96, pp. 805-809, ISSN 0090-8258

*Medicine,* 340, pp.1144-1153, ISSN 0028-4793

*Oncology letters,* 1, pp. 515-519, ISSN1792-1074.

*Journal of Cancer*, 81, pp. 95-98, ISSN 0007-0920

*Gynecologic Oncology,* 49, pp. 48-50, ISSN 0090-8258

*Cancer Treatment Reports,* 70, pp. 1097-1100, ISSN 0361-5960

0732-183X

ISSN 0090-8258

0090-8258

8258

for Stage Ib2-II Squamous Cell Carcinoma and Non-Squamous Cell Carcinoma of the Cervix 91

Nagao, S. Fujiwara, K. & Oda, T. et al. (2005). Combination chemotherapy of docetaxel and

Neoadjuvant Chemotherapy for Cervical Cancer Meta-analysis Collaboration. (2003).

Pearcey, R.; Brundage, M. & Drouin, P. et al. (2002). Phase III trial comparing radical

Rose, PG.; Blessing, JA. & Buller, RE. et al. Prolonged oral etoposide in recurrent or

Rose, PG.; Bundy, BN. & Watkins, EB. et al. (1999). Concurrent cisplatin-based radiotherapy

Sardi, JE.; Giaroli, A. & Sananes, C. et al. (1997). Long-term follow-up of the first

Serur, E.; Mathews, RP. & Gates, J. et al. (1997). Neoadjuvant chemotherapy in stage IB2

Shoji, T.; Takatori, E. & Hatayama, S. et al. (2010). Phase II Study of Tri-weekly Cisplatin and

Shoji, T.; Sugiyama, T. & Yamaguchi, S. et al. (2010). Phase II Study of Neoadjuvant

Sugiyama, T.; Nishida, T. & Kumagai, S. et al. (1999). Combination chemotherapy with

Sutton, GP.; Blessing, JA. & DiSaia, PJ. et al. (1993). Phase II study of ifosfamide and mesna

Thigpen, JT.; Blessing, JA. & Fowler, WC Jr. et al. (1986). Phase II trials of cisplatin and

Umesaki, N.; Fujii, T. & Nishimura, R. et al. (2004). Phase II study of irinotecan combined

*Proceedings of European Society of Medical Oncology*, #993 2010.

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*European Journal of Cancer*, 39, pp. 2470-2486, ISSN 0959-8049

carboplatin in advanced or recurrent cervix cancer. A pilot study. *Gynecologic* 

Neoadjuvant chemotherapy for locally advanced cervical cancer. a systematic review and meta-analysis of individual patient data from 21 randomised trials.

radiotherapy with and without cisplatin chemotherapy in patients with advanced squamous cell cancer of the cervix. *Journal of Clinical Oncology*, 20, pp. 966-972, ISSN

advanced non-squamous cell carcinoma of the cervix: a Gynecologic Oncology

and chemotherapy for locally advanced cervical cancer. *The New England journal of* 

randomised trial using neoadjuvant chemotherapy in stage Ib squamous carcinoma of the cervix: The final results. *Gynecologic Oncology,* 67, pp. 61-69,

squamous cell carcinoma of the cervix. *Gynecologic Oncology,* 65, pp. 348-356, ISSN

Irinotecan as Neoadjuvant Chemotherapy for Locally Advanced Cervical Cancer.

Chemotherapy with CPT-11 and Nedaplatin (CPT-11/NDP) for Stage Ib2/II Carcinoma of the Cervix (Japanese Gynecologic Oncology Group 1065 Study).

irinotecan and cisplatin as neoadjuvant in locally advanced cervical cancer. *British* 

in nonsquamous carcinoma of the cervix: a Gynecologic Oncology Group study.

piperazinedione as single agents in the treatment of advanced or recurrent nonsquamous cell carcinoma of the cervix: a Gynecologic Oncology Group Study.

with mitomycin-C for advanced or recurrent squamous cell carcinoma of the uterine cervix: the JGOG study. *Gynecologic Oncology,* 95, pp. 127-132, ISSN 0090-


Curtin, JP.; Blessing, JA. & Webster, KD. et al. (2001). Paclitaxel, an active agent in

Study. *Journal of Clinical Oncology*, 19, pp. 1275-1278, ISSN 0732-183X D'Agostino, G.; Distefano, M. & Greggi, S. et al. (2003). Neoadjuvant treatment of locally

*Cancer Chemotherapy and Pharmacology,* 49, pp. 256-260, ISSN 0344-5704 Di Vagno, G.; Cormio, G. & Pignata, S. et al. (2003). Cisplatin and vinorelbine as

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advances carcinoma of the uterine cervix with epirubicin, paclitaxel and cisplatin.

neoadjuvant chemotherapy in locally advanced cervical cancer: a phase II study.

study of gemcitabine and cisplatin combination as induction chemotherapy for untreated locally advanced cervical carcinoma. *Annals of Oncology*, 12, pp. 541-547,

study of multimodality treatment for locally advanced cervical cancer: neoadjuvant carboplatin and paclitaxel followed by radical hysterectomy and adjuvant cisplatin

chemotherapy versus pelvic and para-aortic irradiation for high-risk cervical cancer: an update of radiation therapy oncology group trial (RTOG) 90-01. *Journal* 

concomitant chemotherapy and radiotherapy for cancer of the uterine cervix: a systematic review and meta-analysis. *Lancet*, 358, pp. 781-786, ISSN 0140-6736 Hwang, YY.; Moon, H. & Cho, SH. et al. (2001). Ten-year survival of patients with locally

advanced, stage ib-iib cervical cancer after neoadjuvant chemotherapy and radical

neoadjuvant chemotherapy (NAC) followed by radical hysterectomy (RH) versus RH for bulky stage I/II cervical cancer (JCOG0102). *American Society of Clinical* 

versus radiotherapy for stage Ib-IIa cervical cancer. *Lancet,* 350, pp. 535-540, ISSN

leucovorin in recurrent adenocarcinoma of the cervix: a Gynecologic Oncology

radiotherapy for cervical cancer-a meta analysis. *Journal of Clinical Oncology*, 14,

compared with pelvic and para-aortic radiation for high risk cervical cancer. *The*

*International Journal of Gynecological Cancer* , 13, pp. 308-312, ISSN 1048-891X. Dueñas-Gonzalez, A.; Lopez-Graniel, C. & Gonzalez-Enciso, A. et al. (2001). A phase II

Dueñas-Gonzalez, A.; López-Graniel, C. & González-Enciso, A. et al. (2003). A phase II

Green, JA.; Kirwan, JM. & Tierney, JF. et al. (2001). Survival and recurrence after

Katsumata, N.; Yoshikawa, H. & Hirakawa, T. et al. (2006). Phase III randomized trial of

Landoni, F.; Maneo, A. & Colombo, A. et al. (1997). Randomised study of radical surgery

Look, KY.; Blessing, JA. & Valea, FA. (1997). Phase II trial of 5-fluorouracil and high-dose

Lukka, H.; Hirte, H. & Fyles, A. et al. (2002). Concurrent cisplatin-based chemotherapy plus

Morris, M.; Eifel, PJ. & Lu, J. et al. (1999). Pelvic radiation with concurrent chemotherapy

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hysterectomy. *Gynecologic Oncology,* 83, pp. 88-93, ISSN 0090-8258

chemoradiation. *Annals of Oncology,* 14, pp.1278-1284, ISSN 0923-7534 Eifel, PJ.; Winter, K. & Morris, M. et al. (2004). Pelvic irradiation with concurrent

*of Clinical Oncology*, 22, pp. 872-880, ISSN 0732-183X

*Oncology Annual Meeting Proceedings*, #5013, 2006


**6** 

**Combined Therapy For Squamous Carcinoma** 

**Cells: Application of Porphyrin-Alkaloid** 

Jarmila Králová1, Kamil Záruba2, Pavel Řezanka1, Pavla Poučková3,

Photodynamic therapy (PDT) is an established and useful modality for the clinical noninvasive treatment of cancer. This therapy requires a photosensitizing agent (photosensitizer) selectively taken up by tumor cells, visible light, and molecular oxygen to generate highly reactive oxygen species (ROS), which ultimately cause tumor destruction. The specificity achieved from drug uptake selectivity combined with light targeting makes

PDT consists of three phases: excitation of photosensitizers (PS) by light, production of ROS, and induction of cell death (Triesscheijn et al., 2006). In the first phase, irradiated light of a suitable wavelength, typically visible or near-infrared, excites the PS molecules. The light is generally selected to correspond with the maximum absorption wavelength of the PS. The PS molecules then absorb light energy and change to an excited singlet state. These excited molecules can fall back to their native state with emission of fluorescence. Thus, all PS molecules are also examples of fluorescent molecules. On the other hand, the molecules also have the ability to undergo an electron spin conversion to their triplet state followed by the transfer of this energy to oxygen molecules or to other substrate molecules in the

The fact that sunlight can be used to treat a variety of diseases such as rickets, psoriasis, and skin cancer is known from ancient civilizations, i.e. Egyptian, Chinese and Indian (Ackroyd et al., 2001; Daniell & Hill, 1991; Fitzpatrick & Pathak, 1959). At the beginning of the 20th century the term "photodynamic action" was used by Tappeiner et al. to explain the oxygen-consuming chemical reactions induced by photosensitization (Moan & Peng, 2003; Szeimies et al., 2001). Tappeiner, in cooperation with Jesionek, successfully treated patients

**1. Introduction**

PDT an appealing approach.

**1.1 History of PDT** 

surroundings which then react with oxygen.

**Modified Gold Nanoparticles** 

*4Zentiva Development (Part of Sanofi-Aventis Group)* 

Lenka Veverková1 and Vladimír Král1,4 *1Academy of Sciences of the Czech Republic, 2Institute of Chemical Technology Prague,* 

*3Charles University in Prague,* 

*Czech Republic* 

Whitney, CW.; Sause, W. & Bundy, BN. et al. (1999). Randomized comparison of fluorouracil plus cisplatin versus hydroxyurea as an adjunct to radiation therapy in stage II B-IV A carcinoma of the cervix with negative para-aortic lymph nodes: a Gynecologic Oncology Group and Southwest Oncology Group study. *Journal of Clinical Oncology*, 17, pp.1339-1348, ISSN 0732-183X

## **Combined Therapy For Squamous Carcinoma Cells: Application of Porphyrin-Alkaloid Modified Gold Nanoparticles**

Jarmila Králová1, Kamil Záruba2, Pavel Řezanka1, Pavla Poučková3, Lenka Veverková1 and Vladimír Král1,4

*1Academy of Sciences of the Czech Republic, 2Institute of Chemical Technology Prague, 3Charles University in Prague, 4Zentiva Development (Part of Sanofi-Aventis Group) Czech Republic* 

## **1. Introduction**

92 Squamous Cell Carcinoma

Whitney, CW.; Sause, W. & Bundy, BN. et al. (1999). Randomized comparison of

*Clinical Oncology*, 17, pp.1339-1348, ISSN 0732-183X

fluorouracil plus cisplatin versus hydroxyurea as an adjunct to radiation therapy in stage II B-IV A carcinoma of the cervix with negative para-aortic lymph nodes: a Gynecologic Oncology Group and Southwest Oncology Group study. *Journal of* 

> Photodynamic therapy (PDT) is an established and useful modality for the clinical noninvasive treatment of cancer. This therapy requires a photosensitizing agent (photosensitizer) selectively taken up by tumor cells, visible light, and molecular oxygen to generate highly reactive oxygen species (ROS), which ultimately cause tumor destruction. The specificity achieved from drug uptake selectivity combined with light targeting makes PDT an appealing approach.

> PDT consists of three phases: excitation of photosensitizers (PS) by light, production of ROS, and induction of cell death (Triesscheijn et al., 2006). In the first phase, irradiated light of a suitable wavelength, typically visible or near-infrared, excites the PS molecules. The light is generally selected to correspond with the maximum absorption wavelength of the PS. The PS molecules then absorb light energy and change to an excited singlet state. These excited molecules can fall back to their native state with emission of fluorescence. Thus, all PS molecules are also examples of fluorescent molecules. On the other hand, the molecules also have the ability to undergo an electron spin conversion to their triplet state followed by the transfer of this energy to oxygen molecules or to other substrate molecules in the surroundings which then react with oxygen.

#### **1.1 History of PDT**

The fact that sunlight can be used to treat a variety of diseases such as rickets, psoriasis, and skin cancer is known from ancient civilizations, i.e. Egyptian, Chinese and Indian (Ackroyd et al., 2001; Daniell & Hill, 1991; Fitzpatrick & Pathak, 1959). At the beginning of the 20th century the term "photodynamic action" was used by Tappeiner et al. to explain the oxygen-consuming chemical reactions induced by photosensitization (Moan & Peng, 2003; Szeimies et al., 2001). Tappeiner, in cooperation with Jesionek, successfully treated patients

Combined Therapy For Squamous Carcinoma Cells:

longer-living excited triplet state (T1).

to cell death as well (Foote, 1991).

**1.2 Mechanism of PDT** 

Application of Porphyrin-Alkaloid Modified Gold Nanoparticles 95

PDT requires an interaction of three key elements: light, a photosensitizer, and oxygen. After exposure to particular wavelengths of light, the photosensitizer is excited from a ground state (S0) to an excited singlet state (S1) (Fig. 3) followed by intersystem crossing to a

Fig. 3. Mechanism of PDT; State energies are represented by thick lines:

porphyrin sensitizer, dioxygen; reactive dioxygen intermediates are in bold

After that, the photosensitizer at T1 state is able to go through two types of reaction with nearby molecules: either a type I reaction through hydrogen or electron transfer generating free radicals, or a type II reaction through energy transfer to oxygen, creating molecular singlet oxygen (1O2). The type I reaction results in generation of reactive free radicals or radical ions, which then react with ground-state molecular oxygen to produce superoxide anion radicals, hydrogen peroxides and hydroxyl radicals (Foote, 1991). The type II reaction produces singlet oxygen which has an important role in the molecular processes initiated by PDT (Foote, 1991; Niedre et al., 2002).The singlet oxygen has a lifetime approx. 3 s and can diffuse no more than 0.07 m in cells (Moan, 1990; Hatz at al., 2007). Therefore, the initial damage is limited to the site of the PS molecule. This is usually the mitochondria, Golgi apparatus, plasma membrane, endosomes, lysosomes, and endoplasmic reticulum (Buytaert et al., 2007). Damage to the subcellular organelles and plasma membrane eventually leads to apoptotic, autophagic and/or necrotic cell death. Generally, PS molecules localized to the mitochondria or the endoplasmic reticulum cause apoptosis, while localization either in the plasma membrane or lysosomes is found to delay or block the apoptotic pathway. On the other hand, if the apoptotic route is blocked, damaged cells still die using the autophagic or necrotic pathways (Buytaert et al., 2007; Oleinick et al., 2002). Latest studies support apoptosis as probably the preferred path to cell death (Buytaert et al., 2007). Even though it is considered that 1O2 is the main cytotoxic species and starts the pathway responsible for the damaging effects of PDT, free radicals formed by type I reactions significantly contribute

suffering from stage II syphilis, lupus vulgaris, and superficial skin cancer with topical eosin red solution (Szeimies et al., 2001). In 1942, Auler and Banzer observed specific uptake and retention of hematoporphyrin in tumors followed by higher fluorescence in cancer cells as compared with the surrounding tissue, and induction of necrosis after irradiation (Szeimies et al., 2001). Afterwards, PDT had not been used until Dougherty initiated revitalization by treating a group of patients suffering from cutaneous and subcutaneous tumors with the injection of photosensitizer dihematoporphyrin and red light produced by laser. The majority of the treated tumors showed either complete or partial remission (Dougherty et al., 1975; Dougherty et al., 1978; Szeimies et al., 2001).

Particularly, PDT has grown in reputation in dermatology, mostly due to the simple accessibility of light exposure for the skin and the simplicity of topical use of photosensitizers. In the late 1970s, Thomas Dougherty initiated human clinical trials of PDT with hematoporphyrin derivative (HpD) for the treatment of cutaneous cancer metastases (Blume & Oseroff, 2007; Dougherty, 1996; Zeitouni, 2003). PDT has been revived and has become more applicable to common dermatology since 1990, when Kennedy et al. introduced 5-aminolevulinic acid (ALA) (Fig. 1), a topical porphyrin precursor causing local accumulation of the endogenous photosensitizer protoporphyrin IX (PpIX) (Fig. 2) with no significant prolonged phototoxicity (Kennedy, 1990). Nowadays, PDT is used to treat diseases in a variety of fields, including respiratory medicine (Ost, 2001; Sutedja & Postmus, 1996), urology (Jichlinski, 2006; Juarranz et al., 2008; Pinthus et al., 2006), ophthalmology (Mittra, 2002), and gastroenterology (Barr et al., 2001; Wiedmann & Caca, 2004), as well as dermatology. Mostly porphyrins or phthalocyanines have been studied (Marmur et al., 2004). On the other hand, for dermatological purposes, only hematoporphyrin derivatives such as porfimer sodium, or PpIX-inducing precursors such as ALA or methyl aminolevulinate (MAL) are of useful concern. As systemic photosensitizing drugs caused extended phototoxicity (Marmur et al., 2004), topical photosensitizers are preferred for the use in dermatology. Several drugs containing ALA or MAL are used for treating epithelial cancers and there is an increasing importance in the use of PDT (Braathen, 2001; Dragieva et al., 2004a; Dragieva et al., 2004b).

$$\underbrace{\ast}\_{\mathbb{R}^{\mathsf{N}}} \asymp\_{\mathbb{R}^{\mathsf{N}}}$$

Fig. 1. Structure of 5-aminolevulinic acid

#### **1.2 Mechanism of PDT**

94 Squamous Cell Carcinoma

suffering from stage II syphilis, lupus vulgaris, and superficial skin cancer with topical eosin red solution (Szeimies et al., 2001). In 1942, Auler and Banzer observed specific uptake and retention of hematoporphyrin in tumors followed by higher fluorescence in cancer cells as compared with the surrounding tissue, and induction of necrosis after irradiation (Szeimies et al., 2001). Afterwards, PDT had not been used until Dougherty initiated revitalization by treating a group of patients suffering from cutaneous and subcutaneous tumors with the injection of photosensitizer dihematoporphyrin and red light produced by laser. The majority of the treated tumors showed either complete or partial remission (Dougherty et

Particularly, PDT has grown in reputation in dermatology, mostly due to the simple accessibility of light exposure for the skin and the simplicity of topical use of photosensitizers. In the late 1970s, Thomas Dougherty initiated human clinical trials of PDT with hematoporphyrin derivative (HpD) for the treatment of cutaneous cancer metastases (Blume & Oseroff, 2007; Dougherty, 1996; Zeitouni, 2003). PDT has been revived and has become more applicable to common dermatology since 1990, when Kennedy et al. introduced 5-aminolevulinic acid (ALA) (Fig. 1), a topical porphyrin precursor causing local accumulation of the endogenous photosensitizer protoporphyrin IX (PpIX) (Fig. 2) with no significant prolonged phototoxicity (Kennedy, 1990). Nowadays, PDT is used to treat diseases in a variety of fields, including respiratory medicine (Ost, 2001; Sutedja & Postmus, 1996), urology (Jichlinski, 2006; Juarranz et al., 2008; Pinthus et al., 2006), ophthalmology (Mittra, 2002), and gastroenterology (Barr et al., 2001; Wiedmann & Caca, 2004), as well as dermatology. Mostly porphyrins or phthalocyanines have been studied (Marmur et al., 2004). On the other hand, for dermatological purposes, only hematoporphyrin derivatives such as porfimer sodium, or PpIX-inducing precursors such as ALA or methyl aminolevulinate (MAL) are of useful concern. As systemic photosensitizing drugs caused extended phototoxicity (Marmur et al., 2004), topical photosensitizers are preferred for the use in dermatology. Several drugs containing ALA or MAL are used for treating epithelial cancers and there is an increasing importance in the use of PDT (Braathen, 2001; Dragieva et

H2N OH

O

NH N HN N

H3COOC COOCH3

O

al., 1975; Dougherty et al., 1978; Szeimies et al., 2001).

al., 2004a; Dragieva et al., 2004b).

Fig. 1. Structure of 5-aminolevulinic acid

Fig. 2. Structure of protoporphyrin IX

PDT requires an interaction of three key elements: light, a photosensitizer, and oxygen. After exposure to particular wavelengths of light, the photosensitizer is excited from a ground state (S0) to an excited singlet state (S1) (Fig. 3) followed by intersystem crossing to a longer-living excited triplet state (T1).

After that, the photosensitizer at T1 state is able to go through two types of reaction with nearby molecules: either a type I reaction through hydrogen or electron transfer generating free radicals, or a type II reaction through energy transfer to oxygen, creating molecular singlet oxygen (1O2). The type I reaction results in generation of reactive free radicals or radical ions, which then react with ground-state molecular oxygen to produce superoxide anion radicals, hydrogen peroxides and hydroxyl radicals (Foote, 1991). The type II reaction produces singlet oxygen which has an important role in the molecular processes initiated by PDT (Foote, 1991; Niedre et al., 2002).The singlet oxygen has a lifetime approx. 3 s and can diffuse no more than 0.07 m in cells (Moan, 1990; Hatz at al., 2007). Therefore, the initial damage is limited to the site of the PS molecule. This is usually the mitochondria, Golgi apparatus, plasma membrane, endosomes, lysosomes, and endoplasmic reticulum (Buytaert et al., 2007). Damage to the subcellular organelles and plasma membrane eventually leads to apoptotic, autophagic and/or necrotic cell death. Generally, PS molecules localized to the mitochondria or the endoplasmic reticulum cause apoptosis, while localization either in the plasma membrane or lysosomes is found to delay or block the apoptotic pathway. On the other hand, if the apoptotic route is blocked, damaged cells still die using the autophagic or necrotic pathways (Buytaert et al., 2007; Oleinick et al., 2002). Latest studies support apoptosis as probably the preferred path to cell death (Buytaert et al., 2007). Even though it is considered that 1O2 is the main cytotoxic species and starts the pathway responsible for the damaging effects of PDT, free radicals formed by type I reactions significantly contribute to cell death as well (Foote, 1991).

Combined Therapy For Squamous Carcinoma Cells:

Fig. 6. Structure of lutetium texaphyrin

cutaneous phototoxicity.

**1.4 Nanoparticles in PDT** 

**1.4.1 Active nanoparticles**  Photosensitizer nanoparticles

Application of Porphyrin-Alkaloid Modified Gold Nanoparticles 97

O O

O O

To avoid the prolonged photosensitivity caused by systemic administration, topically applied photosensitizers have been developed for the treatment of skin cancers. The most successful commercially accessible topical drugs are ALA and its methyl ester MAL. Levulan® using ALA and the Blu-U light source was accepted by the U. S. Food and Drug Administration for the treatment of nonhyperkeratotic actinic keratoses of the face and scalp in 1999 (Babilas et al., 2005; Kormeili et al., 2004). MAL was accepted in Europe for topical PDT of actinic keratosis (AK) and basal cell carcinoma (BCC) in 2001 (Morton, 2003; Morton et al., 2002) and for the treatment of AK in the USA in 2004 (Zeitouni et al., 2003; Garcia-Zuazaga et al., 2005). The endogenous photosensitizer PpIX generated from ALA or MAL can be fully metabolized to photodynamically inactive heme over 24–48 h (Blume & Oseroff, 2007; Morton, 2004), which radically decreases the unpleasant side effect of prolonged

In 2002 Konan et al. divided methods of PS molecules delivery into passive and active based on the presence or absence of a targeting molecule on the surface (Konan et al., 2002). The methods employed to bring the PS explicitly into diseased tissues using the target tissue receptors or antigens were designated active, whilst others that enable parenteral administration and passive targeting, such as PS conjugates of oil-dispersions, polymeric particles, liposomes, and hydrophilic polymers, were named passive. Active nanoparticles can be subclassified by their mechanism of activation and passive nanoparticles can be subclassified by material composition into (a) non-polymer-based nanoparticles, e.g. ceramic and metallic nanoparticles, and (b) biodegradable polymer-based nanoparticles.

Quantum dots (QDs) have great photostability, intensive fluorescent emission (high quantum yields) and possible use in specific pathological fields. They can be water soluble, and transfer energy to surrounding oxygen with resulting cellular toxicity. Many studies have been devoted to this field (Bakalova et al., 2004). The first report deals with cadmium selenide (CdSe) QDs and was published by Samia at al. in 2002. The authors presented the possibility to use semiconductor QDs alone to generate 1O2 due to the intercalation of dissolved oxygen at the QD surface (Samia et al., 2003). They predicted a comparable interaction in water-soluble phospholipid-capped QDs. Moreover, they assumed that since the lowest excited state of CdSe QDs is a triplet state, the energy transfer was responsible for

O

O

<sup>O</sup> <sup>O</sup>

O

N N

HO

O

HO

O O

N

Lu

N N

#### **1.3 Photosensitizers in PDT**

The first generation of PS molecules was represented by HpD or its purified version porfimer sodium (Photofrin) (Fig. 4). Primarily, they were used as general PS and tested for cutaneous malignancies. On the other hand, general intravenous administration and the consequential prolonged phototoxicity, which can last 6–10 weeks, restricted their use (Dragieva et al., 2004; Fritsch et al., 1998).

Fig. 4. Structure of Photofrin

Second generation PS molecules such as *m*-tetrahydroxyphenyl-chlorin, tin ethyl etiopurpurin, phthalocyanines, and chlorins (Fig. 5) are pure compounds that can be activated by light wavelengths in the range of 660–690 nm. Most significantly, they all have a lower tendency to cause prolonged photosensitivity compared with the first generation of photosensitizers (Moan & Berg, 1992).

Fig. 5. Structure of *m*-tetrahydroxyphenyl-chlorin (a), tin ethyl etiopurpurin (b), phthalocyanines (c), and chlorins (d)

Third generation PS molecules (not yet approved) consist of antibody-conjugated PS (Josefsen & Boyle, 2008) and lutetium texaphyrin (Fig. 6) (Woodburn et al., 1998; Young et al., 1996). These drugs supporting deeper penetration into tissue with absorptions of 700– 800 nm accumulate in tumor tissues with high selectivity.

Fig. 6. Structure of lutetium texaphyrin

The first generation of PS molecules was represented by HpD or its purified version porfimer sodium (Photofrin) (Fig. 4). Primarily, they were used as general PS and tested for cutaneous malignancies. On the other hand, general intravenous administration and the consequential prolonged phototoxicity, which can last 6–10 weeks, restricted their use

> NH N HN N

NaOOC <sup>n</sup>

Second generation PS molecules such as *m*-tetrahydroxyphenyl-chlorin, tin ethyl etiopurpurin, phthalocyanines, and chlorins (Fig. 5) are pure compounds that can be activated by light wavelengths in the range of 660–690 nm. Most significantly, they all have a lower tendency to cause prolonged photosensitivity compared with the first generation of

n = 0 to 6

R

O

O

N N

Sn Cl

Cl

N

Fig. 5. Structure of *m*-tetrahydroxyphenyl-chlorin (a), tin ethyl etiopurpurin (b),

O O

**a b c d**

Third generation PS molecules (not yet approved) consist of antibody-conjugated PS (Josefsen & Boyle, 2008) and lutetium texaphyrin (Fig. 6) (Woodburn et al., 1998; Young et al., 1996). These drugs supporting deeper penetration into tissue with absorptions of 700–

NH N HN N

R

COONa

NH N HN

COONa

O

NH N N

<sup>N</sup> <sup>N</sup>

N

N HN N

N

**1.3 Photosensitizers in PDT** 

(Dragieva et al., 2004; Fritsch et al., 1998).

NaOOC

NaOOC

R =

Fig. 4. Structure of Photofrin

NH N HN N

OH

HO

NH N HN N

R

and / or

OH

photosensitizers (Moan & Berg, 1992).

HO

phthalocyanines (c), and chlorins (d)

OH

800 nm accumulate in tumor tissues with high selectivity.

To avoid the prolonged photosensitivity caused by systemic administration, topically applied photosensitizers have been developed for the treatment of skin cancers. The most successful commercially accessible topical drugs are ALA and its methyl ester MAL. Levulan® using ALA and the Blu-U light source was accepted by the U. S. Food and Drug Administration for the treatment of nonhyperkeratotic actinic keratoses of the face and scalp in 1999 (Babilas et al., 2005; Kormeili et al., 2004). MAL was accepted in Europe for topical PDT of actinic keratosis (AK) and basal cell carcinoma (BCC) in 2001 (Morton, 2003; Morton et al., 2002) and for the treatment of AK in the USA in 2004 (Zeitouni et al., 2003; Garcia-Zuazaga et al., 2005). The endogenous photosensitizer PpIX generated from ALA or MAL can be fully metabolized to photodynamically inactive heme over 24–48 h (Blume & Oseroff, 2007; Morton, 2004), which radically decreases the unpleasant side effect of prolonged cutaneous phototoxicity.

## **1.4 Nanoparticles in PDT**

In 2002 Konan et al. divided methods of PS molecules delivery into passive and active based on the presence or absence of a targeting molecule on the surface (Konan et al., 2002). The methods employed to bring the PS explicitly into diseased tissues using the target tissue receptors or antigens were designated active, whilst others that enable parenteral administration and passive targeting, such as PS conjugates of oil-dispersions, polymeric particles, liposomes, and hydrophilic polymers, were named passive. Active nanoparticles can be subclassified by their mechanism of activation and passive nanoparticles can be subclassified by material composition into (a) non-polymer-based nanoparticles, e.g. ceramic and metallic nanoparticles, and (b) biodegradable polymer-based nanoparticles.

## **1.4.1 Active nanoparticles**

Photosensitizer nanoparticles

Quantum dots (QDs) have great photostability, intensive fluorescent emission (high quantum yields) and possible use in specific pathological fields. They can be water soluble, and transfer energy to surrounding oxygen with resulting cellular toxicity. Many studies have been devoted to this field (Bakalova et al., 2004). The first report deals with cadmium selenide (CdSe) QDs and was published by Samia at al. in 2002. The authors presented the possibility to use semiconductor QDs alone to generate 1O2 due to the intercalation of dissolved oxygen at the QD surface (Samia et al., 2003). They predicted a comparable interaction in water-soluble phospholipid-capped QDs. Moreover, they assumed that since the lowest excited state of CdSe QDs is a triplet state, the energy transfer was responsible for

Combined Therapy For Squamous Carcinoma Cells:

Fig. 7. Structure of zinc pthalocyanine

Non-biodegradable nanoparticle carriers

**1.4.2 Passive nanoparticles** 

Application of Porphyrin-Alkaloid Modified Gold Nanoparticles 99

the red emission peak for the upconversion nanoparticles. Creation of 1O2 by irradiation of the ZnPC-nanoparticle complex with 980 nm light was confirmed through the

N

N N N Zn

In 2003 Roy et al. first reported ceramic-based nanoparticles used as a new drug-carrier system for PDT. It utilizes 30-nm silica-based spherical particles doped with the anticancer

drug 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (Fig. 8) (Roy et al., 2003).

NH N H N N

Fig. 8. Structure of 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide

O

Irradiation of the nanoparticles with light of appropriate wavelength led to efficient creation of singlet oxygen. On the other hand, noncovalent adsorption of PS into porous silica nanoparticles led to drug leakage. Covalent bonding of the PS into organically modified silica nanoparticles produced more stable material (Ohulchanskyy et al., 2007). Organically modified silica nanoparticles were also used for two-photon dye encapsulation (Kim et al., 2007). Cinteza et al. described a combination of magnetism and PDT using micellar polymeric diacylphospholipid-poly(ethylene glycol) capsules for encapsulation of the 2-devinyl-2-(1 hexyloxyethyl)pyropheophorbide PS and magnetic Fe3O4 nanoparticles (Cinteza et al., 2006). In contrast to the previous report (Kim et al., 2007), the magnetic nanoparticles were used for targeted delivery of PS to tumor cells and increased imaging (Cinteza et al., 2006). Wieder at el. described a delivery system consisting of gold nanoparticles modified with phthalocyanine (Wieder et al., 2006). Phthalocyanine derivative-modified gold nanaoparticles have 2-4 nm in diameter and have a maximum absorption peak at 695 nm. They generated 1O2 catalytically with high efficiency. Upon irradiation of these nanoparticles, significant improvement in PDT

OH O

O

photobleaching of disodium 9,10-anthracenedipropionic acid (Wieder et al., 2006).

N N N

N

the generation of singlet oxygen (1O2) from triplet oxygen (3O2). On the other hand, the efficiency of generation of 1O2 was about 5% (with 65% emission quantum yield of QDs) as compared to 43% for the PS only. It may be due to carrier trapping and nonradiative carrier recombinations occurring on the early picosecond time scale and the very small fraction of QD - 3O2 pairs created at any moment (Samia et al., 2003). To avoid the ineffectiveness of QDs alone to produce singlet oxygen, several experiments have been carried out to covalently conjugate PSs to CdSe/ZnS via organic bridges (Hsieh et al., 2006; Samia et al., 2003). These experiments have a frequent problem with lower water solubility.

Self-lighting nanoparticles

Scintillation or persistent luminescence nanoparticles with attached PS molecules such as porphyrins were applied as *in vivo* agents for PDT (Auzel, 2004). After exposure to ionizing radiation such as X-rays, scintillation luminescence is produced from the nanoparticles and stimulates the photosensitizers, followed by production of singlet oxygen that increases the destruction of cancer cells by ionizing radiation. Employment of common radiation therapy with PDT allows application of lower doses of radiation. Using BaFBr:Eu+,Mn+ nanoparticles displaying luminescence, short X-ray exposures could be applied followed by extended PS excitation. The period of phosphorescent decay is increased *in vivo* due to higher local temperatures (Chen et al., 2006).

Upconversion nanoparticles

Upconversion and simultaneous two-photon absorption occurs in luminescent materials with triplet excitation states (Auzel, 2004). Upconverting nanoparticles are modified nanometer-sized composites that generate higher energy light from lower energy radiation typically near or middle infrared (anti-Stokes emission) using transition metal ions doped into a solid-state host (Boyer et al., 2006; Pires et al., 2006). For biological use, the desired nanocrystalline core should have morphological and optical features that are appropriate for conjugation with biological molecules and exhibit high intensity emission as well (Pires et al., 2006). Preparation of high-quality nanocrystals is needed, and the surface properties and growth dynamics must be precisely controlled (Wang et al., 2006). Upconversion nanoparticles can be prepared via numerous different ionic materials – typically rare earth ions such as lanthanides and actinides doped in a suitable crystalline matrix (Zijlmans et al., 1999). Micrometer-sized Er3+/Yb3+ or Tm3+/Yb3+ co-doped hexagonal NaYF4 are examples of nanoparticles that exhibit the highest upconversion efficiencies (Heer et al., 2004) and are precursors of upconverting nanoparticles with biological applications (Zhang et al., 2006). In 2006, the NaYF4 nanocrystals doped with Er and Yb and coated with organic polymers were prepared and strong emission upon activation with 980 nm NIR laser was shown (Feng et al., 2006). One year later, Zhang et al. used upconverting nanoparticles (nanoparticles of NaYF4:Yb3+,Er3+ coated with a porous thin layer of silica doped with merocyanine and functionalized with a tumor-targeting antibody) in PDT, but these nanoparticles were not activated in depth in animal tissue and the efficiency in killing cancer cells was very low (Zhang et al., 2007).

Another class of employed upconversion nanoparticles consists of zinc pthalocyanine (ZnPC) (Fig. 7) physically adsorbed to the surface of the nanoparticles with the encapsulation efficiency of 98 % (Ricci-Junior & Marchetti, 2006b). The fluorescence excitation spectrum of ZnPC exhibits an excitation maximum at 670 nm and greatly overlaps the red emission peak for the upconversion nanoparticles. Creation of 1O2 by irradiation of the ZnPC-nanoparticle complex with 980 nm light was confirmed through the photobleaching of disodium 9,10-anthracenedipropionic acid (Wieder et al., 2006).

Fig. 7. Structure of zinc pthalocyanine

## **1.4.2 Passive nanoparticles**

98 Squamous Cell Carcinoma

the generation of singlet oxygen (1O2) from triplet oxygen (3O2). On the other hand, the efficiency of generation of 1O2 was about 5% (with 65% emission quantum yield of QDs) as compared to 43% for the PS only. It may be due to carrier trapping and nonradiative carrier recombinations occurring on the early picosecond time scale and the very small fraction of QD - 3O2 pairs created at any moment (Samia et al., 2003). To avoid the ineffectiveness of QDs alone to produce singlet oxygen, several experiments have been carried out to covalently conjugate PSs to CdSe/ZnS via organic bridges (Hsieh et al., 2006; Samia et al.,

Scintillation or persistent luminescence nanoparticles with attached PS molecules such as porphyrins were applied as *in vivo* agents for PDT (Auzel, 2004). After exposure to ionizing radiation such as X-rays, scintillation luminescence is produced from the nanoparticles and stimulates the photosensitizers, followed by production of singlet oxygen that increases the destruction of cancer cells by ionizing radiation. Employment of common radiation therapy with PDT allows application of lower doses of radiation. Using BaFBr:Eu+,Mn+ nanoparticles displaying luminescence, short X-ray exposures could be applied followed by extended PS excitation. The period of phosphorescent decay is increased *in vivo* due to

Upconversion and simultaneous two-photon absorption occurs in luminescent materials with triplet excitation states (Auzel, 2004). Upconverting nanoparticles are modified nanometer-sized composites that generate higher energy light from lower energy radiation typically near or middle infrared (anti-Stokes emission) using transition metal ions doped into a solid-state host (Boyer et al., 2006; Pires et al., 2006). For biological use, the desired nanocrystalline core should have morphological and optical features that are appropriate for conjugation with biological molecules and exhibit high intensity emission as well (Pires et al., 2006). Preparation of high-quality nanocrystals is needed, and the surface properties and growth dynamics must be precisely controlled (Wang et al., 2006). Upconversion nanoparticles can be prepared via numerous different ionic materials – typically rare earth ions such as lanthanides and actinides doped in a suitable crystalline matrix (Zijlmans et al., 1999). Micrometer-sized Er3+/Yb3+ or Tm3+/Yb3+ co-doped hexagonal NaYF4 are examples of nanoparticles that exhibit the highest upconversion efficiencies (Heer et al., 2004) and are precursors of upconverting nanoparticles with biological applications (Zhang et al., 2006). In 2006, the NaYF4 nanocrystals doped with Er and Yb and coated with organic polymers were prepared and strong emission upon activation with 980 nm NIR laser was shown (Feng et al., 2006). One year later, Zhang et al. used upconverting nanoparticles (nanoparticles of NaYF4:Yb3+,Er3+ coated with a porous thin layer of silica doped with merocyanine and functionalized with a tumor-targeting antibody) in PDT, but these nanoparticles were not activated in depth in animal tissue and the efficiency in killing cancer cells was very low

Another class of employed upconversion nanoparticles consists of zinc pthalocyanine (ZnPC) (Fig. 7) physically adsorbed to the surface of the nanoparticles with the encapsulation efficiency of 98 % (Ricci-Junior & Marchetti, 2006b). The fluorescence excitation spectrum of ZnPC exhibits an excitation maximum at 670 nm and greatly overlaps

2003). These experiments have a frequent problem with lower water solubility.

Self-lighting nanoparticles

higher local temperatures (Chen et al., 2006).

Upconversion nanoparticles

(Zhang et al., 2007).

Non-biodegradable nanoparticle carriers

In 2003 Roy et al. first reported ceramic-based nanoparticles used as a new drug-carrier system for PDT. It utilizes 30-nm silica-based spherical particles doped with the anticancer drug 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (Fig. 8) (Roy et al., 2003).

Fig. 8. Structure of 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide

Irradiation of the nanoparticles with light of appropriate wavelength led to efficient creation of singlet oxygen. On the other hand, noncovalent adsorption of PS into porous silica nanoparticles led to drug leakage. Covalent bonding of the PS into organically modified silica nanoparticles produced more stable material (Ohulchanskyy et al., 2007). Organically modified silica nanoparticles were also used for two-photon dye encapsulation (Kim et al., 2007). Cinteza et al. described a combination of magnetism and PDT using micellar polymeric diacylphospholipid-poly(ethylene glycol) capsules for encapsulation of the 2-devinyl-2-(1 hexyloxyethyl)pyropheophorbide PS and magnetic Fe3O4 nanoparticles (Cinteza et al., 2006). In contrast to the previous report (Kim et al., 2007), the magnetic nanoparticles were used for targeted delivery of PS to tumor cells and increased imaging (Cinteza et al., 2006). Wieder at el. described a delivery system consisting of gold nanoparticles modified with phthalocyanine (Wieder et al., 2006). Phthalocyanine derivative-modified gold nanaoparticles have 2-4 nm in diameter and have a maximum absorption peak at 695 nm. They generated 1O2 catalytically with high efficiency. Upon irradiation of these nanoparticles, significant improvement in PDT

Combined Therapy For Squamous Carcinoma Cells:

**1.5 Combined therapy** 

problematic side effects.

et al., 2002; Zhou et al., 2005).

nanotechnology.

**1.6 Light sources in PDT** 

parts of tumors (Juzeniene et al., 2004).

Application of Porphyrin-Alkaloid Modified Gold Nanoparticles 101

Even if PDT has been used effectively for treating various tumors, it still has several restrictive factors for a target-specific response, such as an observed angiogenic effect and pronounced inflammatory reaction after PDT treatment (Pervaiz & Olivo, 2006). PDT in combination with other types of therapy is an attractive approach to suppress these

PDT-induced hypoxia has been associated with an increase in the expression of many angiogenic growth factors, such as hypoxia-inducible factor 1 (HIF-1), fibroblast growth factor receptor-1(FGFR-1), cyclooxigenase-2 (COX-2), and vascular endothelial growth factor (VEGF). Combination therapy using antiangiogenic agents (e.g., COX-2 or VEGF inhibitors) with PDT led to a significant decrease of PDT-induced expression of prostaglandin E2 and VEGF, as well as a marked improvement in tumoricidal response (Akita et al., 2004; Ferrario

In contrast to radiotherapy, surgery or chemotherapy, PDT can lead to a strong acute inflammatory response, generally as tumor-localized edema. This PDT-induced immune activation makes it possible to positively reverse the tumor–host relationship from one that is tumor dominated to one that is oriented against the tumor. The combination with immunotherapy can reinforce the immune response triggered by PDT and thus significantly improve the anti-tumor immune response (Pervaiz & Olivo, 2006). Numerous recent clinical trials conclude that enhanced clinical outcomes can be achieved by a combination of ALA-PDT and immunomodulation therapy for the treatment of premalignant skin diseases, such

In several cases, combination therapy can be done by linking the photosensitizer directly to an anticancer drug or to a specific antibody to target highly tumor-expressed receptors (Palumbo, 2007). It would also be easily accomplished by combining them using

A variety of light sources that are used in PDT consist of light-emitting diodes (LEDs), filtered xenon arc and metal halide lamps, fluorescent lamps, and lasers. Lasers and filtered broadband sources provide comparable efficacy in topical PDT (Clark et al., 2003). Non-laser light sources are also important in topical PDT, because in contrast to lasers they are stable, cheap, and offer broad-area illumination fields. Recently, LEDs showed significant progress in design, creating these low-cost sources suitable for broad-area irradiation, and were accepted for patient use. These LEDs are focused on the 630-to-635-nm activation peak of PpIX while excluding the inappropriate wavelengths present in broadband sources, thus allowing shorter irradiation times. Biophysical calculations show that LEDs with peak emission of 631 ± 2 nm can have a deeper PDT action in tissue than filtered halogen lamps with 560–740 nm emission, and hence LEDs may be more successful in treating the deeper

PpIX has its main absorption peak in the blue region at 410 nm (Soret band) with smaller absorption peaks at 505, 540, 580 and 630 nm. Most light sources for PDT seek to utilize the 630-nm absorption peak, in order to improve tissue penetration. On the other hand, a blue fluorescent lamp (peak emission 417 nm) is usually used. Nowadays, there are several reports

as Bowen's disease (BD), BCC and AK (Wang et al., 2007; Wang et al., 2008).

efficiency was observed, probably thanks to 50% increase of 1O2 quantum yields as compared to the free PS. In the same year El-Sayed reported efficient conversion of strongly absorbed light by plasmonic gold nanoparticles to heat energy. Easy bioconjugation of nanoparticles used suggests their application as selective photothermal agents in molecular cancer cell targeting (El-Sayed et al., 2006).

Two-photon dyes have received attention lately because of their ability to convert absorbed low-energy radiation to higher energy emissions. Dyes that can direct transfer of the higher energy to molecular oxygen for generation of 1O2 can be very useful in PDT because they can be activated in deep tissues. The first use of two-photon dyes that are able to convert absorbed low-energy radiation to higher-energy emissions was recently reported using microemulsion to incorporate the two-photon dye porphyrin tetra(*p*-toluenesulfonate) into polyacrylamide nanoparticles (Gao et al., 2006).

Biodegradable nanoparticle carriers

Biodegradable polymeric nanoparticles allow high drug loading and controlled drug release. They exist in a large variety of materials (Konan et al., 2002). Modifying the surface of nanoparticles with polymers such as poly(ethylene glycol) and poly(ethylene oxide) increases circulation times (McCarthy et al., 2005). Brasseur et al. described hematoporphyrin adsorbed in polyalkylcyanoacrylate nanoparticles (Brasseur et al., 1991), but the resulting materials showed poor carrier capacity and rapid drug release. Encapsulation of tetrasulfonated zinc phthalocyanine or aluminium naphthalocyanine into poly(isobutylcyanoacrylate) or poly(ethylbutylcyanoacrylate) nanocapsules or nanosphere was published in the same year (Labib et al., 1991). Then, second generation phthalocyanine derivatives were used in PEG-poly(lactic acid) nanoparticles (Allemann et al., 1995). The results showed that immobilization in the biodegradable nanoparticle improved PDT response of the tumor in contrast to conventional Cremophor EL emulsion by providing prolonged tumor sensitivity towards PDT (Allemann et al., 1995). After a few years, Konan et al. developed polyester poly(D,L-lactide-coglycolide) and poly(D,L-lactide) doped with PS with much higher loading than ever published before (Konan et al., 2003a; Konan et al., 2003b). In order to further investigate these nanoparticles, the efficacy of the encapsulated drug was assessed on the chick embryo chorioallantoic membrane model (Vargas et al., 2004). In another work the *in vitro* and *in vivo* photodynamic activities of verteporfin-loaded poly(D,L-lactide-coglycolide) nanoparticles were studied. The results showed improved photodynamic activity of PS (Konan-Kouakou et al., 2005).

The problem with side photosensitivity due to non-specific localization of the PS into healthy tissue or skin was studied by McCarthy et al., who developed a new nano-agent that has several desirable properties for use as photodynamic drug including no toxicity in extracellular spaces and time-dependent intracellular release of PS (McCarthy et al., 2005). They demonstrated in cell culture that the phototoxicity caused by non-internalized nanoparticles is minimal (9% cell death) in contrast to the effect of internalized nanoparticles (95% cell death under identical testing conditions) (Dougherty et al., 1978). In another study Ricci-Junior et al. reported the preparation, characterization, and results of the phototoxicity assay of poly(D,L-lactide-coglycolide) nanoparticles containing ZnPC for PDT use (Ricci-Junior & Marchetti, 2006a). Other photosensitizers that have been studied consist of Indocyanine green (Saxena et al., 2006) and Hypericin (Zeisser-Labouebe et al., 2006). These compounds have the potential to be used for both diagnostic and therapeutic purposes.

## **1.5 Combined therapy**

100 Squamous Cell Carcinoma

efficiency was observed, probably thanks to 50% increase of 1O2 quantum yields as compared to the free PS. In the same year El-Sayed reported efficient conversion of strongly absorbed light by plasmonic gold nanoparticles to heat energy. Easy bioconjugation of nanoparticles used suggests their application as selective photothermal agents in molecular cancer cell

Two-photon dyes have received attention lately because of their ability to convert absorbed low-energy radiation to higher energy emissions. Dyes that can direct transfer of the higher energy to molecular oxygen for generation of 1O2 can be very useful in PDT because they can be activated in deep tissues. The first use of two-photon dyes that are able to convert absorbed low-energy radiation to higher-energy emissions was recently reported using microemulsion to incorporate the two-photon dye porphyrin tetra(*p*-toluenesulfonate) into

Biodegradable polymeric nanoparticles allow high drug loading and controlled drug release. They exist in a large variety of materials (Konan et al., 2002). Modifying the surface of nanoparticles with polymers such as poly(ethylene glycol) and poly(ethylene oxide) increases circulation times (McCarthy et al., 2005). Brasseur et al. described hematoporphyrin adsorbed in polyalkylcyanoacrylate nanoparticles (Brasseur et al., 1991), but the resulting materials showed poor carrier capacity and rapid drug release. Encapsulation of tetrasulfonated zinc phthalocyanine or aluminium naphthalocyanine into poly(isobutylcyanoacrylate) or poly(ethylbutylcyanoacrylate) nanocapsules or nanosphere was published in the same year (Labib et al., 1991). Then, second generation phthalocyanine derivatives were used in PEG-poly(lactic acid) nanoparticles (Allemann et al., 1995). The results showed that immobilization in the biodegradable nanoparticle improved PDT response of the tumor in contrast to conventional Cremophor EL emulsion by providing prolonged tumor sensitivity towards PDT (Allemann et al., 1995). After a few years, Konan et al. developed polyester poly(D,L-lactide-coglycolide) and poly(D,L-lactide) doped with PS with much higher loading than ever published before (Konan et al., 2003a; Konan et al., 2003b). In order to further investigate these nanoparticles, the efficacy of the encapsulated drug was assessed on the chick embryo chorioallantoic membrane model (Vargas et al., 2004). In another work the *in vitro* and *in vivo* photodynamic activities of verteporfin-loaded poly(D,L-lactide-coglycolide) nanoparticles were studied. The results showed improved

The problem with side photosensitivity due to non-specific localization of the PS into healthy tissue or skin was studied by McCarthy et al., who developed a new nano-agent that has several desirable properties for use as photodynamic drug including no toxicity in extracellular spaces and time-dependent intracellular release of PS (McCarthy et al., 2005). They demonstrated in cell culture that the phototoxicity caused by non-internalized nanoparticles is minimal (9% cell death) in contrast to the effect of internalized nanoparticles (95% cell death under identical testing conditions) (Dougherty et al., 1978). In another study Ricci-Junior et al. reported the preparation, characterization, and results of the phototoxicity assay of poly(D,L-lactide-coglycolide) nanoparticles containing ZnPC for PDT use (Ricci-Junior & Marchetti, 2006a). Other photosensitizers that have been studied consist of Indocyanine green (Saxena et al., 2006) and Hypericin (Zeisser-Labouebe et al., 2006). These compounds have the potential to be used for both diagnostic and therapeutic purposes.

targeting (El-Sayed et al., 2006).

polyacrylamide nanoparticles (Gao et al., 2006).

photodynamic activity of PS (Konan-Kouakou et al., 2005).

Biodegradable nanoparticle carriers

Even if PDT has been used effectively for treating various tumors, it still has several restrictive factors for a target-specific response, such as an observed angiogenic effect and pronounced inflammatory reaction after PDT treatment (Pervaiz & Olivo, 2006). PDT in combination with other types of therapy is an attractive approach to suppress these problematic side effects.

PDT-induced hypoxia has been associated with an increase in the expression of many angiogenic growth factors, such as hypoxia-inducible factor 1 (HIF-1), fibroblast growth factor receptor-1(FGFR-1), cyclooxigenase-2 (COX-2), and vascular endothelial growth factor (VEGF). Combination therapy using antiangiogenic agents (e.g., COX-2 or VEGF inhibitors) with PDT led to a significant decrease of PDT-induced expression of prostaglandin E2 and VEGF, as well as a marked improvement in tumoricidal response (Akita et al., 2004; Ferrario et al., 2002; Zhou et al., 2005).

In contrast to radiotherapy, surgery or chemotherapy, PDT can lead to a strong acute inflammatory response, generally as tumor-localized edema. This PDT-induced immune activation makes it possible to positively reverse the tumor–host relationship from one that is tumor dominated to one that is oriented against the tumor. The combination with immunotherapy can reinforce the immune response triggered by PDT and thus significantly improve the anti-tumor immune response (Pervaiz & Olivo, 2006). Numerous recent clinical trials conclude that enhanced clinical outcomes can be achieved by a combination of ALA-PDT and immunomodulation therapy for the treatment of premalignant skin diseases, such as Bowen's disease (BD), BCC and AK (Wang et al., 2007; Wang et al., 2008).

In several cases, combination therapy can be done by linking the photosensitizer directly to an anticancer drug or to a specific antibody to target highly tumor-expressed receptors (Palumbo, 2007). It would also be easily accomplished by combining them using nanotechnology.

## **1.6 Light sources in PDT**

A variety of light sources that are used in PDT consist of light-emitting diodes (LEDs), filtered xenon arc and metal halide lamps, fluorescent lamps, and lasers. Lasers and filtered broadband sources provide comparable efficacy in topical PDT (Clark et al., 2003). Non-laser light sources are also important in topical PDT, because in contrast to lasers they are stable, cheap, and offer broad-area illumination fields. Recently, LEDs showed significant progress in design, creating these low-cost sources suitable for broad-area irradiation, and were accepted for patient use. These LEDs are focused on the 630-to-635-nm activation peak of PpIX while excluding the inappropriate wavelengths present in broadband sources, thus allowing shorter irradiation times. Biophysical calculations show that LEDs with peak emission of 631 ± 2 nm can have a deeper PDT action in tissue than filtered halogen lamps with 560–740 nm emission, and hence LEDs may be more successful in treating the deeper parts of tumors (Juzeniene et al., 2004).

PpIX has its main absorption peak in the blue region at 410 nm (Soret band) with smaller absorption peaks at 505, 540, 580 and 630 nm. Most light sources for PDT seek to utilize the 630-nm absorption peak, in order to improve tissue penetration. On the other hand, a blue fluorescent lamp (peak emission 417 nm) is usually used. Nowadays, there are several reports

Combined Therapy For Squamous Carcinoma Cells:

**2.1 Preparation of modified gold nanoparticles** 

NH N HN N

Br Br

Br Br

**2.2 Cell culture and** *in vitro* **experiments** 

N O

H3CO OCH3

N O

H3CO OCH3

O

N O

N

Br

H3CO OCH3

O

H3CO OCH3

N O

O

**1 2**

4T1 (mouse mammary carcinoma) and A431 (epidermal squamous carcinoma) cells were purchased from ATCC and PE/CA-PJ34 (human basaloid squamous cell carcinoma) cells were purchased from ETCC. As described before (Králová et al., 2006), all cells were grown exponentially in RPMI 1640 medium with 10% fetal calf serum. For photodynamic experiments, 1–1.5 x 105 cells were seeded into 1.8 cm-2 wells and incubated overnight with the porphyrin–brucine conjugates or their counterparts immobilized on gold nanoparticles (1 and 2.5 M). After incubation, cells were rinsed with PBS, cultured for 1 h in fresh medium without phenol red and illuminated with a 75 W halogen lamp with a band-pass filter (Andover, Salem, NH) that emitted light at wavelengths between 500–520 nm. The

N

Br

NH N HN N

N O

H3CO OCH3

O

N O

H3CO OCH3

N

Br

O

N

Br

O

N

N

*vitro* and *in vivo* studies.

**2. Experimental** 

N O

H3CO OCH3

O

N O

H3CO OCH3

O

N

N

Fig. 9. The structure of **1** and **2**

Application of Porphyrin-Alkaloid Modified Gold Nanoparticles 103

combination of PDT and thermo-effect, and iii) verification of the biological activity by *in* 

Porphyrin–brucine conjugates **1** and **2** (Fig. 9) were prepared according to the procedure described previously (Král et al., 2005). Gold nanoparticles (14.7 nm) were prepared by citrate reduction of potassium tetrachloroaurate(III) (**Au-citr**). After modification with 3- mercaptopropanoic acid, derivatives **1** and **2** were immobilized as described elsewhere (Řezanka et al., 2008). Here, a solution of **1** or **2** (5 mg) in methanol was added to 50 ml of **Aucitr**. Modified nanoparticles (**Au-1** and **Au-2**, respectively) were isolated by centrifugation after three days of incubation. Using redispersion in methanol, methanol–water, water and dimethylsulfoxide, unbound porphyrin derivatives were removed and **Au-1** and **Au-2** molecules were concentrated to a volume of 1 ml. According to the spectral analysis of supernatants, 0.8 mg of **1** or **2** was present in the final 1 ml solution of **Au-1** and **Au-2** nanoparticles. The core of modified nanoparticles was characterized by transmission electron microscopy and photon cross-correlation spectroscopy (Nanophox). The chemical modification, ligand, was analyzed by absorption and fluorescence spectrometry. Fluorescence spectra were recorded using a Fluoromax spectrometer (Jobin-Yvon, Japan). A volume of 1 ml of sample was placed into 1 cm plastic cuvettes. The excitation wavelength was 520 nm.

that blue, green, and red light itself can be efficient in topical PDT of AK; however, the more deeply penetrating red light is better when treating BD and BCC (Morton et al., 2002).

The concept of ambulatory PDT to decrease hospital attendance for PDT was described by Moseley et al. (Moseley et al., 2006). In a study of five patients with BD, PDT was carried out with ALA and a portable LEDs device. Current studies have suggested that pulsed light therapy may be helpful for treatment in topical PDT of acne, AK and photorejuvenation. On the other hand, a recent controlled investigative study carried out in healthy human skin *in vivo* demonstrated that two pulsed light sources formerly reported in PDT brought evidence of minimal activation of photosensitizer, with a significantly smaller photodynamic reaction than observed with a conventional continuous wave broadband source (Strasswimmer & Grande, 2006). These sources deliver intense light in short periods (< 20 ms), which might suppress oxygen consumption (Kawauchi et al., 2004). Unplanned ambient light exposure may have considerably contributed to the clinical effect. However, three studies have recently addressed the possibility of using ambient light for ALA-PDT of AK (Batchelor et al., 2007; Marcus et al., 2007; Strasswimmer & Grande, 2006). Two of them report on therapeutic advantage. Nevertheless, the randomized ambient light-controlled study using ALA demonstrated no significant effect on lesion ablation. A randomized rightleft intrapatient evaluation of conventional MAL-PDT combined with LEDs device *versus* daylight (for 2.5 h) for the treatment of AK of face and scalp demonstrated corresponding reduction in AK and significantly less pain with daylight (Wiegell et al., 2008).

Total effective light dosage is proposed as a concept for optimizing the accuracy of light dosimetry in PDT considering incident spectral irradiance and optical transmission through tissue and absorption by PS (Moseley, 1996). Actually, light dosimetry is explained as the irradiance rate (mW cm–2) at the skin surface and the total dosage (J cm–2) distributed to the surface, the second being a product of irradiance and time of exposure.

It has been suggested that lower fluence rates and fractionation of light exposure can improve lesional reaction by promotion of the photodynamic reaction (Henderson et al., 2004). A study of superficial BCC illuminated with 45 J cm–2 at 4 h and repeated at 6 h with 633-nm laser light at 50 mW cm–2 showed a total response of 84 % after a mean of 59 months (Star at el., 2006). Newer studies support advantages of the fractionation approach in BCC, although not in BD (Haas et al., 2006; Haas et al., 2007).

## **1.7 Synthetic** *meso***-tetraphenylporphyrins in PDT**

Extensive information about the application of various porphyrins and their derivatives in PDT has been published (Král et al., 2006). Accordingly, our laboratory synthesized porphyrin conjugates with glycol (Králová et al., 2008a), bile acid (Králová et al., 2008b), and cyclodextrins (Králová et al., 2006) and their *in vitro* and *in vivo* PDT activity has been tested. It was shown that these porphyrin conjugates are taken up preferentially by tumor cells and have the potential to be used for PDT to selectively ablate tumors (Králová et al., 2006; Králová et al., 2008a; Králová et al., 2008b).

Our contemporary strategy is to combine favorable features of gold nanoparticles mediating the photothermal effect with a photosensitizing compound mediating the photodynamic effect into one combined modality and thus introduce a therapeutic protocol efficient against SCC.

The key steps in our strategy are: i) generation of a synthetic ligand with photosensitizing properties, ii) ligand immobilization on the surface of modified gold nanoparticles to enable combination of PDT and thermo-effect, and iii) verification of the biological activity by *in vitro* and *in vivo* studies.

## **2. Experimental**

102 Squamous Cell Carcinoma

that blue, green, and red light itself can be efficient in topical PDT of AK; however, the more

The concept of ambulatory PDT to decrease hospital attendance for PDT was described by Moseley et al. (Moseley et al., 2006). In a study of five patients with BD, PDT was carried out with ALA and a portable LEDs device. Current studies have suggested that pulsed light therapy may be helpful for treatment in topical PDT of acne, AK and photorejuvenation. On the other hand, a recent controlled investigative study carried out in healthy human skin *in vivo* demonstrated that two pulsed light sources formerly reported in PDT brought evidence of minimal activation of photosensitizer, with a significantly smaller photodynamic reaction than observed with a conventional continuous wave broadband source (Strasswimmer & Grande, 2006). These sources deliver intense light in short periods (< 20 ms), which might suppress oxygen consumption (Kawauchi et al., 2004). Unplanned ambient light exposure may have considerably contributed to the clinical effect. However, three studies have recently addressed the possibility of using ambient light for ALA-PDT of AK (Batchelor et al., 2007; Marcus et al., 2007; Strasswimmer & Grande, 2006). Two of them report on therapeutic advantage. Nevertheless, the randomized ambient light-controlled study using ALA demonstrated no significant effect on lesion ablation. A randomized rightleft intrapatient evaluation of conventional MAL-PDT combined with LEDs device *versus* daylight (for 2.5 h) for the treatment of AK of face and scalp demonstrated corresponding reduction

Total effective light dosage is proposed as a concept for optimizing the accuracy of light dosimetry in PDT considering incident spectral irradiance and optical transmission through tissue and absorption by PS (Moseley, 1996). Actually, light dosimetry is explained as the irradiance rate (mW cm–2) at the skin surface and the total dosage (J cm–2) distributed to the

It has been suggested that lower fluence rates and fractionation of light exposure can improve lesional reaction by promotion of the photodynamic reaction (Henderson et al., 2004). A study of superficial BCC illuminated with 45 J cm–2 at 4 h and repeated at 6 h with 633-nm laser light at 50 mW cm–2 showed a total response of 84 % after a mean of 59 months (Star at el., 2006). Newer studies support advantages of the fractionation approach in BCC,

Extensive information about the application of various porphyrins and their derivatives in PDT has been published (Král et al., 2006). Accordingly, our laboratory synthesized porphyrin conjugates with glycol (Králová et al., 2008a), bile acid (Králová et al., 2008b), and cyclodextrins (Králová et al., 2006) and their *in vitro* and *in vivo* PDT activity has been tested. It was shown that these porphyrin conjugates are taken up preferentially by tumor cells and have the potential to be used for PDT to selectively ablate tumors (Králová et al., 2006;

Our contemporary strategy is to combine favorable features of gold nanoparticles mediating the photothermal effect with a photosensitizing compound mediating the photodynamic effect into one combined modality and thus introduce a therapeutic protocol efficient against SCC. The key steps in our strategy are: i) generation of a synthetic ligand with photosensitizing properties, ii) ligand immobilization on the surface of modified gold nanoparticles to enable

deeply penetrating red light is better when treating BD and BCC (Morton et al., 2002).

in AK and significantly less pain with daylight (Wiegell et al., 2008).

surface, the second being a product of irradiance and time of exposure.

although not in BD (Haas et al., 2006; Haas et al., 2007).

**1.7 Synthetic** *meso***-tetraphenylporphyrins in PDT** 

Králová et al., 2008a; Králová et al., 2008b).

#### **2.1 Preparation of modified gold nanoparticles**

Porphyrin–brucine conjugates **1** and **2** (Fig. 9) were prepared according to the procedure described previously (Král et al., 2005). Gold nanoparticles (14.7 nm) were prepared by citrate reduction of potassium tetrachloroaurate(III) (**Au-citr**). After modification with 3- mercaptopropanoic acid, derivatives **1** and **2** were immobilized as described elsewhere (Řezanka et al., 2008). Here, a solution of **1** or **2** (5 mg) in methanol was added to 50 ml of **Aucitr**. Modified nanoparticles (**Au-1** and **Au-2**, respectively) were isolated by centrifugation after three days of incubation. Using redispersion in methanol, methanol–water, water and dimethylsulfoxide, unbound porphyrin derivatives were removed and **Au-1** and **Au-2** molecules were concentrated to a volume of 1 ml. According to the spectral analysis of supernatants, 0.8 mg of **1** or **2** was present in the final 1 ml solution of **Au-1** and **Au-2** nanoparticles. The core of modified nanoparticles was characterized by transmission electron microscopy and photon cross-correlation spectroscopy (Nanophox). The chemical modification, ligand, was analyzed by absorption and fluorescence spectrometry. Fluorescence spectra were recorded using a Fluoromax spectrometer (Jobin-Yvon, Japan). A volume of 1 ml of sample was placed into 1 cm plastic cuvettes. The excitation wavelength was 520 nm.

Fig. 9. The structure of **1** and **2**

#### **2.2 Cell culture and** *in vitro* **experiments**

4T1 (mouse mammary carcinoma) and A431 (epidermal squamous carcinoma) cells were purchased from ATCC and PE/CA-PJ34 (human basaloid squamous cell carcinoma) cells were purchased from ETCC. As described before (Králová et al., 2006), all cells were grown exponentially in RPMI 1640 medium with 10% fetal calf serum. For photodynamic experiments, 1–1.5 x 105 cells were seeded into 1.8 cm-2 wells and incubated overnight with the porphyrin–brucine conjugates or their counterparts immobilized on gold nanoparticles (1 and 2.5 M). After incubation, cells were rinsed with PBS, cultured for 1 h in fresh medium without phenol red and illuminated with a 75 W halogen lamp with a band-pass filter (Andover, Salem, NH) that emitted light at wavelengths between 500–520 nm. The

Combined Therapy For Squamous Carcinoma Cells:

Application of Porphyrin-Alkaloid Modified Gold Nanoparticles 105

spectra significantly decreased (Fig. 10B) despite the concentration of porphyrins remained the same. The weak quantum yield may be attributed to that: (1) both porphyrins and nanoparticles absorb light at approximately 520 nm, (2) fluorescence quenching by porphyrinto-metal energy transfer, (3) partial aggregation of the modified nanoparticles. In the case of **Au-1**, aggregation seems to be the cause (Fig. 10B, compare traces "**Au-1/water**" and "**Au-1/medium**"), as the intensity of emitted fluorescence was several times higher in cell culture medium compared to water only. These results demonstrate that both para- (**1**) and meta- (**2**) derivatives aggregate in a solution-dependent manner that is not affected by the presence of PS or immobilization on gold nanoparticles. Importantly, the presence of model plasma proteins present in the cell medium dramatically reduced the aggregation of modified nanoparticles. This observation led us to further test these compounds for *in vivo* PDT efficacy.

Fig. 10. The fluorescence emission spectra of porphyrins **1** and **2** (left) and porphyrinmodified nanoparticles **Au-1** and **Au-2** (right) in water and cell culture media. Excitation was performed at 520 nm. Porphyrin–brucine conjugates were used at a concentration of 3.5 M. The concentration of human serum albumin used in growth medium was 50 mg ml–1.

The porphyrin–brucine conjugates (**1** and **2**) were next analyzed for tumor cell uptake and intracellular distribution. The mammary carcinoma cell line, 4T1 was cultivated in the presence of the conjugates for 16 h, during which time the cells were well-dispersed and growing mostly as planar sheets, enabling focused images of fluorescence to be recorded.

**3.3 Intracellular localization** 

A

B

These cells exhibited punctate red fluorescence (Fig. 11).

fluence rate at the level of the cell monolayer was 1 mW cm-2, and the total light dose was 7.2 J cm-2. Twenty-four hours post irradiation, the viability of PDT-treated cultures was determined by the Trypan blue exclusion method. In parallel, control "dark" experiments (without illumination) were performed.

#### **2.3 Microscopic studies**

Cells grown on coverslips in 35 mm Petri dishes were incubated with 2.5 M porphyrin– brucine conjugates in culture medium for 16 h. After washing, porphyrin fluorescence was observed with a DM IRB Leica microscope equipped with a DFC 480 camera using a x63 oil immersion objective and Leica filter cube N2.1 (excitation filter BP 515–560 nm and long pass filter LP 590 nm for emission). To label lysosomes, 500 nM LysoTracker Green (Molecular Probes) was added to the culture media for 30 min. Cells were washed and examined by fluorescence microscopy using the Leica filter cube I3 (excitation filter BP 450– 490 nm and long pass filter LP 515 nm for emission).

## **2.4** *In vivo* **experiments**

For *in vivo* experiments, the immuno-compromised nude mice with subcutaneously implanted human SCC tumors were used. When the tumor mass reached a volume of 100 mm3 (10–14 days after injection), mice were intravenously injected with porphyrin-brucine conjugates (5 mg kg-1) resuspended in a volume of 0.1 ml per 20 g mice and six hours later the tumor area (2 cm2) was irradiated with a 500–700 nm xenon lamp ONL051 (maximum at 635 nm, Preciosa Crytur, Turnov, Czech Republic) with a total impact energy of 100 J cm-2 and fluence rate of 200 mW cm-2. Each experimental group consisted of five or eight mice. The tumor size was measured repeatedly and the tumor volume was determined (Králová et al., 2006). All aspects of animal experimentation and husbandry were carried out in compliance with national and European regulations and were approved by the institutional committee.

## **3. Results and discussion**

#### **3.1 Modification by gold nanoparticles**

Gold nanoparticles (14.7 nm) prepared by citrate reduction of potassium tetrachloroaurate(III) (**Au-citr**) were modified with 3-mercaptopropanoic acid, and the derivatives **1** and **2** were immobilized. Gold nanoparticles modified with **1** and **2** are designated **Au-1** and **Au-2**, respectively.

#### **3.2 Fluorescence spectra**

The fluorescence intensity of **1** and **2** was strongly dependent on the solvent used. The influence of additional compounds on the intensity of emitted fluorescence wavelengths was tested by measuring the emission spectra (excitation of the first Q-band of porphyrins at 520 nm) of **1** and **2** in water, an inorganic salt solution (corresponding to the cell culture media) supplemented with a 50 mg ml-1 solution of human serum albumin (HSA) (Fig. 10A). In comparison with water, the emission bands of **1** and **2** measured in the media were red-shifted (for **1**, from 638 and 700 nm to 644 and 709 nm, and for **2**, from 643 and 707 nm to 647 and 710 nm) and the fluorescence intensity of **1** increased slightly whilst that of **2** decreased. After immobilizing the porphyrin conjugates on nanoparticles, the intensity of fluorescence emission

fluence rate at the level of the cell monolayer was 1 mW cm-2, and the total light dose was 7.2 J cm-2. Twenty-four hours post irradiation, the viability of PDT-treated cultures was determined by the Trypan blue exclusion method. In parallel, control "dark" experiments

Cells grown on coverslips in 35 mm Petri dishes were incubated with 2.5 M porphyrin– brucine conjugates in culture medium for 16 h. After washing, porphyrin fluorescence was observed with a DM IRB Leica microscope equipped with a DFC 480 camera using a x63 oil immersion objective and Leica filter cube N2.1 (excitation filter BP 515–560 nm and long pass filter LP 590 nm for emission). To label lysosomes, 500 nM LysoTracker Green (Molecular Probes) was added to the culture media for 30 min. Cells were washed and examined by fluorescence microscopy using the Leica filter cube I3 (excitation filter BP 450–

For *in vivo* experiments, the immuno-compromised nude mice with subcutaneously implanted human SCC tumors were used. When the tumor mass reached a volume of 100 mm3 (10–14 days after injection), mice were intravenously injected with porphyrin-brucine conjugates (5 mg kg-1) resuspended in a volume of 0.1 ml per 20 g mice and six hours later the tumor area (2 cm2) was irradiated with a 500–700 nm xenon lamp ONL051 (maximum at 635 nm, Preciosa Crytur, Turnov, Czech Republic) with a total impact energy of 100 J cm-2 and fluence rate of 200 mW cm-2. Each experimental group consisted of five or eight mice. The tumor size was measured repeatedly and the tumor volume was determined (Králová et al., 2006). All aspects of animal experimentation and husbandry were carried out in compliance with national and

Gold nanoparticles (14.7 nm) prepared by citrate reduction of potassium tetrachloroaurate(III) (**Au-citr**) were modified with 3-mercaptopropanoic acid, and the derivatives **1** and **2** were immobilized. Gold nanoparticles modified with **1** and **2** are

The fluorescence intensity of **1** and **2** was strongly dependent on the solvent used. The influence of additional compounds on the intensity of emitted fluorescence wavelengths was tested by measuring the emission spectra (excitation of the first Q-band of porphyrins at 520 nm) of **1** and **2** in water, an inorganic salt solution (corresponding to the cell culture media) supplemented with a 50 mg ml-1 solution of human serum albumin (HSA) (Fig. 10A). In comparison with water, the emission bands of **1** and **2** measured in the media were red-shifted (for **1**, from 638 and 700 nm to 644 and 709 nm, and for **2**, from 643 and 707 nm to 647 and 710 nm) and the fluorescence intensity of **1** increased slightly whilst that of **2** decreased. After immobilizing the porphyrin conjugates on nanoparticles, the intensity of fluorescence emission

European regulations and were approved by the institutional committee.

(without illumination) were performed.

490 nm and long pass filter LP 515 nm for emission).

**2.3 Microscopic studies** 

**2.4** *In vivo* **experiments** 

**3. Results and discussion** 

**3.2 Fluorescence spectra** 

**3.1 Modification by gold nanoparticles** 

designated **Au-1** and **Au-2**, respectively.

spectra significantly decreased (Fig. 10B) despite the concentration of porphyrins remained the same. The weak quantum yield may be attributed to that: (1) both porphyrins and nanoparticles absorb light at approximately 520 nm, (2) fluorescence quenching by porphyrinto-metal energy transfer, (3) partial aggregation of the modified nanoparticles. In the case of **Au-1**, aggregation seems to be the cause (Fig. 10B, compare traces "**Au-1/water**" and "**Au-1/medium**"), as the intensity of emitted fluorescence was several times higher in cell culture medium compared to water only. These results demonstrate that both para- (**1**) and meta- (**2**) derivatives aggregate in a solution-dependent manner that is not affected by the presence of PS or immobilization on gold nanoparticles. Importantly, the presence of model plasma proteins present in the cell medium dramatically reduced the aggregation of modified nanoparticles. This observation led us to further test these compounds for *in vivo* PDT efficacy.

Fig. 10. The fluorescence emission spectra of porphyrins **1** and **2** (left) and porphyrinmodified nanoparticles **Au-1** and **Au-2** (right) in water and cell culture media. Excitation was performed at 520 nm. Porphyrin–brucine conjugates were used at a concentration of 3.5 M. The concentration of human serum albumin used in growth medium was 50 mg ml–1.

#### **3.3 Intracellular localization**

The porphyrin–brucine conjugates (**1** and **2**) were next analyzed for tumor cell uptake and intracellular distribution. The mammary carcinoma cell line, 4T1 was cultivated in the presence of the conjugates for 16 h, during which time the cells were well-dispersed and growing mostly as planar sheets, enabling focused images of fluorescence to be recorded. These cells exhibited punctate red fluorescence (Fig. 11).

Combined Therapy For Squamous Carcinoma Cells:

**3.4** *In vitro* **phototoxicity** 

independent experiments is shown.

**3.5 In vivo PDT efficacy** 

occurs in the aqueous cell growth media (Fig. 12).

(Fig. 13).

Application of Porphyrin-Alkaloid Modified Gold Nanoparticles 107

To investigate the photodynamic potential of the free porphyrin–brucine conjugates or those immobilized on gold nanoparticles, we incubated PE/CA-PJ34 cells in the presence of the conjugates for 16 h and subjected them to PDT. In parallel, cells were incubated with porphyrins without illumination to serve as dark controls. Twenty-four hours following the illumination of cells with filtered light, the mortality of post-PDT cultures was determined

Fig. 13. The effect of free or immobilized porphyrin–brucine conjugates on the induction of cell death via PDT. PE/CA-PJ34 cells were incubated with either 1 or 2.5 M of **1** and **2** or their modified Au-nanoparticles for 16 h. Cells were then illuminated with filtered light (500–520 nm, 7.2 J cm–2). The percentage of dead cells was established the following day by using the Trypan blue exclusion method. The average and standard deviation for three

Satisfyingly, the induction of cell death was both light and drug-dose dependent. Control cells incubated with unconjugated gold nanoparticles (**Au-citr**) did not display any increase in cell death after illumination. Thus, under these *in vitro* conditions we can exclude the possibility that any case of cell death is due to the photothermal activity of the gold nanoparticles. Interestingly, the phototoxicities of unbound porphyrin–brucine conjugates **1**  and **2** were higher than those immobilized on gold nanoparticles. This reduction of photodynamic efficacy is likely to be a consequence of **Au-1** and **Au-2** aggregation that

Using an *in vivo* mouse cancer model, the PDT effectiveness of the unbound porphyrin– brucine conjugates **1** and **2** was compared with those immobilized on gold nanoparticles (**Au-1**, **Au-2**). Nude mice (NuNu) bearing basaloid squamous cell carcinoma PE/ CA-PJ34 cells received by intravenous injection either unmodified porphyrins or their gold nanoparticle-modified counterparts. Six hours post injection, tumors were illuminated

Fig. 11. The intracellular localization of porphyrin–brucine conjugates in 4T1 cells. The middle panels show the red fluorescence of **1** and **2** and co-staining with the lysosomal specific probe (LysoTracker Green); right panels represent an overlay of the green and red images and demonstrate co-localization (shown in orange/yellow). Porphyrin–brucine conjugates were used at a concentration of 2.5 M.

To identify the intracellular compartment where **1** and **2** accumulate, co-staining with the LysoTracker Green fluorescence probe was performed. The merged images revealed that **1** and **2** colocalized to a subset of LysoTracker-stained structures that represent lysosomes. Similar localization was also observed in PE/CA-PJ34 basaloid squamous cell carcinoma cells and A431 epidermal squamous carcinoma, cell lines that were predominantly used in our study (data not shown). Upon addition of gold nanoparticle-conjugated **1** and **2** to cell culture media, aggregates formed, which were visible as a reddish precipitate that covered parts of the cell. These were particularly abundant in the case of **Au-1** (Fig. 12).

Fig. 12. Difference in aggregation behavior of porphyrin–brucine conjugates immobilized on gold nanoparticles (left panels). 4T1 cells were incubated with **Au-1** and **Au-2** at a concentration of 2.5 M for 4 h before pictures were taken. Aggregates are highlighted by arrows.

#### **3.4** *In vitro* **phototoxicity**

106 Squamous Cell Carcinoma

Fig. 11. The intracellular localization of porphyrin–brucine conjugates in 4T1 cells. The middle panels show the red fluorescence of **1** and **2** and co-staining with the lysosomal specific probe (LysoTracker Green); right panels represent an overlay of the green and red images and demonstrate co-localization (shown in orange/yellow). Porphyrin–brucine

parts of the cell. These were particularly abundant in the case of **Au-1** (Fig. 12).

To identify the intracellular compartment where **1** and **2** accumulate, co-staining with the LysoTracker Green fluorescence probe was performed. The merged images revealed that **1** and **2** colocalized to a subset of LysoTracker-stained structures that represent lysosomes. Similar localization was also observed in PE/CA-PJ34 basaloid squamous cell carcinoma cells and A431 epidermal squamous carcinoma, cell lines that were predominantly used in our study (data not shown). Upon addition of gold nanoparticle-conjugated **1** and **2** to cell culture media, aggregates formed, which were visible as a reddish precipitate that covered

Fig. 12. Difference in aggregation behavior of porphyrin–brucine conjugates immobilized on

gold nanoparticles (left panels). 4T1 cells were incubated with **Au-1** and **Au-2** at a concentration of 2.5 M for 4 h before pictures were taken. Aggregates are highlighted by

conjugates were used at a concentration of 2.5 M.

arrows.

To investigate the photodynamic potential of the free porphyrin–brucine conjugates or those immobilized on gold nanoparticles, we incubated PE/CA-PJ34 cells in the presence of the conjugates for 16 h and subjected them to PDT. In parallel, cells were incubated with porphyrins without illumination to serve as dark controls. Twenty-four hours following the illumination of cells with filtered light, the mortality of post-PDT cultures was determined (Fig. 13).

Fig. 13. The effect of free or immobilized porphyrin–brucine conjugates on the induction of cell death via PDT. PE/CA-PJ34 cells were incubated with either 1 or 2.5 M of **1** and **2** or their modified Au-nanoparticles for 16 h. Cells were then illuminated with filtered light (500–520 nm, 7.2 J cm–2). The percentage of dead cells was established the following day by using the Trypan blue exclusion method. The average and standard deviation for three independent experiments is shown.

Satisfyingly, the induction of cell death was both light and drug-dose dependent. Control cells incubated with unconjugated gold nanoparticles (**Au-citr**) did not display any increase in cell death after illumination. Thus, under these *in vitro* conditions we can exclude the possibility that any case of cell death is due to the photothermal activity of the gold nanoparticles. Interestingly, the phototoxicities of unbound porphyrin–brucine conjugates **1**  and **2** were higher than those immobilized on gold nanoparticles. This reduction of photodynamic efficacy is likely to be a consequence of **Au-1** and **Au-2** aggregation that occurs in the aqueous cell growth media (Fig. 12).

#### **3.5 In vivo PDT efficacy**

Using an *in vivo* mouse cancer model, the PDT effectiveness of the unbound porphyrin– brucine conjugates **1** and **2** was compared with those immobilized on gold nanoparticles (**Au-1**, **Au-2**). Nude mice (NuNu) bearing basaloid squamous cell carcinoma PE/ CA-PJ34 cells received by intravenous injection either unmodified porphyrins or their gold nanoparticle-modified counterparts. Six hours post injection, tumors were illuminated

Combined Therapy For Squamous Carcinoma Cells:

nanoparticles without porphyrin.

**4. Conclusion** 

with nanoparticles will be the subject of future work.

Application of Porphyrin-Alkaloid Modified Gold Nanoparticles 109

Fig. 15. The PDT effectiveness of Au-2 against fast progressing epidermal squamous carcinoma A431. Nude mice (NuNu) bearing subcutaneous A431 tumors (*n* = 5 per each group) received an intravenous dose of the drug (5 mg kg–1). Tumors were illuminated with light (100 J cm–2) six hours after injection. The tumor size was measured repeatedly and the tumor volume was determined. Control mice were exposed to illumination but did not receive the porphyrin drug. The **Au-citr** group represents mice injected with Au

The apparent discrepancy in the *in vitro* and *in vivo* performance of unbound porphyrin– brucine conjugates **1** and **2** and those immobilized on gold nanoparticles (**Au-1** and **Au-2**) is likely to be due to the differing environmental conditions to which the porphyrin conjugates were exposed. The fluorescence data revealed that conjugates **1** and **2** were efficiently taken up by cells under the *in vitro* conditions tested. However, in culture media, **Au-1** and **Au-2** tended to aggregate, which resulted in their lower intracellular availability (Fig. 12) and lower PDT efficacy (Fig. 13). Under the *in vivo* conditions tested, the gold nanoparticleimmobilized conjugates were more effective than free conjugates alone. Both spectroscopic and ECD studies demonstrated that conjugated nanoparticles exhibited a strong interaction with plasma proteins (mainly HSA), which led to their self-assembly and to generation of supramolecular complexes. Subsequently, thanks to the enhanced permeability and retention (EPR) effect resulting in potent accumulation of **Au-1** and **Au-2** in the tumors, their PDT efficacy was increased. Moreover, the direct lethal effect of PDT on tumor cells combines well with the nanoscale size of gold-immobilized porphyrins that may limit the local blood supply (vascular impairment). This hypothesis of vascular damage after PDT

The spectroscopic studies demonstrated that fluorescence intensity of free and immobilized conjugates were strongly dependent on the solvent used. After immobilizing the porphyrin

with light at a dose of 100 J cm–2. Mice not injected with unmodified porphyrins or nanoparticles served as controls. Tumor size was measured after PDT at regular intervals (Fig. 14).

Fig. 14. The PDT effectiveness of **1** and **2** and their respective Au-immobilized nanoparticle counterparts to eradicate mouse tumors. Nude mice (NuNu) bearing subcutaneous PE/CA-PJ34 tumors (*n* = 8 per each group) received an intravenous dose of the drug (5 mg kg–1). Tumors were illuminated with light (100 J cm–2) six hours after injection. The tumor size was measured repeatedly and the tumor volume was determined. Control mice were exposed to illumination but did not receive the porphyrin drug. The **Au-citr** group represents mice injected with Au nanoparticles, **1** and **2** groups received porphyrin conjugates, **Au-1** and **Au-2** groups received porphyrin-modified Au nanoparticles.

We observed the greatest reduction in tumor growth in mice treated with **Au-1** and **Au-2**. All tumors were eliminated in animals that received these conjugated porphyrins and importantly, no detectable relapse of the primary tumor was observed. In contrast, animals treated with unbound **1** and **2** exhibited only a transient regression in tumor size that lasted until day 18, when the primary tumors began to gradually regrow. Presumably, this relapse in tumor growth comes from the small population of tumor cells that survived the PDT. Interestingly, mice treated with unconjugated gold nanoparticles exhibited slight tumor retardation in growth, which is most likely due to the photothermal effect described in other systems (Gamaleia et al., 2010; O'Neal et al., 2010; Řezanka et al., 2008).

These results clearly show that porphyrin alkaloid-modified gold nanoparticles are very effective against basaloid SCC *in vivo.* To verify more general applicability of porphyrin alkaloid-modified gold nanoparticles, the same approach was used against epidermal SCC tumors (Fig. 15). A431 cells formed fast progressing subcutaneous tumors, which were completely eradicated after **Au-2**-mediated PDT treatment in 60% mice or their growth was substantially reduced. These results demonstrate a high potential of porphyrin alkaloidmodified gold nanoparticles to fight SCC.

with light at a dose of 100 J cm–2. Mice not injected with unmodified porphyrins or nanoparticles served as controls. Tumor size was measured after PDT at regular intervals

Fig. 14. The PDT effectiveness of **1** and **2** and their respective Au-immobilized nanoparticle counterparts to eradicate mouse tumors. Nude mice (NuNu) bearing subcutaneous PE/CA-PJ34 tumors (*n* = 8 per each group) received an intravenous dose of the drug (5 mg kg–1). Tumors were illuminated with light (100 J cm–2) six hours after injection. The tumor size was measured repeatedly and the tumor volume was determined. Control mice were exposed to illumination but did not receive the porphyrin drug. The **Au-citr** group represents mice injected with Au nanoparticles, **1** and **2** groups received porphyrin conjugates, **Au-1** and

We observed the greatest reduction in tumor growth in mice treated with **Au-1** and **Au-2**. All tumors were eliminated in animals that received these conjugated porphyrins and importantly, no detectable relapse of the primary tumor was observed. In contrast, animals treated with unbound **1** and **2** exhibited only a transient regression in tumor size that lasted until day 18, when the primary tumors began to gradually regrow. Presumably, this relapse in tumor growth comes from the small population of tumor cells that survived the PDT. Interestingly, mice treated with unconjugated gold nanoparticles exhibited slight tumor retardation in growth, which is most likely due to the photothermal effect described in other

These results clearly show that porphyrin alkaloid-modified gold nanoparticles are very effective against basaloid SCC *in vivo.* To verify more general applicability of porphyrin alkaloid-modified gold nanoparticles, the same approach was used against epidermal SCC tumors (Fig. 15). A431 cells formed fast progressing subcutaneous tumors, which were completely eradicated after **Au-2**-mediated PDT treatment in 60% mice or their growth was substantially reduced. These results demonstrate a high potential of porphyrin alkaloid-

**Au-2** groups received porphyrin-modified Au nanoparticles.

systems (Gamaleia et al., 2010; O'Neal et al., 2010; Řezanka et al., 2008).

modified gold nanoparticles to fight SCC.

(Fig. 14).

Fig. 15. The PDT effectiveness of Au-2 against fast progressing epidermal squamous carcinoma A431. Nude mice (NuNu) bearing subcutaneous A431 tumors (*n* = 5 per each group) received an intravenous dose of the drug (5 mg kg–1). Tumors were illuminated with light (100 J cm–2) six hours after injection. The tumor size was measured repeatedly and the tumor volume was determined. Control mice were exposed to illumination but did not receive the porphyrin drug. The **Au-citr** group represents mice injected with Au nanoparticles without porphyrin.

The apparent discrepancy in the *in vitro* and *in vivo* performance of unbound porphyrin– brucine conjugates **1** and **2** and those immobilized on gold nanoparticles (**Au-1** and **Au-2**) is likely to be due to the differing environmental conditions to which the porphyrin conjugates were exposed. The fluorescence data revealed that conjugates **1** and **2** were efficiently taken up by cells under the *in vitro* conditions tested. However, in culture media, **Au-1** and **Au-2** tended to aggregate, which resulted in their lower intracellular availability (Fig. 12) and lower PDT efficacy (Fig. 13). Under the *in vivo* conditions tested, the gold nanoparticleimmobilized conjugates were more effective than free conjugates alone. Both spectroscopic and ECD studies demonstrated that conjugated nanoparticles exhibited a strong interaction with plasma proteins (mainly HSA), which led to their self-assembly and to generation of supramolecular complexes. Subsequently, thanks to the enhanced permeability and retention (EPR) effect resulting in potent accumulation of **Au-1** and **Au-2** in the tumors, their PDT efficacy was increased. Moreover, the direct lethal effect of PDT on tumor cells combines well with the nanoscale size of gold-immobilized porphyrins that may limit the local blood supply (vascular impairment). This hypothesis of vascular damage after PDT with nanoparticles will be the subject of future work.

## **4. Conclusion**

The spectroscopic studies demonstrated that fluorescence intensity of free and immobilized conjugates were strongly dependent on the solvent used. After immobilizing the porphyrin

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In contrast, when the PDT effectiveness was tested *in vivo*, the greatest reduction in tumor growth was observed in mice treated with porphyrin conjugates immobilized on gold nanoparticles. All tumors were eliminated and no detectable relapse of the primary tumor was observed. When animals were treated with unbound conjugates, they exhibited only a transient regression in tumor size that lasted until day 18, and then the primary tumors began to gradually re-grow. Importantly, mice treated with gold nanoparticles without porphyrin exhibited slight tumor retardation in growth that is most likely attributed to the photothermal effect described in other systems. Thus, under the *in vivo* conditions tested, the gold nanoparticle-immobilized conjugates were more effective than free conjugates alone. In addition, both spectroscopic and ECD studies demonstrated that conjugated nanoparticles exhibited a strong interaction with plasma proteins (mainly serum albumin), which led to their self-assembly and generation of supramolecular complexes, and thereby to the enhanced permeability and retention effect. It further contributed to potent accumulation of immobilized conjugates in tumors leading to increased PDT efficacy. Moreover, the direct lethal effect of PDT on tumor cells combines well with the nanoscale size of gold-immobilized porphyrins that may limit the local blood supply (vascular impairment).

## **5. Acknowledgements**

This work was funded by grants from the Grant Agency of the Czech Republic (Grant No. 203/09/1311 and P303/11/1291), supported in part by projects LC06077 and 512 awarded by the Ministry of Education of the Czech Republic, by project AV0Z50520514 awarded by the Academy of Sciences of the Czech Republic to J. Králová, and by projects MSM6046137307, BIOMEDREG CZ. 1.05./2.1.00/01.0030, and KAN2001008016.

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The evaluation of the biological activity of porphyrin-brucine conjugates, either free or immobilized to gold nanoparticles, started with determination of their intracellular uptake. It was shown that both forms were effectively taken into the cell, although a lower level was observed for immobilized forms. To investigate the photodynamic potential of the conjugates, SCC were exposed *in vitro* to photodynamic treatment and cell mortality of post-PDT cultures was determined. The phototoxicities of unbound porphyrin-brucine conjugates were higher than those of conjugates immobilized on gold nanoparticles. This reduction of photodynamic efficacy is likely to be a consequence of nanoparticle aggregation

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