Introduction

Pancreatic adenocarcinoma (PAC) is the eighth leading cause of cancer-related death in men and the ninth in women, with estimated 500,000 new cases diagnosed worldwide in 2020 [1,2]. PAC is usually diagnosed at advanced stages and has poor prognosis due to the extremely aggressive behaviour of the tumor. Surgical resection offers the single chance of cure for PAC, but only 15-20% of cases are potentially resectable at presentation and the prognosis is poor even for those in whom complete resection was successful [3]. In this setting, the overall survival is short and most patients are expected to die from the disease.

In respect to first-line chemotherapy, the FOLFIRINOX regimen is primarily recommended and yields better overall survival, but the toxicity and rate of adverse events are high [4]. In case of intolerance or lack of clinical status to carry out the FOLFIRINOX scheme, the use of gemcitabine can be offered as first line [5]. Use of gemcitabine together with paclitaxel associated to albumin (nab-paclitaxel) is also a first line treatment choice [6]. Second or third lines of chemotherapy may eventually prolong the global survival but are often limited by the low tolerability in those patients with deteriorating clinical state [7].

In pioneer studies, our laboratory has developed formulations of anticancer drugsassociated to artificially made lipid nanoparticles that resemble the lipid structure of low-density lipoprotein (LDL) [8,9]. The quasi-spherical nanoparticles are constituted by a monolayer of phospholipids and unesterified cholesterol surrounding a core of esterified cholesterol with small amounts of triglycerides. The nanoparticles, termed LDE, are made without apolipoprotein (apo) B, the protein moiety of native LDL that binds LDL particles to specific receptors on the cell membrane. However, when injected into the bloodstream, LDE acquires apo E which is also recognized by LDL receptors. LDL receptors are strongly upregulated in cancer cells [8,9]. This allows LDE and drugs carried in the nanoparticles to concentrate in different malignant neoplastic tissues, since LDL receptor upregulation is an ubiquitous phenomenon in cancer [10-14]. In experiments with animals with implanted melanoma B17 tumor, the formulation of paclitaxel incorporated to LDE showed increased antitumoral action, as documented by the reduction of tumor growth and of metastases and by prolongation of survival rates of the treated mice, compared with conventional paclitaxel [15]. The toxicity of paclitaxel associated to LDE was pronouncedly reduced, with LD50 ninefold higher than that of paclitaxel using Cremophor L as vehicle [15]. LDE-paclitaxel are stable formulations, with shelftime up to 4 months without addition of antioxidant compounds when stored at 4 ^oC.

The pharmacokinetic studies of LDE-paclitaxel in humans confirmed that dissociation of the drug from the nanoparticle vehicle does not occur in the bloodstream and showed that the residence time of the formulation is markedly longer than that of paclitaxel-Cremophor L, which per se is a consistent pharmacological advantage over the commercial formulation [12,13]. LDE-paclitaxel had no observable laboratorial or clinical toxicity used as 3^rd or later lines of patients with ovarian adenocarcinoma [16] and in end-of-life patients with metastatic cancers [17], as administered as intravenous infusions at 175 mg/ m² body surface dose every 3 weeks. Uninterrupted treatments up to one year were performed without occurrence of cumulative toxicity [16,17].

In view of the supportive data on the use of LDE-paclitaxel in patients with advanced cancers, as described above, this study was primarily designed to test the toxicity and tolerability of this preparation in the second or third line settings in patients with PAC. As secondary end-points, it was also aimed the evaluation of life quality and patient survival after initiation of treatment.

Materials and Methods

Study design and patients

This was a prospective one-arm, open-label study enrolling consecutive volunteer patients with advanced PAC already submitted to first or second line of chemotherapy. Treatment with LDE-paclitaxel was evaluated by determining the toxicity profile of the drug by clinical and laboratorial exams, by determining the survival period after initiation of the treatment and by application of a quality of life questionnaire (EORTC QLQ-C30).

The patient inclusion criteria were as follows: confirmed histologic diagnosis of advanced  PAC; both genders, aged 18-75 years;  Eastern Cooperative Oncology Group (ECOG) performance status score 0–2; and adequate haematologic, renal, and hepatic function; having already performed one or more standard chemotherapy regimens for the treatment of the disease. Entry data were collected from clinical records at the participant institutions.

A washout period of at least 15 days after completion of the previous chemotherapy line was kept before starting the LDEpaclitaxel treatment. LDE-paclitaxel at paclitaxel dose 85 mg/m²body surface was weekly administered I.V. diluted in 200 mL saline solution. The infusion was made over 90 min, with a two-week interval in the 7^th week of treatment. It was previously established that the treatment would be discontinued upon disease progression or at patient request. Patients were submitted to clinical interview and physical examination before every two chemotherapy cycles by one attending oncologist.

The study was in accordance with the Declaration of Helsinki principles and was approved by the Ethics Committee of the Federal University of São Paulo (Protocol n^o. 4237198).

Preparation of paclitaxel oleate associated with LDE

To increase the lipophilicity of paclitaxel and thereby to improve the yield of association to LDE and stability of LDEpaclitaxel, we synthesized a paclitaxel derivative, paclitaxel oleate, as previously described [16,17]. The LDE-paclitaxel formulation was prepared from a lipid mixture composed of 135 mg cholesteryl oleate, 333 mg egg phosphatidylcholine, 132 mg Miglyol 812 N, 6 mg cholesterol and 60 mg of paclitaxel and the aqueous phase comprised 100 mg of polysorbate 80 and 10 ml tris–HCl buffer, pH 8.05. Emulsification of all lipids, functionalized drug and the aqueous phase was obtained by high-pressure homogenization using an Emulsiflex C5 homogenizer (Avestin, Ottawa, Canada). After homogenization cycles, the formed nanoparticle was centrifuged and the nanoparticles are sterilized by passage through 0.22-lm pore polycarbonate filter (Millipore, Darmstadt, Germany). The preparations are stored at 4 °C until use. The quantification of paclitaxel in the preparations was performed by high- performance liquid chromatography (Shimadzu, Columbia, MD) developed in isocratic mode, mobile phase 100% methanol and UV–visible detector (227 nm).

Results

Total 9 consecutive patients were studied, 5 of the female and 4 of the male gender, with 63 year average age. Three were at clinical stage II, 3 at stage III and 3 at stage IV of the disease. Six out of the 9 patients had been submitted to resection surgery and none to radiotherapy. Seven of the participants had FOLFORINOX and 2 had gemcitabine as first line chemotherapy. None of the 9 patients used a previous Taxane-based treatment. Three patients had zero ECOG performance status, one ECOG 1 and one had ECOG 2. Four of the participants had been previously treated in first line chemotherapy scheme only and 3 had been treated in second line.

Table 1 shows the toxicity profile of LDE-paclitaxel at the adopted 85mg/m² body surface weekly dose level. Each chemotherapy cycle referred in Table 1 encompasses 6 consecutive weekly LDE-paclitaxel infusions followed by a two-week pause until the next infusion. Maximal 5 chemotherapy cycles were performed, with only one participant reaching the 5^th cycle. Altogether, total 24 cycles were performed and in none it was not necessary to reduce the medication dose. 

During the treatment with LDE-paclitaxel of the nine patients with PAC no CTCAE grade 3 and 4 toxicities were observed, with exception of one patient who had grade 3 renal toxicity in the first treatment cycle. In fact, it was unlikely that this renal disease event that was diagnosed 15 days after the first LDE-paclitaxel infusion could be accounted for the treatment but rather to the clinical status of the patient. As shown on Table 1, only grade 1 and grade 2 laboratorial toxicities, namely hematologic toxicities were registered in 9 from the total 24 cycles. Hepatic toxicity was not observed. Events of CTCAE grade 1 or 2 clinical toxicities, namely nausea and vomiting, diarrhoea and neuropathy were registered in 13 among the total 24 performed cycles.  Alopecia, fever, dyspnea, systemic arterial hypertension and mucositis were not observed.

As shown in Figure 1,the results of the questionnaire evaluation show that the quality of life score remained unchanged during the treatment. 

Figure 1: EORTC QLQ-C30 scores as measured at each treatment cycle.

Figure 2 presents the Kaplan-Meier plot of the survival of the patients since the beginning of the treatment with LDE-paclitaxel. The median survival was 4.73 months (CI 95% 0.013; 37.83). Figure 3 shows the survival periods of the patients treated with LDE-paclitaxel in either 2^nd or 3^rd lines. Apparently, there was no difference between 2^nd and 3^rd line-treated patients regarding the duration of the treatment periods.

Figure 2: Kaplan-Meier plots of the overall survival of the patients since the beginning of the LDE-paclitaxel treatment (in weeks).

Figure 3: Survival periods of each patient since the beginning of the LDE-paclitaxel treatments, discriminated as 2^nd or 3^rd line therapy (in months).

The median overall survival since the diagnosis of the disease was 20.14 months (95% CI 16.87; 23.40).