Pauline Roussille

Marie Auvray

Damien Vansteene

Thierry Lecomte

Eugénie Rigault

Marianne Maillet

Christophe Locher

Marie Dior

Vincent Hautefeuille

Pascal Artru

May Mabro

Yann Touchefeu

Lysiane Marthey

Valérie Moulin

Samy Louafi

Cédric Lecaille

Romain Chautard

Astrid Lièvre

Aziz Zaanan

Jaafar Bennouna

Antoine Berger

Sheik Emambux

Violaine Randrian

David Tougeron
email: davidtougeron@hotmail.fr

Prognostic factors of colorectal cancer patients with brain metastases

Keywords: Colorectal cancer, Brain metastases, Prognostic score, Radiosurgery, Whole brain radiation therapy

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The incidence of brain metastases (BM) from colorectal cancer (CRC) ranges from 0.6 to 3.2% of all CRC and is increasing [START_REF] Barnholtz-Sloan | Incidence proportions of brain metastases in patients diagnosed (1973 to 2001) in the Metropolitan Detroit Cancer Surveillance System[END_REF][START_REF] Christensen | Systematic review: brain metastases from colorectal cancer--Incidence and patient characteristics[END_REF][START_REF] Schouten | Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma[END_REF][START_REF] Tanriverdi | The clinical and pathological features of 133 colorectal cancer patients with brain metastasis: a multicenter retrospective analysis of the Gastrointestinal Tumors Working Committee of the Turkish Oncology Group (TOG)[END_REF]. Improvements in the treatment of metastatic CRC (mCRC) with prolonged survival of more than 30 months probably explain this phenomenon as it corresponds to the mean interval between primary tumor and BM diagnosis [START_REF] Tanriverdi | The clinical and pathological features of 133 colorectal cancer patients with brain metastasis: a multicenter retrospective analysis of the Gastrointestinal Tumors Working Committee of the Turkish Oncology Group (TOG)[END_REF][START_REF] Berghoff | Descriptive statistical analysis of a real life cohort of 2419 patients with brain metastases of solid cancers[END_REF]. Few small series of patients with BM from CRC have identified a specific profile of these patients having predominant primary tumor site in the rectum and sigmoid colon, frequent lung metastases and high rate of KRAS-mutated tumors [START_REF] Christensen | Systematic review: brain metastases from colorectal cancer--Incidence and patient characteristics[END_REF][START_REF] Tanriverdi | The clinical and pathological features of 133 colorectal cancer patients with brain metastasis: a multicenter retrospective analysis of the Gastrointestinal Tumors Working Committee of the Turkish Oncology Group (TOG)[END_REF][START_REF] Sundermeyer | Changing patterns of bone and brain metastases in patients with colorectal cancer[END_REF][START_REF] Yaeger | RAS mutations affect pattern of metastatic spread and increase propensity for brain metastasis in colorectal cancer[END_REF][START_REF] Christensen | Risk factors for brain metastases in patients with metastatic colorectal cancer[END_REF].

BMs from CRC are associated with a poor prognosis with a median overall survival (OS) of about 5.0 months but with a high heterogeneity essentially due to extracranial involvement [START_REF] Tanriverdi | The clinical and pathological features of 133 colorectal cancer patients with brain metastasis: a multicenter retrospective analysis of the Gastrointestinal Tumors Working Committee of the Turkish Oncology Group (TOG)[END_REF][START_REF] Jung | Brain metastases from colorectal carcinoma: prognostic factors and outcome[END_REF]. The treatment modalities include local treatments (surgery and radiosurgery), whole brain radiation therapy (WBRT), systemic treatments and best supportive care (BSC). The treatment objectives are local control of the BM, control of the non-tumoral cerebral parenchyma (prevention of other BM) and improving the patient's symptoms and quality of life [START_REF] Gu | Prognostic factors and multidisciplinary treatment modalities for brain metastases from colorectal cancer: analysis of 93 patients[END_REF]. Therapeutic strategy is based on patients' characteristics (age and performance status), BM (size and number), extracranial metastases (ECM) and prognosis. Indeed, it is important to identify prognostic factors to help treatment decisionmaking and prognostic scores could be useful. The most used scores are the Recursive Partitioning Analysis (RPA) classification [START_REF] Gaspar | Recursive partitioning analysis (RPA) of prognostic factors in three Radiation Therapy Oncology Group (RTOG) brain metastases trials[END_REF], the Graded Prognostic Assessment (GPA) score [START_REF] Sperduto | A New Prognostic Index and Comparison to Three Other Indices for Patients With Brain Metastases: An Analysis of 1,960 Patients in the RTOG Database[END_REF] and the Diagnosis Specific Graded Prognostic Assessment (DS-GPA) score [START_REF] Sperduto | Summary Report on the Graded Prognostic Assessment: An Accurate and Facile Diagnosis-Specific Tool to Estimate Survival for Patients With Brain Metastases[END_REF].

The proportion of CRC patients included in the studies developing these scores is unknown.

Recently, a Gastro-Intestinal Graded Prognostic Assessment (GI-GPA) was published for allcomers GI cancers. Although tumor site had a major prognostic impact (p<0.001), it was not retained in the final model [START_REF] Sperduto | Estimating survival in patients with gastrointestinal cancers and brain metastases: An update of the graded prognostic assessment for gastrointestinal cancers (GI-GPA)[END_REF]. Indeed, these prognostic scores are not designed specifically for patients with mCRC and are not validated in this specific population. A recent Italian retrospective study of 119 patients with BM from CRC developed a nomogram to estimate OS [START_REF] Pietrantonio | A new nomogram for estimating survival in patients with brain metastases secondary to colorectal cancer[END_REF]. This tool also takes into account the subtentorial or supratentorial location of BM.

The main objective of this study was to identify the clinico-pathological prognostic factors in CRC patients with BM. We also analyzed the distribution of the patients according to the different prognostic scores and evaluated their relevance to predict patient's prognosis.

PATIENTS AND METHODS

Population

This retrospective multicenter study included patients with BM from CRC. Patients were diagnosed between January 2000 and January 2017, in 17 French hospitals using local clinical database. The local Ethics Committee approved the study and, due to the retrospective analysis with a majority of deceased patients, no patient consent was required. The study was performed according to the principles of the Declaration of Helsinki.

Inclusion criteria were age over 18 years old, histologically confirmed CRC and histologically or radiologically confirmed BM by computed tomography-scan (CT-scan) and/or magnetic resonance imaging (MRI). This study did not need an informed consent because of its retrospective nature and most of the patients had died (84.3%). Exclusion criteria were non-adenocarcinoma tumors and leptomeningeal carcinomatosis.

Patient and tumor characteristics

Patients' characteristics were described (age, sex and WHO performance status), as well as characteristics of the primary tumor (site, stage at CRC diagnostic, grade and treatment) and ECM (number, site and treatment). When available, KRAS/NRAS (codons 12, 13, 61, 146) and BRAF (V600E) mutations and mismatch repair status (MMR) were collected.

Characteristics of BM were analyzed, including delay between CRC diagnosis and

(supratentorial, subtentorial or both) and treatment (surgery, WBRT, radiosurgery and/or BSC).

Prognostic factors

We tested different scores in predicting the OS of our CRC-derived BM cohort. The RPA and GPA scores are both based on 4 prognostic factors, with 3 factors in common: Karnofsky index, age, presence of ECM. The RPA also incorporates the control of the primary tumor and the GPA the number of BM. Both scores do not consider primary tumor site and are essentially based on patients with BM from lung and breast cancers and are therefore perhaps not transposable to CRC. The DS-GPA classification for gastrointestinal tumors retains only the Karnofsky index to stratify patients into 4 prognostic classes [START_REF] Sperduto | Summary Report on the Graded Prognostic Assessment: An Accurate and Facile Diagnosis-Specific Tool to Estimate Survival for Patients With Brain Metastases[END_REF].

Recently, the same team developed the GI-GPA that included Karnofsky index, age, ECM and number of BM [START_REF] Sperduto | Estimating survival in patients with gastrointestinal cancers and brain metastases: An update of the graded prognostic assessment for gastrointestinal cancers (GI-GPA)[END_REF]. Both of these scores are not specific to CRC. The nomogram is specific to CRC and included Karnofsky index, age, BM site, number of BM as prognostic criteria but excluded patients with a Karnofsky index ≤ 50% [START_REF] Pietrantonio | A new nomogram for estimating survival in patients with brain metastases secondary to colorectal cancer[END_REF].

The distribution of patients according to the different prognostic scores RPA, GPA, DS-GPA, GI-GPA and the nomogram was also evaluated to test their relevance [START_REF] Gaspar | Recursive partitioning analysis (RPA) of prognostic factors in three Radiation Therapy Oncology Group (RTOG) brain metastases trials[END_REF][START_REF] Sperduto | A New Prognostic Index and Comparison to Three Other Indices for Patients With Brain Metastases: An Analysis of 1,960 Patients in the RTOG Database[END_REF][START_REF] Sperduto | Summary Report on the Graded Prognostic Assessment: An Accurate and Facile Diagnosis-Specific Tool to Estimate Survival for Patients With Brain Metastases[END_REF][START_REF] Sperduto | Estimating survival in patients with gastrointestinal cancers and brain metastases: An update of the graded prognostic assessment for gastrointestinal cancers (GI-GPA)[END_REF][START_REF] Pietrantonio | A new nomogram for estimating survival in patients with brain metastases secondary to colorectal cancer[END_REF]. OS was also determined according to BM treatment using four groups: BSC alone, focal treatment alone (surgery and/or radiosurgery), WBRT alone and focal treatment plus WBRT.

Statistical analysis

The main objective of the study was to identify prognostic factors of OS. OS was calculated from the date of BM diagnosis to the date of death from any cause. Survival curves were determined using the Kaplan-Meier method. Patients known to be alive were censored at the date of their last follow-up. Prognostic factors of OS were evaluated using the log-rank test for univariate analysis and statistically significant variables with p<0.10 and/or clinically relevant variables were included in multivariate analysis using a Cox regression model. The level of significance was set at a p value of 0.05 (one-sided). Statistical analyses were performed using XLSTAT 2017 © software (Addinsoft, USA).

RESULTS

A total of 358 patients with BM from CRC were included. Among them 93.4% had neurologic symptoms that led to the BM diagnosis and 6.6% had non-symptomatic lesions diagnosed on brain imaging. Median age at BM diagnosis was 65.5 years (Table 1). The primary tumors were located in the rectum, left colon and right colon in respectively 42.4%, 37.2% and 20.3% of cases. Tumors were predominantly well or moderately differentiated (89.2%). Most patients had a metastatic disease at diagnosis (58.0%), 26.0% with lung metastases and 37.7% with liver metastases. Most patients had a surgical resection of the primary tumor (82.4%).

KRAS mutations (codons 12-13) were found in 56.9% of cases. Of the 94 wild-type KRAS tumors (codons 12-13), 43 had complete RAS testing (codons 12, 13, 61 and 146 of KRAS and NRAS) of which 6 tumors were mutated. Indeed, rate of complete RAS mutation were 59.4% (56.0% KRAS and 3.4% NRAS). BRAF mutation was observed in 6.5% and tumors were mostly MMR-proficient (pMMR) (93.7%).

Only 16.2% of patients had synchronous BM and CRC diagnosis. The median time from primary tumor diagnosis to BM diagnosis was 29.2 ± 3.2 months and the median time from ECM diagnosis to BM diagnosis was 18.5 ± 2.5 months. Patients were mostly in good general condition (57.0% ECOG performance status 0-1). The majority of BM was single (56.9%) and only supratentorial (54.4%). At BM diagnosis, 86.1% of patients had ECM, mostly lung (70.1%) or liver (47.0%) metastases. Patients had a median of 2 chemotherapy lines before BM diagnostic (range 0-9).

The median follow-up from BM diagnosis was 44.2 ± 7.4 months and 84.3% of patients had died. Median OS from BM diagnosis was 5.1 ± 0.3 months, ranged from 0.1 to 158.7 months (1-year and 2-year OS were 23.7% and 9.5% respectively) (Supp Figure 1).

Median OS from CRC diagnosis was 43.0 ± 1.7 months and median OS from ECM diagnosis was 31.9 ± 2.9 months. Prognostic scores RPA, GPA, DS-GPA, GI-GPA, as well as the nomogram, were calculated in the whole population. A significant OS difference between prognostic groups of each score was observed (p<0.0001) (Table 2).

In multivariate analysis, age < 65 years, ECOG performance status 0-1 (p<0.0001), single BM (p<0.0001), and 0 to 2 chemotherapy lines before BM diagnosis (p<0.0001) were associated with better OS (Table 3). Surgical resection of BM was performed in 33.0% (n=118) of patients, 73.2% (n=262) received radiotherapy alone or after surgery [21.2% radiosurgery (n=76) and 43.6% WBRT (n=156)].

Patients were divided into four groups according to the treatment received (Fig. 1): BSC alone (n=58), focal treatments alone (n=93), WBRT alone (n=114) and focal treatments plus WBRT (n=44). Patients with different or unknown treatment (n=49) were excluded.

Patient and tumor characteristics are different according to treatment groups (Supp Table 1).

Compared to focal treatments alone and focal treatments plus WBRT groups, BSC alone and WBRT alone groups had more unfit patients who were older, with poor performance status and with more aggressive disease (multiple BM and more frequently ECM).

OS was significantly different depending on the BM treatment. Median OS was 13.0 ±1.0 months in patients with BM surgery compared to 3.4 ± 0.2 months in non-operated patients (p<0.0001). In patients with radiosurgery or WBRT median OS was 5.9 ± 0.3 months compared to 2.1 ± 0.4 months in non-irradiated patients (p=0.0003). Median OS was 12.0 ± 0.7 months in patients with BM surgery alone or with WBRT compared to 8.5 ± 7.3 months in patients with radiosurgery alone or with WBRT (p=0.20).

OS was also different according to the four pre-defined groups: BSC alone (1.3 ± 0.2 months), WBRT alone (3.6 ± 0.2 months), local treatments plus WBRT (9.8 ± 1.4 months) and local treatments alone (17.1 ± 2.4 months) (p<0.0001) (Fig. 2). Patients with BSC alone (1.3 ± 0.2 months) had a shorter OS as compared patients with specific treatments (6.0 ± 0.4 months) (Fig. 3).

Patients in BSC alone and WBRT alone groups were more frequently in the poor prognosis subgroups of each score (RPA, GPA, DS-GPA, GI-GPA and nomogram) compared to local treatments alone and local treatments plus WBRT groups (Supp Table 2). In each subgroup, prognostic scores were correlated with OS, i.e. BSC alone (RPA, GPA, DS-GPA and nomogram), WBRT alone (RPA and DS-GPA), local treatments plus WBRT (RPA, GPA, DS-GPA, GI-GPA and nomogram) and local treatments alone (RPA and GI-GPA) (data not shown).

Concerning prognostic factors, in multivariate analysis, number of previous chemotherapy lines < 3 (p=0.03) was associated with better OS in local treatment alone group and age < 65 years (p=0.01), female (p=0.01), ECOG performance status 0-1 (p=0.01) and single BM (p=0.0008) were associated with better OS in local treatment plus WBRT group. ECOG performance status 0-1 (p=0.04) was associated with better OS in WBRT alone group. KRAS WT status (p=0.03) and number of previous chemotherapy lines < 3 (p=0.04) was associated with better OS in BSC alone group.

DISCUSSION

In this large cohort, prognosis of CRC patients with BM remains poor but with a high heterogeneity of OS ranging from 0 to 159 months. CRC patients with BM had frequently a rectum tumor, lung metastases and a KRAS-mutated tumor. The median time from metastatic CRC diagnosis to BM diagnosis was 18 months. We identified age < 65 years, ECOG performance status 0-1, single BM and less than 3 chemotherapy lines before BM diagnosis as good prognostic factors. Among the common prognostic scores available in the literature, the DS-GPA and GI-GPA gave a more homogenous repartition among different subgroups and the closest predictions of OS to the observed OS in our series.

In our study, as already described in other studies, young age, rectal or sigmoid tumor site and lung metastases seemed to be more frequent in mCRC patients with BM compared to all-comers mCRC patients [START_REF] Christensen | Systematic review: brain metastases from colorectal cancer--Incidence and patient characteristics[END_REF][START_REF] Tanriverdi | The clinical and pathological features of 133 colorectal cancer patients with brain metastasis: a multicenter retrospective analysis of the Gastrointestinal Tumors Working Committee of the Turkish Oncology Group (TOG)[END_REF][START_REF] Christensen | Risk factors for brain metastases in patients with metastatic colorectal cancer[END_REF][START_REF] Jung | Brain metastases from colorectal carcinoma: prognostic factors and outcome[END_REF][START_REF] Pietrantonio | A new nomogram for estimating survival in patients with brain metastases secondary to colorectal cancer[END_REF][START_REF] Qiu | Pattern of distant metastases in colorectal cancer: a SEER based study[END_REF][START_REF] Hugen | Metastatic pattern in colorectal cancer is strongly influenced by histological subtype[END_REF]. Moreover, most of the patients had synchronous metastatic disease at primary tumor diagnosis (58.0%), which probably reflected an "aggressive" disease that would later develop BM, since only ≈25% of patients diagnosed with CRC present synchronous BM. In accordance with the literature, most BMs from CRC were metachronous to CRC diagnostic (≈80%). In addition rate of KRAS mutations was high (56.9%) when compared with all-comers mCRC (≈40%) [START_REF] Christensen | Systematic review: brain metastases from colorectal cancer--Incidence and patient characteristics[END_REF][START_REF] Yaeger | RAS mutations affect pattern of metastatic spread and increase propensity for brain metastasis in colorectal cancer[END_REF][START_REF] El-Deiry | Molecular profiling of 6,892 colorectal cancer samples suggests different possible treatment options specific to metastatic sites[END_REF][START_REF] Suzuki | Prognostic Factors and Treatment Effects in Patients With Curatively Resected Brain Metastasis From Colorectal Cancer[END_REF]. In our cohort, BM diagnosis occurred with a median time of 30 months after the primary tumor diagnosis and about 20 months after the metastatic disease diagnosis which is in accordance with the literature [START_REF] Tanriverdi | The clinical and pathological features of 133 colorectal cancer patients with brain metastasis: a multicenter retrospective analysis of the Gastrointestinal Tumors Working Committee of the Turkish Oncology Group (TOG)[END_REF][START_REF] Berghoff | Descriptive statistical analysis of a real life cohort of 2419 patients with brain metastases of solid cancers[END_REF]. Due that most patients were at the terminal phase of the disease with both brain and extracranial metastatic sites, we were not able to distinguish whether death was due to BM or ECM. In the same way, most patients died soon after BM diagnosis without further imaging then it is difficult to know if the BM treatment carried out allow a disease control or not beyond analyzing the OS. Some prognostic scores exist to guide treatment strategies for patients with BM but, except for the nomogram, they are not designed specifically for mCRC patient's [START_REF] Gaspar | Recursive partitioning analysis (RPA) of prognostic factors in three Radiation Therapy Oncology Group (RTOG) brain metastases trials[END_REF][START_REF] Sperduto | Summary Report on the Graded Prognostic Assessment: An Accurate and Facile Diagnosis-Specific Tool to Estimate Survival for Patients With Brain Metastases[END_REF][START_REF] Sperduto | Estimating survival in patients with gastrointestinal cancers and brain metastases: An update of the graded prognostic assessment for gastrointestinal cancers (GI-GPA)[END_REF].

The aim of our study was not to develop a new score but to evaluate specifically the already existing scores in the subgroup of CRC. RPA and GPA do not consider primary tumor site and are essentially based on patients with BM from lung and breast cancers. The DS-GPA classification for gastrointestinal tumors retains only the Karnofsky index to stratify patients into 4 prognostic classes with an OS ranging from 3.1 to 13.5 months. The GI-GPA classification for gastrointestinal tumors developed in 2019 is dedicated to GI tumors but the authors did not include the primary tumor site although it was significantly associated with OS (p<0.001) [START_REF] Sperduto | Estimating survival in patients with gastrointestinal cancers and brain metastases: An update of the graded prognostic assessment for gastrointestinal cancers (GI-GPA)[END_REF]. As reported by the authors, median OS ranged from 2 months for gastric cancer to 14 months for anal cancer. Most of the patients in our study were in the "intermediate" or "poor" prognostic groups of RPA, GPA, DS-GPA and GI-GPA scores and few in the category with "good" prognosis. This is probably related to the late diagnosis of BM in CRC. The repartition of patients throughout the four categories seems better for the GI-GPA and DS-GPA scores. RPA and GPA predict worse OS than those observed in our cohort but DS-GPA and GI-GPA were better to predict the observed OS. Although, DS-GPA seems to predict more accurately the observed OS in our cohort, both DS-GPA and GI-GPA scores displayed a balanced repartition of patients throughout the four prognostic groups.

The Nomogram proposed in a recent Italian retrospective study on 227 patients with BM from CRC, treated between 2000 and 2013, excluded patients with a Karnofsky index ≤ 50%, which induces a major selection bias [START_REF] Pietrantonio | A new nomogram for estimating survival in patients with brain metastases secondary to colorectal cancer[END_REF]. In our cohort, this Nomogram is associated with OS. Nevertheless, the Nomogram estimated a median OS of approximately 9.0 months compared to 5.7 months really observed in our cohort. The Nomogram is difficult to use in routine clinical practice with multiple cut offs for each four prognostic factors. The Nomogram has been also evaluated on a cohort of 64 patients and in 46 patients (72%) the observed survival was shorter than the predicted median (median deviation of -1.4 months) [START_REF] Nieder | Predicted survival in patients with brain metastases from colorectal cancer: Is a current nomogram helpful?[END_REF]. Moreover, the Nomogram overestimated OS in patients treated with WBRT by a median of 2.1 months. Our population seemed similar to both studies in terms of primary tumor location with 42% of rectal tumours (34% in the Italian study [START_REF] Pietrantonio | A new nomogram for estimating survival in patients with brain metastases secondary to colorectal cancer[END_REF] and 48% in the Norwish study [START_REF] Nieder | Predicted survival in patients with brain metastases from colorectal cancer: Is a current nomogram helpful?[END_REF]) and in terms of ECM at BM diagnosis with 13.6% of patients without ECM (0% in the Italian study and 27% in the Norwish study). Our cohort was larger than both studies and demonstrated that the Nomogram has some limits to predict OS. DS-GPA provided the closest estimations of OS to the OS we observed in the three categories. This confirmed that ECOG performance status, which we identified as a prognostic factor, was the most important one. It was included with a bigger weight in the GI-GPA. In our study, age, which had a low weight in the GI-GPA, is an independent prognostic factor but with a p value near 0.05. Our large cohort of 358 patients identified other prognostic factors in multivariate analysis: number of BM and number of previous chemotherapy lines before BM diagnosis. The number of previous chemotherapy lines is not included in any of the existent scores but one study found that patients with BM from CRC receiving ≥ 2 lines of chemotherapy had lower OS compared to other patients [START_REF] Jung | Brain metastases from colorectal carcinoma: prognostic factors and outcome[END_REF]. The delay between BM and CRC diagnosis (i.e. synchronous versus metachronous) could be a surrogate marker for the number of previous chemotherapy lines. As confounding factors cannot be considered independent from each other we assumed they could not be included in the same multivariate analysis and we chose to keep delay between BM and CRC diagnosis from the analysis.

Nowadays, the therapeutic management of BM depends on the performance status of the patient, the expected OS, the number, size and location of BM that is to say prognostic factors, rather than tumor location. Identifying the most efficient prognostic factors is of great importance to decide the most adequate BM treatment. In case of a single, large and symptomatic lesion in a patient whose general condition is preserved, surgery is often preferred in order to improve the quality of life of the patients [START_REF] Soffietti | Diagnosis and treatment of brain metastases from solid tumors: guidelines from the European Association of Neuro-Oncology (EANO)[END_REF]. Stereotactic radiosurgery can be an alternative treatment option to surgery in case of a single asymptomatic lesion of less than three centimetres, or in case of plurifocal involvement but with a limited volume of parenchyma reached. In other cases with multiple BMs and/or poor performance status (Karnofsky index <70%) and/or poor prognosis (<3 months), WBRT is the best option. Future directions include novel radiation technique such as ultra-high dose rate radiotherapy, which can provide adequate tumor control and spare cognitive decline. The role of systemic therapy in the management of BMs is currently evolving. Most tyrosine kinase inhibitors, e.g. BRAF inhibitors, are able to penetrate the blood-brain barrier. Several studies have demonstrated promising activity of immune checkpoint inhibitors in BMs and trials combining immune checkpoint inhibitors with stereotactic radiosurgery are ongoing [START_REF] Palmer | Multidisciplinary patient-centered management of brain metastases and future directions[END_REF].

In our study, as in other series, there was a high heterogeneity of BM treatment.

Currently, there is no specific consensus on the therapeutic management of BM from CRC.

We split the cohort in 4 groups with different treatments (BSC alone, local treatments alone, local treatments plus WBRT and WBRT alone). Consequently, a great difference in term of prognosis was observed with an OS from 1.3 months to 17.1 months according the subgroups.

Consistent with the literature, patients with radiation have a better prognosis compared to patients with no radiation (≈6 months versus ≈3 months), as well as patients with local treatments plus WBRT compared to WBRT alone (≈12 months versus ≈3 months) [START_REF] Jung | Brain metastases from colorectal carcinoma: prognostic factors and outcome[END_REF][START_REF] Suzuki | Prognostic Factors and Treatment Effects in Patients With Curatively Resected Brain Metastasis From Colorectal Cancer[END_REF][START_REF] Patchell | A randomized trial of surgery in the treatment of single metastases to the brain[END_REF].

In this retrospective study, it is not possible to determine the best treatment option for CRC patients with BM due to the great difference in patients and tumours' characteristics according to treatment subgroups and obvious selection bias. Nevertheless, we analyzed the proposed prognostic factors according to the four treatment subgroups [START_REF] Sperduto | A New Prognostic Index and Comparison to Three Other Indices for Patients With Brain Metastases: An Analysis of 1,960 Patients in the RTOG Database[END_REF][START_REF] Nieder | Colorectal cancer metastatic to the brain: time trends in presentation and outcome[END_REF]. The significant prognostic factors obtained were the previously identified variables, strengthening the relevance to stratify patients with BM derived from CRC. The RPA correlated with OS in each subgroup of treatment but did not differentiate patients correctly and could not predict OS in our cohort when compared to DS-GPA. It is worth noting that in these subgroups we are not able to differentiate factors identified as prognostic factors and/or predictive factors of treatment efficacy.

Despite its retrospective nature and the prolonged period of patient's inclusion, there are few missing clinical data (≈10%) in our study. Nevertheless, we cannot exclude a selection bias in our population, particularly an over-representation of patients with radiotherapy and/or surgery compared to BSC alone. Another limitation is the heterogeneity of BM treatment but this reflects routine clinical practice. Finally, this is the largest published series so far of CRC patients with BM and, in our opinion, it allows a highly accurate determination of prognostic factors in this subgroup of CRC patients.

In conclusion, we found age < 65 years, ECOG performance status 0 or 1, single BM and less than 3 previous chemotherapy lines clinically relevant good prognostic factors. All these factors can guide treatment decision-making in this particular population of metastatic CRC with BM. The GPA classification scores 3 and 3.5-4 were grouped together for the OS analysis in view of the small number of patients in each of these groups. ** For the DS-GPA classification, the scores 2 (Karnosfsky at 80) and 3 (Karnosfky at 90) of the classification were grouped together, as they both corresponded to an ECOG performance status at 1. # For the Nomogram we performed a correlation between the score as continuous variable and OS using a Cox model. § Median OS observed in the princeps publication. DS-GPA: Diagnosis-Specific Graded Prognostic Assessment, GPA: Graded Prognostic Assessment, DS-GI-GPA: Gastro-Intestinal Graded Prognostic Assessment, RPA: Recursive Partitioning Analysis

Figure captions Figure 1 .Figure 2 .Figure 3 .

 captions123 Figure captions

Table 1 . Patient and tumor characteristics (n=358)

 1

	n (%)

Table 2 . Overall survival according the prognostic scores (n=358) n (%)

 2

	Median OS	Median OS in
	observed with the score §	our cohort (months)	p
	(months)

Acknowledgement

The authors thank all of the clinical research associates who participated in the data collection (Aziza Abaza Nedhif, Claire Jollivet, Magali Balhadere). Finally, the authors would like to thank Vanessa Le Berre for her assistance in preparing the submission of the article.

Conflicts of interest: None

Funding: None

Conflicts of interest

None.


domain: chim chim.anal sdv

text:
Aml M Asran

Mona A Mohamed

Ghada M G Eldin

Rupesh K Mishra

Abdelhamid Errachid
email: abdelhamid.errachid-el-salhi@univ-lyon1.fr

ruthenium decorated electrochemical platform for sensitive and selective determination of amisulpride in presence of co-administered drugs using safranin as a mediator

Keywords: Amisulpride, Antipsychotic medications, Ruthinum nanoparticles, Safranin, modified electrochemical sensors, Real samples analysis

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Depression is considered as a common mental disorder, which influences over 264 million people worldwide and becomes a critical health problem; approximately 800,000 persons have been died because of suicide every year [START_REF] Elgawish | Toxicity Profile, Pharmacokinetic, and Drug-Drug Interaction Study of Citalopram and Sertraline Following Oral Delivery in Rat: An LC-MS/MS Method for the Simultaneous Determination in Plasma[END_REF]. Recently, the psychiatric consequences of the covid-19 infections to manifest are receiving high attention [START_REF] Chaturvedi | Covid-19 Related Psychiatric Disorders and the New Psychosocial Rehabilitation[END_REF]. Historically, many of psychiatric situations have been described due to viral infections [START_REF] Chaturvedi | Covid-19 Related Psychiatric Disorders and the New Psychosocial Rehabilitation[END_REF]. According to the recent studies [START_REF] Chaturvedi | Covid-19 Related Psychiatric Disorders and the New Psychosocial Rehabilitation[END_REF], the epidemic covid-19 is likely to generate related or may be different psychiatric disorders.

Therefore, more attention is directed to antipsychotic drugs due to the recent epidemic circumstances and the increase use of these kinds of drug. Serotonergic antidepressants are one of the first line agents for treatment of depression [START_REF] Carvalho | Amisulpride augmentation after the failure of citalopram for depression: a case report[END_REF]. Amisulpride (AMS) is a substituted benzamide atypical antipsychotic that has high affinity for dopamine receptors toward management of psychoses such as schizophrenia [START_REF] Sweetman | Martindale: the complete drug reference[END_REF][START_REF] Salama | Thermal behavior study and decomposition kinetics of amisulpride under non-isothermal and isothermal conditions[END_REF]. AMS obviously has the clinical profile of an atypical antipsychotic [START_REF] Möller | Amisulpride: limbic specificity and the mechanism of antipsychotic atypicality[END_REF], known by its lower propensity to induce extrapyramidal side effects as well as its greater efficacy in treating negative symptoms compared to classical neuroleptics, particularly haloperidol which was used as comparative substance in most studies [START_REF] Möller | Amisulpride: limbic specificity and the mechanism of antipsychotic atypicality[END_REF]. The mixture of an antidepressant with an antipsychotic agent has been proved to be effective for the treatment of psychotic depression [START_REF] Politis | Combination therapy with amisulpride and antidepressants: Clinical observations in case series of elderly patients with psychotic depression[END_REF]. Low-dose of AMS augmentation resulted in a significant clinical improvement along with serotonergic antidepressant Citalopram (CIT) [START_REF] Carvalho | Amisulpride augmentation after the failure of citalopram for depression: a case report[END_REF]. AMS has IUPAC name; 4-amino-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(ethylsulfonyl)-2methoxybenzamide. Citalopram (CIT), chemically 1- propyl]-1-(4fluorophenyl)-1,3-dihydro-5-isobenzofurancarbonitrile, belongs to selective serotonin reuptake inhibitors (SSRIs) family [START_REF] Salama | Thermal behavior study and decomposition kinetics of amisulpride under non-isothermal and isothermal conditions[END_REF]. About 30%-40% of Alzheimer's disease patients have depressive and psychotic symptoms [START_REF] Sweetman | Martindale: The Complete Drug Reference[END_REF]. Patient with dementia described by Alzheimer had psychotic symptoms, including paranoid delusions and hallucinations. Consequently, the term "senile psychosis" was usually used to denote to psychosis in old patients with dementia [START_REF] Jeste | Psychosis of Alzheimer's disease and related dementias: diagnostic criteria for a distinct syndrome[END_REF], so there is a high need for sensitive selective determination of AMS alongside with administered Memantine hydrochloride (MEM). MEM is a 1-amino-3,5-dimethyladamantane derivative. MEM has low affinity, uncompetitive NMDA receptor antagonist used for the treatment of dementia. Lately, MEM was approved by FDA for the moderate treatment of serious Alzheimer's disease [START_REF] Sonkusare | Dementia of Alzheimer's disease and other neurodegenerative disorders-memantine, a new hope[END_REF].

Various analytical methods have been described for the determination of AMS including spectrofluorimetric [START_REF] Walash | Spectrofluorimetric determination of amisulpride and bumidazone in raw materials and tablets[END_REF], spectrophotometric [START_REF] Sharma | Development and validation of spectrophotometric methods for estimating amisulpride in pharmaceutical preparations[END_REF][START_REF] Humaira | Development and validation of spectrophotometric method for determination of amisulpride in pharmaceutical dosage forms[END_REF], chromatographic [START_REF] Neog | Isocratic liquid chromatographic method for analysis of amisulpride in pharmaceutical preparations[END_REF][START_REF] Remane | Fast and simple procedure for liquid-liquid extraction of 136 analytes from different drug classes for development of a liquid chromatographic-tandem mass spectrometric quantification method in human blood plasma[END_REF][START_REF] Kudris | Analysis of amisulpride in human plasma by SPE and LC with fluorescence detection[END_REF], and electrochemical methods [START_REF] Özkan | Electroanalytical characteristics of amisulpride and voltammetric determination of the drug in pharmaceuticals and biological media[END_REF].

Electrochemical sensors, especially carbon paste electrode (CPE), is considered as a distinctive kind of heterogeneous carbonic electrochemical platforms including a mixture of graphitic carbon powder with a non-conductive and water immiscible binder [START_REF] Nossol | Carbon paste electrodes made from novel carbonaceous materials: Preparation and electrochemical characterization[END_REF][START_REF] Mohamed | Electroanalytical sensing of the antimicrobial drug linezolid utilising an electrochemical sensing platform based upon a multiwalled carbon nanotubes/bromocresol green modified carbon paste electrode[END_REF]. The easy functionalization, surface modification is the main feature of CPE, which makes them to offer higher electrocatalytic activity by increasing the electron transfer mechanism [START_REF] Nossol | Carbon paste electrodes made from novel carbonaceous materials: Preparation and electrochemical characterization[END_REF][START_REF] Asran | An innovative electrochemical platform for the sensitive determination of the hepatitis B inhibitor Entecavir with ionic liquid as a mediator[END_REF][START_REF] Saleh | A novel and ultrasensitive electrochemical biosensor based on MnO2-V2O5 nanorods for the detection of the antiplatelet prodrug agent Cilostazol in pharmaceutical formulations[END_REF][START_REF] Mohamed | Smart electrochemical sensing platform for the simultaneous determination of psychotic disorder drugs isopropamide iodide and trifluoperazine hydrochloride[END_REF].

Therefore, fabrication of CPE by employing appropriate mediators, and nanomaterials like carbon nanotubes [START_REF] Mohamed | Smart electrochemical sensing platform for the simultaneous determination of psychotic disorder drugs isopropamide iodide and trifluoperazine hydrochloride[END_REF], graphene derivatives [START_REF] Mohamed | 3D spongy graphene-modified screen-printed sensors for the voltammetric determination of the narcotic drug codeine[END_REF][START_REF] Mohamed | One-pot green synthesis of reduced graphene oxide decorated with β-Ni(OH)2-nanoflakes as an efficient electrochemical platform for the determination of antipsychotic drug sulpiride[END_REF], metal and metal oxide nanoparticles [START_REF] Mohamed | Magnetic cobalt ferrite nanoparticles CoFe2O4 platform as an efficient sensor for trace determination of Cu(ii) in water samples and different food products[END_REF], or their composites [START_REF] Mohamed | One-pot green synthesis of reduced graphene oxide decorated with β-Ni(OH)2-nanoflakes as an efficient electrochemical platform for the determination of antipsychotic drug sulpiride[END_REF][START_REF] Khalifa | Graphene-Based Sensor for Voltammetric Quantification of Dapoxetine Hydrochloride: A Drug for Premature Ejaculation in Formulation and Human Plasma[END_REF], have attracted an increasing interest to be utilized in different electrochemical analyses.

Metal-oxide film based electrochemical platforms have been widely used for their high electrocatalytic activity and excellent perspective of different application as electrochemical sensors [START_REF] Mohamed | Incorporation of Tetrazolium Blue (TB)/Gold Nanoparticles (GNPs) into Carbon Paste Electrode: Application as an Electrochemical Sensor for the Sensitive and Selective Determination of Sotalol in Micellar Medium[END_REF]. Metallic nanoparticles and metal oxide particles have received significant attention due to their unique properties such as conductivity and excellent electrocatalytic and electroanalytical properties due to their nano-size structures and effective surface area that in turns enhance the rate of electron exchange between the sensor surface and the different analytes [START_REF] Asran | An innovative electrochemical platform for the sensitive determination of the hepatitis B inhibitor Entecavir with ionic liquid as a mediator[END_REF][START_REF] Mohamed | Cobalt ferrite magnetic nanoparticles as a highly efficient electrochemical platform for simultaneous determination of dexlansoprazole and granisetron hydrochloride[END_REF][START_REF] Rahman | Electrodeposited Ru nanoparticles for electrochemical reduction of NAD? to NADH[END_REF]. Due to different oxidation states of ruthenium derivatives and their electrochemical reversibility makes them as excellent electron transfer mediators for modification of different electrochemical sensors surfaces [START_REF] Rahman | Electrodeposited Ru nanoparticles for electrochemical reduction of NAD? to NADH[END_REF][START_REF] Zare | Electrodeposited nano-scale islands of ruthenium oxide as a bifunctional electrocatalyst for simultaneous catalytic oxidation of hydrazine and hydroxylamine[END_REF]. Safranin (SAF) is lipophilic electro active poly aromatic cationic dye [START_REF] Herrero | Adsorption and reduction kinetics of safranine T in self-assembled phospholipid monolayers deposited on mercury[END_REF], which is commonly used in cytology, histology and as a redox indicator in analytical chemistry [START_REF] Lavanya | Selective determination of dopamine in presence of ascorbic acid by using Triton X-100 poly (safranin) modified carbon paste electrode[END_REF].

In this work, we develop a self-assembled Ru decorated electrochemical sensing platform based on SPE, for sensitive and selective determination of AMS in the presence of coadministered drugs; MEM and CIT using SAF as a mediator. For additional enrichments on the sensor surface, , Ru nanoparticles were integrated due to their reported excellent physicochemical characters like strong adsorptive ability, good biocompatibility, thermal stability, high surface area, and electrical/electrochemical properties [START_REF] Zare | Electrodeposited nano-scale islands of ruthenium oxide as a bifunctional electrocatalyst for simultaneous catalytic oxidation of hydrazine and hydroxylamine[END_REF]. To the best of our knowledge, there is no work reported to date that employ Ru-SAF for the electrochemical determination of AMS. We therefore design the first ever example of using Ru-SAF/CPE sensor for bio monitoring of AMS in human urine and pharmaceutical products. The Ru-SAF/CPE constructed sensor offers a selective, simple and consistent electrochemical platform for simultaneous determination of AMS with a detection limit of 8.80×10 -9 M and other common molecules like MEM and CIT.

Experimental

All details of the present experimental procedures, instrumentation and reagents are included in the Electronic Supplementary Information (ESI) part.

Results and discussion

Morphologic and Electrochemical characterization of the fabricated sensors

The scanning electron microscope (SEM) image of the bare unmodified sensor is shown in Fig. 1SA as the electrode surface was dominated by smooth and uniformly designed carbon flakes and divided sheets whereas, Figure 1SB clearly exhibited that the Saf particles were incorporated into the carbon paste and hence the captured image is relatively different. Next, Energydispersive X-ray (EDX) and SEM analysis are used to reveal the conformation of Ru nanoparticles formation on electrode surface. As illustrated in Figure 1A, the EDX analysis indicates the creation of Ru-nanoparticles over the sensor surface. As observed in the SEM images (Figure 1B), Ru nanoparticles are randomly distributed on the sensor surface, which increases the effective surface area and eventually improves the microscopic area of Ru-Saf/CPE surface and in turns increases the electrode response and sensitivity of voltammetric determination. Cyclic voltammograms (CVs) for the electrochemical oxidation of AMS (1.0 x 10 -3 M) was performed using bare CPE, Saf/CPE, Ru/CPE and Ru-Saf/CPE sensors at a pH 7.0 using B-R buffer. The corresponding voltammograms are illustrated in figure 1C. Figure 1C shows the typical cyclic voltammograms for all the fabricated sensors. At the CPE unmodified sensor, the obtained anodic peak current (Ip) with a value of 35.0 μA at 0.915 V coincide with the electrochemical oxidation of AMS. As Saf is considered as a powerful redox material with high electrochemical activity, it is predictable to assist and improve the AMS electrochemical oxidation at the Saf/CPE sensor surface. The anodic peak current Ip value for the oxidation of AMS was elevated to 39.0 μA and moved to a less positive potential (0.907 V). Next, upon the deposition of Ru nanoparticles at fixed potential (Ru/CPE; exhibited in Figure 1C), anodic current Ip is further increased. The anodic peak appears at 0.988 mV with a current value of 52.0 μA, which is attributed to the high conductivity and greater surface area of Ru nanoparticles that can act as a promoter for the electron transfer process that facilitates the AMS oxidation at the fabricated electrochemical platform. Surprisingly, companying the deposited Ru nanoparticles with the Saf material inside the sensor composite resulted in more enrichment to the AMS anodic current (e.g. Ip is raised up to 67.0 μA at 0.980 V). Because of the incorporation of Ru nanoparticles with Saf, anodic peak current Ip much enhanced comparing to other sensors that may be attributed to the enhancement of electrochemical properties, ease of electrons transfer and surface area.

Based on the above discussion, CV was exploited to detect the electrochemical behavior of different bare and Ru-Saf/CPE using the redox couple of [Fe(CN)6] 3-/4-in f 5.0 mM [Fe(CN)6] 3- /4-and 0.1 M KCl solution. Figure 1d shows a CV response of bare CPE along with Ru-Saf/CPE in a 5.0 mM [Fe(CN)6]3-/4-and 0.1 M KCl solution with a scan rate of 100 mV s -1 . The AMS voltammetric behavior was scanned in the range between +0.4 to +1.2 V. The electroactive area was calculated from the slope of the plot of Ip versus ν 1/2 and the calculated areas were found to correspond to 0.054 and 0.173 cm 2 for CPE, Ru-Saf/CPE, respectively. Fig. 1D shows the comparatively weak redox current of CPE. Upon modifying the CPE with Ru-Saf, the redox current significantly increased to ~ 2.6 times as against of the CPE. This indicates that the synergetic effect of involving both Ru nanoparticles and Saf in the sensor composite might offer the essential conduction pathway for the electrocatalytic mechanism.

Optimization of the experimental parameters

The electrochemical behavior of AMS is dependent on the pH of buffer. In the pH investigation, cyclic voltammetry is done on the Ru-Saf/CPE using 1.0 x10 -3 M of AMS in different pH values (3.0 -9.0 of 0.04 M B-R buffer; SR=100 mVs -1 ). This this scan rate was selected due to the high sensitivity with well-shaped voltammograms and a relatively narrow peak width at this value. By the inspection of the suggested oxidation mechanism, AMS oxidation is may be due the amino group oxidation that exists in primary amine group on the m-anisidine ring [START_REF] Özkan | Electroanalytical characteristics of amisulpride and voltammetric determination of the drug in pharmaceuticals and biological media[END_REF][START_REF] Wang | Determination of trace metoclopramide by anodic stripping voltammetry with nafion modified glassy carbon electrode[END_REF].

Next, the mechanism of the electrochemical reaction can be assessed through the By changing the scan rate (ʋ), electron-transmission kinetic parameters of AMS at Ru-Saf/CPE sensor can be determined. Applying log ʋ and anodic peak potential (Ep), a linear performance was detected (Fig. S2) and can be described by the following equation: E/mV =0.633 +0.1252 log ʋ (V/s) (R 2 =0.9781) Furthermore, kinetic parameters were calculated for the electrochemical process on Ru-Saf/CPE using the theory of Laviron for irreversible reactions [START_REF] Laviron | General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems[END_REF]. For a totally irreversible reaction, α (electron transfer coefficient) was assumed as 0.5, then n (the number of electrons transferred) was determined to be 0.944, suggesting that one electron was involved in the oxidation of AMS.

Applying Laviron equation, k o (the standard heterogeneous rate constant of the surface reaction) was found to be 1.177 s -1 .

Last, the influence of the amount of Saf, Ru deposition potential and time used in the construction of the Ru-Saf/CPE based on the value of the voltammetric response / peak sharpness are tested. The Ip for AMS was tested towards varying the amount of Saf over the range 3.0 mg to 25.0 mg.

10.0 mg of Saf was found to be the most appropriate amount as any addition cause a background augmentation with poor AMS detection repeatability.

Different deposition potentials are studied in the range -0.1 to -0.4 V. Upon the highest anodic peak current and stability, -0.4 V is chosen. The Ru electrodeposition at -0.4 V offers the development of Ru dense film that displays great electrocatalytic activity and enhances the AMS oxidation [START_REF] Jaksic | Electrocatalysis for hydrogen electrode reactions in the light of fermi dynamics and structural bonding FACTORS-I. individual electrocatalytic properties of transition metals[END_REF]. Furthermore, the deposition time is depicted at different time intervals (from 10 sec to 50 sec) and 40 sec was chosen due to the highest current response.

Optimization of developed electrochemical platform

The procedures delivered by the International Conference on Harmonization (ICH) for methods validation were followed for the validation of our developed protocol [START_REF] Kan | Composites of multiwalled carbon nanotubes and molecularly imprinted polymers for dopamine recognition[END_REF]. The SWV experiments were performed using Ru-Saf/CPE in B-R buffer at pH 7.0 solution containing different concentrations of AMS. The calibration curve was drawn based on the necessary practical consideration, to offer accurate, precise and linear results. Figure S3 illustrates the SWV of AMS at Ru-Saf/CPE in B-R buffer at pH 7.0 containing different concentrations, for each at scan rate: 0.1 V s -1 , PH= 0.25 V, PW=0.50 V, SH=0.02 V. Insets: the plot of the peak current as a function of AMS in concentration range 9.5 × 10 -8 -7.0 × 10 -5 M. The plot of the peak current vs. AMS concentration showed wide linear for AMS concentrations: for 9.8 × 10 -8 -7.0 × 10 -5 M, the regression equation was I(μA)=0.385 C + 0.13 (R 2 =0.9992). Lower limit of detection (LOD) and lower limit of quantification (LOQ) for linear calibration curve were calculated and found to be 8.80×10 -9 and 2.93×10 -8 , respectively. In addition, the repeatability and intermediate precisions were calculated and found to be 0.78 -0.92%, respectively.

Moreover, stability factor was tested for Ru-Saf/CPE sensor, where the anodic peak current (Ip)

was not found to variate upon the storage in air at room temperature for 7 days. The modified sensor reserved 97.90 -98.87% of its initial response.

Comparing to the previously published electrochemical determination of AMS, Ozkan et al [START_REF] Özkan | Electroanalytical characteristics of amisulpride and voltammetric determination of the drug in pharmaceuticals and biological media[END_REF] reported a bare unmodified stationary glassy carbon electrode with a linearity range 4.0x10 -6 -6.0x 10 -4 M and detection limit of 1.9×10 -7 M. While the present protocol offers wider linearity range and lower detection limits. The co-administered drug MEM displayed no electrochemical activity at the suggested electrochemical platform Ru-Saf/CPE under the optimized experimental conditions. Paracetamol (PAR ; acetaminophen) is one of the most common pharmaceutical medication used as painkiller globally due to its safety and efficiency [START_REF] Aghababian | Essentials of emergency medicine[END_REF]. PAR can be prescribed as analgesic coadministered medication with AMS. Furthermore, inflammation is accompanying with psychological distress and depression, so nonsteroidal anti-inflammatory drugs like PAR might be prescribed as associate therapy with antidepressants [START_REF] Perera | Cohort profile of the South London and Maudsley NHS Foundation Trust Biomedical Research Centre (SLaM BRC) Case Register: current status and recent enhancement of an electronic mental health record-derived data resource[END_REF]. The SWVs of both AMS, PAR, ascorbic acid (ASA) and Caffeine (CAF) were performed under the optimized experimental conditions showing an excellent peak separation that allows the precise selective determination of AMS, Figure 4A andB.

E/V(vs

Levodopa (LEV) is a catecholamine class pharmaceutical, precursor of dopamine and is used in the treatment of Parkinson's disease [START_REF] Cerri | An update on the use of non-ergot dopamine agonists for the treatment of Parkinson's disease[END_REF] was also checked with AMS at Ru-Saf/CPE. High resolution was observed along with good sharpness were achieved. The oxidation signal of LEV was recorded at 0.4 V while the anodic peak of AMS was achieved at 0.83 V, Figure 4C.

Reproducibility study and stability of developed electrochemical sensor

The electrochemical responses of 1.0 mM AMS at Ru-Saf/CPE at the optimum conditions for five consecutive measurements were measured to test the reproducibility factor. The relative standard deviation (RSD) for the Ip of AMS in five consecutive experiments was calculated and found to be 1.12 %. Furthermore, the fabrication protocol reproducibility was tested by preparing independently three modified sensors with the same way. The RSD was found to be 1.15%. In addition, inter-day precision was also calculated using different drug concentrations over a period of three days. Intra-day and inter-day precision were examined as relative standard deviation (RSD %) and presented in Table S1. The %RSD of all tested concentration levels was below1.64% which confirms a good precision.

Analysis of AMS in real samples

The feasibility of Ru-Saf/CPE for analysis of AMS in real human fluids like human plasma, urine and evaluation of dosage form were examined using SWV. The standard addition methods were applied to assess the sensor performance for AMS analysis and compared the obtained data with the calibration plot, Table 1. The values of the recovery display a high accuracy of the developed electrochemical platform. Referring to the experiments outcomes, it is evident that this sensor has extreme potential for the determination of trace amounts of these compounds in biological fluids. Moreover, the dosage form was analyzed by keeping the similar parameters of SWV experiment applied before. The recovery experiment was presented in Table 1, which confirm that, the Ru-Saf/CPE is very sensible for the determination of low and high concentrations of AMS in different real samples.

Interference studies

Under optimized conditions, the interference from selected metal ions and organic compounds on the determination of 1.0 x 10 -5 M AMS was evaluated. Numerous potential interferents were chosen, which are commonly present in the pharmaceutical dosage forms (cations, anions and organic compounds). As can be seen in Table S2, the attained RSD values were low in all cases, indicating the Ru-Saf/CPE can be successfully used for the selective determination of AMS in pharmaceutical, environmental and biologic samples.

Conclusion

This

Figure 1 .

 1 Figure 1. A) EDX of Ru-Saf/CPE; B) SEM of Ru-Saf/CPE; C) Cyclic voltammograms of different studied sensors recorded in 1.0 x 10 -3 M AMS (pH 7.0; B-R buffer; Scan rate: 100 mV s -1 ); D) CVs of CPE and Ru-Saf/CPE in a solution of 5.0 mM [Fe(CN)6] 3-/4-and 0.1 M KCl with a scan rate of 100 mV s -1 .

Fig 2 .

 2 Fig 2. A, signifies the performance of AMS due to changing the pH as the peak potentials (Ep) are moved to a lower positive potential upon rising the pH value. The highest anodic peak

Figure 2 .

 2 Figure 2. A) Voltammetric responses of 1.0 mM AMS over a range of pH values using Ru-Saf/CPE sensing platforms. Scan rate: 0.1 V s -1 . B) The oxidation peak current and peak potential AMS as a function of pH variations. C) Suggested electrochemical oxidation of AMS.

  investigation of the influence of scan rate (v) of the oxidation peak signal by Ru-Saf/CPE in 1.0×10 -3 M AMS in B-R buffer (pH 7.0). The result is demonstrated in Figure S2. Upon increasing the potential scan rate in the 20-100 mV s -1 range, the current value increased, and the peak potential shifted to more positive values with no noticeable reverse reduction peak, suggesting that the oxidation of AMS is an irreversible process. By plotting the peak current (Ipa) against the square root of the scan rate (v 1/2 ), a linear straight line was obtained (Figure S2, inset A), indicating that the reaction is controlled by diffusion, according to: Ip (µA) = 8.176√v -16.45; R 2 =0.9982. Direct relation was acquired between log Ip and log v in the 20 -100 mVs -1 range as the following: log I(µA) = 0.314 + 0.7317 log v; R 2 =0.9956.

Figure 3 .

 3 Figure 3. Electrochemical determination of AMS and CIT. (A) Square wave voltammograms using a Ru-Saf/CPE sensing platform in a pH 7.0 B-R buffer containing different concentrations of AMS and CIT correspond to 3.0 x 10 -7 to 2.9 x 10 -5 M, for each at scan rate: 0.1 V s -1 , PH= 0.25 V, PW=0.50 V, SH=0.02 V. (B) Corresponding current response of AMS and CIT oxidation (analytical curves). Insets B: plots of Ip versus AMS and CIT concentrations.

Fig. 4 .

 4 Fig. 4. Square wave voltammograms using a Ru-Saf/CPE sensing platform in a pH 7.0 B-R buffer containing 3.5 x 10 -6 M AMS, 1.0 x 10 -3 M ASA, 1.0 x 10 -4 M PAR and CAF, and 1.0 x 10 -4 LEV, for each at scan rate: 0.1 V s -1 , PH= 0.25 V, PW=0.50 V, SH=0.02 V.

  study shows the fabrication of a chemically modified carbon paste electrode (CPE) by the combination of Ru nanoparticles and Saf. Through the cooperation of the proposed modifiers, excellent electrocatalytic activity and voltammetric behavior are achieved for sensing of AMS. The electrochemical oxidation behavior of AMS on the new electrochemical platform was depicted. The established sensor showed high selectivity, reproducibility, and sensitivity with other advantages like time simplicity, saving and cost-effectiveness. Additionally, the proposed electrochemical sensor was utilized for the quantitative detection of AMS in tablets, urine and plasma.

Table 1 .

 1 Determination of AMS in human urine and synthetic tablets using the proposed sensing protocol Ru-Saf/CPE

	Sample	Amount added standard	Amount found	Apparent recovery%
		(µM)	(µM)
	Urine	0.50	0.48	96.00
		10.00	9.80	98.00
		40.00	39.29	98.22
		70.00	69.62	99.45
		100.00	98.98	98.98
	Recovery%±R.S.D			98.13±1.30
	Plasma	0.50	0.49	98.00
		10.00	9.78	97.80
		40.00	39.36	98.40
		70.00	69.51	99.30
		100.00	99.70	99.70
	Recovery%±R.S.D			98.80±0.76
	Amipride ® Tablets	10.00	9.84	98.37

ACKNOWLEDGMENT

The authors acknowledge the partial funding from KardiaTool, H2020, under the grant number 768686 and POC Allergies through the ERA PerMed program.

Determination of AMS in the presence of co-administered drugs

To check the selectivity of Ru-Saf/CPE based sensing platform for AMS detection, other co-existing significant analytes or frequently co-formulated drugs CIT and MEM were tested in the presence of AMS. So, simultaneously varying drugs concentrations with recording the voltammetric responses at the fabricated electrochemical platform. Referring to Fig. 3, it is clear the presence of well sharp separated anodic peaks at potentials of +1.117 and +0.830 V (vs. Ag/AgCl), corresponding to the oxidation of CIT and AMS, respectively. This clarifies the high accuracy and precision of the developed sensor for the simultaneous determination of CIT and AMS. Figure 3B displays the current response obtained upon the oxidation of the molecules based on their respective concentrations. The plot of the peak current (Ip) as a function of AMS and CIT concentrations was demonstrated, Figure 3. The calibration plots are linearly for AMS and CIT over the range 3.0×10 -7 to 2.4×10 -5 M for both the drugs. The regression equations were: Ip(μA) = 0.38C (µM) + 0.39, R 2 = 0.9990; and Ip(μA) = 0.481C (µM) + 0.913, R 2 = 0.9987; for AMS and CIT, respectively. The analytical sensitivity of the AMS at Ru-Saf/CPE sensor is found to be 0.38 μA/μM that is very close to the obtained value in the absence of interfering AMS (0.385 μA/μM), signifying that the oxidation processes of both drugs at Ru-Saf/CPE are independent and consequently, the determination AMS of the mixture with CIT is promising without major interference.