PLASMA SAMPLES AND INSTRUMENT FOR REALISATION OF THE METODO

FIELD OF THE INVENTION

The present invention relates to a method for measuring the concentration of 7-ethyl- 10-[4-(piperidino)-l-piperidino]-carbonyloxycamptothecin in plasma samples, which comprises a pre-analytic procedure for isolating the compound from the sample and the subsequent measurement of its concentration by electrochemical means. The invention further relates to an instrument for realizing the analysis method in an automated way.

STATE OF THE ART

The compound 7-ethyl- 10-[4-(piperidino)- 1 -piperidinoj-carbonyloxycamptothecin, having the formula reported below, is known in the medical-pharmaceutical field as CPT- 11 or by the INN name Irinotecan, which will be used in the rest of this text.

Irinotecan

Irinotecan is a cytotoxic prodrug currently used in different chemotherapy regimes, in particular for colorectal cancer. It is activated by the liver carboxylesterase enzyme to provide 7-ethyl-lO-hydroxy-camptothecin (compound known as SN-38), which is a powerful topoisom erase I inhibitor.

Overdoses of Irinotecan are associated with severe side effects, which include amongst others diarrhoea, anaemia, leukopenia, thrombocytopenia and alopecia, amplified by the fact that its pharmacokinetics and metabolism are extremely complex and depend on a series of factors such as the patient’s physical conditions and genetic profile, and concomitant co therapy, such as for example that based on folinic acid (FA) and 5-fluoro-2,4(lH,3H)- pyrimidinedione (5-fluoruracil or 5-FU).

Accordingly, it is important that in the use of the compound the pharmacological treatments are personalized, in order to prevent both excessive dosages and obviously insufficient ones, to the advantage of greater efficacy of the chemotherapy treatment itself.

On this point, therapeutic drug monitoring (technique known in the medical field as TDM) has become essential for helping physicians (clinical, oncologists, etc...) to control the dose of the drug for the individual patient. Furthermore, it is important to underline the fact that TDM in the oncology sector is still not a very developed clinical practice. Its use is often limited to well defined clinical studies within dedicated facilities and laboratories in which the analyses are performed by specialist and highly qualified personnel with instruments mainly based on coupling between high performance liquid chromatography (HPLC) and mass spectrometry (MS).

The therapeutic monitoring of Irinotecan is further complicated by the fact that the compound is subject to a complex metabolic conversion also by enzymatic systems different from those mentioned above and that lead to the formation of different compounds (metabolites), in particular SN-38 and the compounds 7-ethyl- 10-hydroxy-camptothecin glucuronide (SN-38G), camptothecin (CPT), 7-ethyl- 10-[4-N-(5-aminopentanoic acid)-l- piperidinojcarbonyloxycamptothecin (APC) and 7-ethyl- 10-[4-(l-piperi dino)- 1 -amino] - carbonyloxy camptothecin (NPC). These metabolites of Irinotecan, because of the chemical structure which is largely similar to that of the starting compound, can give rise to interferences in the analyses.

Patent application CN 107677734 A describes a method for measuring substances connected to SN-38 in a biological sample. The method is based on the separation of the sample through UPLC (Ultra-Performance Liquid Chromatography) of SN-38 from related substances, and subsequent measurement of the quantity of the species of interest through UV spectrophotometry (wavelength used 254 nm).

Patent EP 2543994 B1 describes an instrument for the high precision measurement of substances in low concentrations in the blood. The instrument is composed of: a disc for the sample provided with containers of samples; a disc of reactants provided with containers of reactants; a first disc provided with first containers in which the purification of the component subject to measurement in the sample is performed; a second disc provided with second containers in which the further purification of the sample purified in the first container is performed; and a mass spectrometry unit that measures the sample purified in the second container. Irinotecan is mentioned among the substances indicated as being measurable with high precision with this instrument.

The methods and instruments described in the last two documents allow the precise measurement of the concentration of metabolites of Irinotecan in biological samples, but are not very suitable for use in a TDM methodology especially because of the difficult portability of the instruments, relatively long analysis times and the need for centralized analysis laboratories and qualified personnel. An effective use of the TDM methodology requires instead simple and quick analytical protocols for the measurement and control of the concentration of the drug in the patient’s plasma.

There is therefore a very perceived need in the field to have available an analytical methodology for the measurement of Irinotecan in biological samples that is completely compatible with the requirements of the TDM technique.

The object of the present invention is to provide an analytical method, and an instrument for the realization thereof, for the quick and accurate measurement of the concentration of Irinotecan in a plasma sample, and to provide a protocol and the analytical set-up for the realization of the method.

SUMMARY OF THE INVENTION

This object is obtained with the present invention, which in a first aspect thereof relates to a method for measuring the concentration of Irinotecan in a plasma sample, which comprises the following steps:

a) buffering the sample of plasma with PBS buffer at physiological pH;

b) loading the sample added with PBS buffer obtained in step a) into a suitably pre conditioned solid phase extraction column;

c) passing through the column a water-methanol mixture, forced with an inert gas flow or by applying vacuum at the column outlet, to remove the polar compounds from said solid phase;

d) passing in the column acetonitrile, forced with an inert gas flow or by applying vacuum at the column outlet, to extract Irinotecan from said solid phase;

e) adding to the Irinotecan in acetonitrile solution obtained in step d) a solution of a base and a solution of a support electrolyte, obtaining a final solution having a concentration between 0.01 and 0.1 M of support electrolyte and between 0.2 and 10 mM of base;

f) analyzing the mixture obtained in step e) by means of a differential pulse voltammetry measurement operating in the range of positive potentials between 0 and +1.5 V with respect to the reference electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

- Fig. 1 shows in a schematic way the steps of the separation of Irinotecan (steps b to d of the method described above), through a solid phase extraction column, from the other components of the plasma and from possible compounds interfering in the subsequent voltammetry analysis;

- Fig. 2 schematically shows an instrument for performing the analysis of Irinotecan in an automated way;

- Fig. 3 shows the voltammetry signals recorded in a series of plasma extracts added with increasing concentrations of Irinotecan;

- Fig. 4 shows the linear regression straight line of the data obtained from the curves of Fig. 3, in particular the data of the peak currents as a function of the concentration.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the following terms and abbreviations have the meanings specified here:

- unless otherwise indicated, all the percentages and concentrations in the following description are molar (M), millimolar (mM) or micromolar (pM);

-“analyte” indicates a substance of which the presence or not in an analysis is to be ascertained, quantifying the concentration thereof; in the present invention the analyte is the compound Irinotecan;

-“LOD”, meaning“limit of detection”, indicates the minimum amount of analyte that can be detected with the method and technique used in the analysis (i.e. that provides a indication different from“absence of the investigated analyte”);

-“LOQ”, meaning“limit of quantification”, indicates the minimum amount of analyte that provides a reliable quantitative measurement of its quantity or concentration; this value is always greater than the LOD;

- the differential pulse voltammetry technique is indicated by the abbreviation“DPV”, commonly used in the field.

The method of the invention comprises two main steps, a first step of selective extraction of Irinotecan from the initial biological sample, and a subsequent electrochemical analysis step of the sample of Irinotecan thus obtained. In the first step of the method, a) the patient’s plasma sample is buffered at the physiological pH of about 7.4 with a phosphate buffered saline solution known as PBS (“phosphate buffer solution”). The PBS solutions are commercially available and have salt concentrations such as to make them isotonic with the human body; a typical PBS solution contains about 8.0 g/L of NaCl, 0.2 g/L of KC1, 1.44 g/L of Na ₂ HP0 ₄ and 0.24 g/L of KH2PO4. The PBS solution is added to the plasma in a volume ratio PBS:plasma between 1 : 1 and 1 :2. The aim of the addition of the PBS solution is to keep the sample at its own physiological pH and to prevent any alterations of the sample itself, of the analyte and of other compounds present in the sample that could lead to deviations in the results of the method.

One of the advantages of the method of the invention is that of being able to work with minimum quantities of plasma, such as to allow limited samples to be taken in patients who are already weak, or the use in other parallel analyses of the taken sample. Typically the volume of plasma used in the method of the invention is variable between 10 and 400 pL, preferably between 100 and 150 pL.

Before loading the sample, according to the manufacturer’s indications, the column (10) is conditioned by wetting the stationary phase (11) first with methanol and then with an equal volume of water (Fig. l .a).

The water used in this step and in all the subsequent steps of the invention is ultrapure water, type I according to standard ASTM D1193-91, having conductability less than or equal to 5.5 1 O ⁶ S/m, water of this quality can be produced in a laboratory from mains water through a Milli-Q ^1® purifier of company Millipore Corp., and ultrapure water type I is often indicated for simplicity in the analytical field as Milli-Q water in the rest of the text and the claims,“water” means Milli-Q water.

In step b) of the method of the invention, the sample prepared in step a) is loaded onto a solid phase extraction column; the step is depicted in Fig. l .b, wherein the black stars represent the molecules of Iriontecan, whereas white triangles, circles and rhombuses represent the molecules of compounds that would interfere in the subsequent voltammetry measurement. Solid phase extraction is known in the field of analytical chemistry by the abbreviation SPE. A SPE column exploits the affinity of the compounds dissolved or suspended in a liquid (known as the mobile phase) towards a solid through which the sample passes (known as stationary phase) to separate a mixture into desired and undesired components. SPE columns are commercially available in optimized form (i.e. containing a specific stationary phase) for the retention of various types of chemical compounds. For the purposes of the present invention, the SPE column must be such as to effectively adsorb neutral compounds containing aromatic rings, with limited solubility in water or strongly polar solvents. Irinotecan has these characteristics, being not very soluble in water or strongly polar solvents when it is in neutral (non-protonated) form. SPE columns suitable for the purpose are those of the Strata ^® series, in particular the Strata ^® -XL, columns, produced by the company Phenomenex of Torrance, California (USA).

After the sample has been loaded into the column, the system is left to equilibrate for a time between 30 and 120 seconds.

Then, in step c) of the method of the invention, two aliquots of a mixture 9: 1 by volume of Milli-Q water: methanol are introduced in succession in the column (Fig. l .c); the volume of each aliquot of this mixture is generally between 0.5 and 1 mL. The passage of this mixture through the stationary phase is accelerated through a flow of inert gas, generally nitrogen, from the head to the outlet of the column; the flow is guaranteed by the application at the head to the column of an overpressure of gas or by applying a vacuum at the outlet. In this step, all the interference compounds are removed almost quantitatively from the stationary phase apart from the Irinotecan analyte; the traces of compounds different from the analyte that remain in the stationary phase after step c), and that can be eluted in the subsequent step d), are at such low concentrations as not to interfere with the final voltammetry analysis. The two aliquots of the Milli-Q water: methanol mixture that have passed through the column, which contain compounds different from Irinotecan, are collected at the outlet of the column and disposed of.

In the subsequent step of the method, d) pure acetonitrile is passed through the column in a volume between 300 and 1000 pL (Fig. 1.d). Also in this case, the passage of the solvent through the stationary phase of the column is promoted by a flow of inert gas (typically nitrogen) through it or by applying vacuum at the outlet, as described in step c). The analyte present in the initial plasma sample is found in a practically quantitative way in the acetonitrile solution recovered in this step.

In step e), the acetonitrile solution recovered in the previous step is then prepared for the voltammetric analysis, through the addition of a solution of a support electrolyte soluble in acetonitrile and of a basic compound. Acetonitrile is used as a solvent for the support electrolyte solution, whereas the solvent for the base solution may be acetonitrile or water. The preferred electrolyte for the purposes of the present invention is tetrabutylammonium hexafluorophosphate (of formula (CFFCFhCFhCFh^NPFe, indicated in the rest of the text by the abbreviation TBAPFr,). The basic compound is preferably selected from sodium bicarbonate (NaHCO,), sodium carbonate (NaiCCh), sodium hydroxide (NaOH), sodium phosphate (NasPCri), disodium hydrogen phosphate (NaiHPCri), sodium dihydrogen phosphate (NaFhPCri) and, preferably, sodium tetraborate (NaiE^Cb). The basic compound is added for transforming the protonated form of Irinotecan (present in the plasma also because the drug is administered as its hydrochloride) into the free base. The final concentration of these two components in the solution varies between 0.01 and 0.1 M, preferably 0.05 M in the case of TBAPF ₆ and from 0.2 to 10 mM, preferably 0.73 mM in the case of Na ₂ B ₄ 07.

Despite the method up to this point involves the realization of five steps, the performance thereof is quick and requires about 4 minutes in total.

In step f) the sample thus prepared is finally analysed through DPV.

The measurement is performed in a three-electrode electrochemical cell, comprising a working electrode, a reference electrode and a counter electrode. As the working electrode it is possible to use a glassy carbon electrode (known in the field as GCE), other carbon based materials (such as graphene and boron-doped diamond) or a platinum electrode; as the reference electrode, a silver wire is used, in the place of the common Ag/AgCl reference electrode (saturated with KC1) to prevent contamination of the sample with chlorine ions; and as the counter electrode a platinum, gold or iridium spiral.

The inventors have verified that by scanning the potential in the positive region with respect to the reference electrode (between 0 and +1.5 V), at a working potential of about +0.90 V, a current peak can be detected attributable to the oxidation at the tertiary amine of the terminal piperidine ring of the molecule. This current peak is in a voltammogram area free from the interference of other compounds, so that its intensity can be attributed univocally to Irinotecan; the inventors have also observed that the intensity of the peak depends linearly on the concentration of the analyte, at least in the range of concentrations from 0.5 to 6 mM, thus allowing the quantitative analysis of the sample. In fact, the inventors have verified, through dedicated tests, that in the range of concentrations indicated above, the measured current is linked to the concentration of the analyte with a relationship of the type:

Im = P ^X C + K

wherein I _m is the measured current intensity (a value that also depends on the geometry and area of the working electrode, and that is generally measured in mA), P is the slope of the experimental data interpolation line, C is the concentration of analyte in pM and K the value of the intercept (a current value) of the interpolation line at analyte concentration equal to 0. The determination coefficient R ² measured in various tests was always greater than 0.98, confirming an excellent linearity of the current measurement response as a function of the analyte concentration.

With these tests, the following analytical performance data were also obtained:

- dynamic range of the method: 5 x 10 ⁷ ÷ 6 x 10 ⁶ M;

- LOD: 1.28 x 10 ⁷ M;

- LOQ: 4.26 x 10 ⁷ M.

In the second aspect thereof, the invention relates to an automated instrument 1 for the realization of the analysis method described above.

The instrument of the invention is schematically represented in Fig. 2. The main elements of the instrument are the solid phase extraction column 10 containing the solid phase (stationary phase) 11, an analyser 12 (which may be a voltmeter or a potentiostat) equipped with a three-electrode measuring head 13, and a microprocessor 14 (which may also be a personal computer) which controls the performance of the different steps of the method, receives the data from the analyser 12, processes them and generates the results of the analysis.

The other parts of the instrument are:

- a chamber 15 for the introduction of the sample (hereinafter defined as injection chamber) connected downstream, through a two-way valve V4, to the inlet opening of the column 10;

- a tank 16 for the PBS physiological solution connected by means of a volumetric valve Vi to the chamber 15;

- a tank 17 for methanol, connected by means of a two-way volumetric valve V2 to the inlet opening of the column 10;

- a tank 17’ for water, connected by means of a two-way volumetric valve V3 to the inlet opening of the column 10; - a tank 18 for acetonitrile, connected by means of a two-way volumetric valve V7 to the inlet opening of the column 10;

- a container 19 for the collection of the sample of Irinotecan extracted from the column 10 by means of the eluent; the container 19 is fixed to a movement means 20 which, at the end of the elution and therefore of the collection of the sample in the container, move the latter below the measuring head 13;

- a tank 21 for a solution of supporting electrolyte in acetonitrile, connected by means of a two-way volumetric valve Vs to a line for the addition of this reactant to the solution in the container 19;

- a tank 22 for a solution of a base in water or in acetonitrile, connected by means of a two-way volumetric valve Vs to a line for the addition of this solution to the solution in the container 19;

- the passage of the water/methanol mixture into the column 10, as previously mentioned, may be promoted by the overpressure of an inert gas in the head to the column 10 or by a depression downstream thereof. These two possibilities correspond to two alternative embodiments of the instrument, both illustrated in Fig. 2 by broken lines. In the first embodiment, to the inlet line of the sample in column 10 is connected, by means of a two-way valve V4, a source 23 of a pressurized gas (preferably nitrogen); in the alternative embodiment, the column 10 is connected downstream by means of a three-way valve V5 to a suction pump 23’. To increase the versatility of the instrument, this could comprise both the source 23 of pressurized gas and the suction pump 23’;

- the valves from Vi to V9 are connected to the microprocessor 14 respectively by means of electrical lines Li to L9; furthermore, electrical and data transfer lines (represented cumulatively in the figure from line L10) connect the microprocessor 14 to the analyser 12.

For the performance of the method of the invention, after the injection of the plasma sample to be analysed in the chamber 15 (for representation clarity, in the figure the inlet line of the sample into the chamber 15 is not shown), the operator can start the automated analysis with a command to the microprocessor 14. Following this command, the microprocessor opens the volumetric valve Vi, allowing the inlet into the chamber 15 of a volume of PBS solution between 1 and 2 times the volume of plasma. At the same time the microprocessor first causes the opening of the volumetric valve V2, letting methanol into the column 10 from the tank 17, and then closes the volumetric valve V2 and opens the volumetric valve V3, letting into the column a volume of water equal to the volume of methanol previously introduced.

Then, the microprocessor closes the valve V3 and opens the valve V4, enabling the introduction of the plasma sample added with the PBS buffer solution into the column 10.

After a balancing time of the sample in the column comprised between 30 and 120 seconds (which can be determined by the operator by acting on the variable parameters of the microprocessor program), the microprocessor closes the valve V4 and opens the volumetric valves V2 and V3, regulating them so that a water: methanol mixture 9: 1 by volume is caused to cross the column; this operation is repeated twice. The passage of the water/methanol mixture into the column 10 is promoted by the overpressure of gas in the head to the column or by a depression downstream thereof; one of these two conditions is determined by the microprocessor 14 through the opening of the valve V5 (action that places in communication the tank 23 of pressurized gas with the inlet line of the sample in the column), or by placing the three-way valve V ₆ in position such as to connect the vacuum pump 23’ with the outlet line from the column. For greater versatility, the instrument 1 can also comprise both the tank 23 and the vacuum pump 23’ (and the related control lines and valves).

Up to this point in the performance of the method, the liquids exiting the column are discharged to the outside of the instrument 1 (the discharge line is not shown in the figure).

Then, the microprocessor closes the valves V2 and V3, through the movement means 20 moves the container 19 below the outlet line from the column 10, and opens the valve V7 allowing the inlet into the column 10 of acetonitrile from tank 18. During this step an overpressure at the head of column 10 is exerted through the opening of the valve V5, while the valve V ₆ is moved into a position such as to direct the flow of liquid exiting the column towards the container 19.

Then the microprocessor determines the opening of the volumetric valves Vs and V9, so that a supporting electrolyte solution and a basic solution are added to the container 19, respectively from the tanks 21 and 22, obtaining a solution that is ready for analysis.

In the subsequent step the microprocessor, through the means 20, moves the container 19 below the analyser 12, and through the line L10 commands the vertical movement of the head 13 and the consequent immersion of the electrodes into the solution to be analysed; in the figure, the dotted lines represent the container 19 and the head 13 in the measurement position. In an alternative embodiment not represented in the figure, it could be the container 19, once positioned below the head 13, that is raised in order to immerse the electrodes into the solution to be analysed. Finally, the measurement is performed and through the line Lio the microprocessor 14 receives and processes the data of the analyser 12 thus generating the result of the analysis.

At the end of the analysis of a sample, the head 13 is extracted from the container 19, the microprocessor (through the means 20) moves a container 19’ below the head and causes the immersion of the electrodes into the liquid contained in the latter container; the liquid in the container 19’ is a washing liquid, which removes traces of the sample just analysed from the surface of the electrodes, for preparing them for a new measurement. The washing liquid in the container 19’ is preferably refreshed after every cleaning of the electrodes, to prevent the accumulation of residues of previous analyses that could lead to cross contamination in a sequence of analyses. The washing liquid can come from the tank 18 or from a dedicated tank (neither of the two options is shown in the figure).

The invention will be further described by the following experimental part.

Instruments

The separations of the plasma for obtaining solutions of Irinotecan in acetonitrile were performed with SPE Strata ^® -XL columns.

For the voltammetry measurements a CHI 920 bipotentiostat produced by the company CH Instruments located in Austin, Texas, USA, was used.

EXAMPLE 1

To confirm the validity of the method of the invention in the quantitative analysis, a series of plasma samples having concentrations between 2 and 24 mM was prepared, dosing measured quantities of Irinotecan. TB APF ₆ and sodium tetraborate at the concentration of 0.05 M and 0.73 mM, respectively, were added to the extract.

The DPV measurements for detecting Irinotecan were performed in a three-electrode cell, using a GCE disc (0 = 3 mm) as the working electrode, a silver wire as the reference electrode and a platinum spiral as the counter electrode. In the electrochemical cell, the measured concentrations were comprised between 0.5 and 6 mM, consistent with the dilution rate undergone by the initial plasma concentration.

The results of the test are reported in Fig. 3, which shows the voltammograms recorded on plasma extracts containing different concentrations of Irinotecan; the broken line represents a typical voltammogram recorded in the absence of Irinotecan.

Fig. 4 shows the calibration curve obtained through linear regression of experimental data and, specifically, the maximum of the intensity of peaks of the curves in Fig. 3 as a function of the concentration of the analyte.

The following linear regression equation is obtained from the test:

I = 0.0332C + 0.003 (eq. 1) wherein I is the current intensity in mA and C the concentration in pM of analyte, with a determination coefficient R ² equal to 0.987. These data confirm that the current intensity of the peak increases linearly with the analyte concentration.

EXAMPLE 2

A blood sample was taken from a patient with colorectal cancer, subjected to FOLFIRI chemotherapy regime (treatment combined with FA, 5-FU and Irinotecan), 30 minutes after the administration of the drug.

From the blood sample, the plasma was separated by centrifugation.

A SPE Strata ^® -XL column was conditioned with 1 mL of MeOH and then with 1 mL of Milli-Q water to wet the stationary phase.

The column was subsequently loaded with 125 pL of plasma and 75 pL of PBS buffer solution (pH 7.4); the latter buffer was used to maintain the plasma samples at their physiological values.

Two aliquots (600 pL each) of a mixture of Milli-Q watenMeOH 9: 1 by volume were forced (through a nitrogen flow of purity 99.99%, provided by SIAD) to pass through the column to remove the polar compounds of the plasma sample.

The analyte was eluted using 475 pL of acetonitrile (anhydrous, purity 99.9%, Sigma- Aldrich) forced to pass through the column by a flow of pure nitrogen.

2 pL of a saturated aqueous solution of sodium tetraborate (used as decahydrate) were added to the acetonitrile solution containing the analyte, obtaining in acetonitrile a concentration equal to 0.73 mM; furthermore, 9.7 mg of TBAPF ₆ were added, taken from a more concentrated solution of the latter salt, to obtain a concentration of this compound in the final solution equal to 0.05 M.

The DPV measurements for detecting Irinotecan were performed in the same three- electrode cell of Example 1.

On the same plasma sample a control test was performed using the validated method, currently used in the field (HPLC-MS test), to verify the accuracy of the results obtained with the method of the invention.

The procedure was then repeated on a plasma sample obtained from a blood sample taken from the patient 180 minutes after the administration of the drug.

To obtain a statistically significant result, each test was repeated at least three times.

The results of the two tests are summarized in the following table:

The table reports:

- in the first column the time of the blood sample after the administration of the drug to the patient;

- in the second column the analyte concentration data (C _Ref ) in the sample obtained with the reference method, and in brackets the standard deviation value as a percentage (DSR%) of the measurement;

- in the third column the analyte concentration data (Ci), and in brackets the DSR% data, obtained on the same plasma samples with the method of the invention;

- in the last column the relative error (ER) of the method of the invention with respect tO CRef.