DE102012014614B4 - Fast and simple method for the determination of chemotherapeutic agents in biological fluids and infusion equipment based on them - Google Patents

Abstract

Method for determining the concentration of a chemotherapeutic agent and its possibly present metabolites in a biological fluid sample of an individual at a point in time between 0 and 60 minutes after the start of administration of the chemotherapeutic agent or one of its salts to the individual, the chemotherapeutic agent being selected from the group consisting of Doxorubicin, epirubicin, idarubicin, daunorubicin, etoposide, 5-fluorouracil, fludarabine and chlorambucil, comprising i) the separation of proteins contained in the liquid sample by precipitating the proteins with a precipitation reagent and centrifugation or filtration of the liquid sample, the precipitation reagent being selected from the group consisting of trichloroacetic acid, methanol, ethanol, acetonitrile, zinc sulfate and mixtures of two or more of these substances, andii) measuring the concentration of a chemotherapeutic agent and its possibly present metabolites in a biological one Liquid sample by means of a fluorescence signal emanating from the liquid sample after protein separation upon irradiation with excitation light, characterized in that the method does not include any chromatographic or electrophoretic process steps and no liquid-liquid extraction.

Description

In 2008, the number of people with cancer in Germany was around 1.33 million. Worldwide, the number of cancer cases in 2008 was estimated at 12.7 million. Chemotherapy is one of the most common methods of treating cancer. Various chemotherapeutic agents, such as anthracyclines, have been used in chemotherapy for years to combat many different tumors. However, the toxicities that are caused by the chemotherapeutic agents used are often limiting for the treatment. One example is the acute and chronic cardiotoxicity caused by anthracyclines used in chemotherapy, the effectiveness and toxicity of which are dependent on the maximum plasma level (Cmax).

The clinical dosage of chemotherapeutic agents is often based on the patient's body surface area (mg / m ² ). The patient's body surface is estimated using an approximate formula. However, this dose calculation does not take into account the individual metabolism of the patient, and the plasma levels between different patients vary considerably, e.g. by a factor of 5 ( Hempel et al, Peak plasma concentrations of doxorubicin in children with acute lymphoblastic leukemia or non-Hodgkin lymphoma. Cancer Chemother Pharmacol. 49 (2), 133-41 (2002) ), despite dose adjustment based on the body surface. Exceeding the toxic plasma concentration can lead to serious side effects; if the dosage falls below the therapeutically necessary dose, the success of the therapy is limited.

Because of these strong fluctuations in the actual individual plasma level, the research area of "personalized medicine" has developed in recent years. More or less comprehensive genetic analysis attempts to extrapolate on the pharmacokinetics and thus ultimately on the plasma level of a patient. The plasma level can, however, fluctuate considerably intra-individually (see e.g. 3 the end Hempel et al, Peak plasma concentrations of doxorubicin in children with acute lymphoblastic leukemia or non-Hodgkin lymphoma. Cancer Chemother Pharmacol. 49 (2), 133-41 (2002)) so that the simulation of the plasma level can be very inaccurate.

In contrast to indirect, genetically based approaches to “personalized medicine”, it would therefore be advantageous to measure the actual plasma level promptly, preferably during the administration (usually as an infusion) of a chemotherapeutic agent, and to determine an optimal individual dosage based on the measurement results. A certain drug level could thus be set as the target parameter for the chemotherapeutic dosage. When establishing the active substance level, serious toxicities could be avoided by using too high a dosage and ineffective therapies by using too low a dosage. A prerequisite for this, however, is the availability of a method with which the plasma concentration of a chemotherapeutic agent can be determined quickly and easily, i.e. with as few and short procedural steps as possible.

Methods described in the literature for, for example, anthracycline determination in samples are lengthy, technically complex and / or show poor yields or high background signals. In an old method for determining adriamycin, various lengthy process steps are described, at the end of which is the measurement of a fluorescence signal. The process includes precipitation, centrifugation, complexing and liquid-liquid extraction steps with incubation times that sometimes last 60 minutes ( Dusonchet et al, Spectrophotofluorometric characterization of adriamycin a new antitumor drug, Pharmacological Research Communications. 3 (1), 55-65 (1971) ). Bachur et al describes a method for determining daunomycin in tissue samples, in which the tissue samples are extracted with large volumes of extractant (ratio of extractant / tissue sample: 20/1). Ethanol, methanol and trichloroacetic acid are described as unsatisfactory extractants due to poor yields or strong background fluorescences. According to Bachur, 0.3N HCl-50% ethanol should be used as the extraction agent. The samples must be kept at 0-4 ° C during the entire procedure and the procedure includes a long centrifugation step of 20 min at high acceleration before daunomycin is determined by means of a fluorescence measurement ( Bachur et al., Tissue distribution and disposition of daunomycin (NSC-82151) in mice: fluorometric and isotopic methods. Cancer Chemother Rep. 1970 Apr; 54 (2): 89-94 ).

At present, the determination of anthracyclines in plasma is usually carried out using chromatographic or electrophoretic separation methods. For example, Mross et al examined the blood plasma of patients who were administered doxorubicin or epirubicin and describes an HPLC-based method with fluorescence detection after prior chromatographic extraction of the blood plasma using a Sep-Pak C18 column ( Mross et al, Pharmacokinetics and Metabolism of Epidoxorubicin and Doxorubicin in Humans J Clin Oncol 6, 517-526 (1988) ). Hempel 1998 and Hempel 2002 examined samples from patients treated with doxorubicin and used an electrophoretic method to separate the samples after deproteinization with acetonitrile or liquid-liquid extraction with chloroform ( Hempel et al, Therapeutic drug monitoring of doxorubicin in pediatric oncology using capillary electrophoresis. Electrophoresis 19, 2939-2943 (1998) ; Hempel et al, Peak plasma concentrations of doxorubicin in children with acute lymphoblastic leukemia or non-Hodgkin lymphoma. Cancer Chemother. Pharmacol. 49 (2), 133-41 (2002) ). However, such methods are expensive, complex with regard to the required equipment, and above all time-consuming, and are therefore not suitable for routine use in everyday clinical practice.

DE 3152907 T5 discloses a therapeutic composition and assembly useful for the extracorporeal treatment of whole blood comprising a dialyzable chemotherapeutic agent and a dialyzable fluorescible tracer.

Vrignaud, P. et al. (European Journal of Cancer and Clinical Oncology, Vol. 21, No. 11, pp. 1307-1313, 1985) describe the pharmacokinetics and metabolism of epirubicin in plasma and urine. The anthracyclines and their metabolites were extracted through a C18 Sep-pak column.

DE 3837298 C1 discloses a device for infusing drugs into the negative pressure area of an extracorporeal blood circuit, in particular an extracorporeal blood circuit of a dialysis machine, with a storage container for the drugs and a supply line from the storage container to the extracorporeal blood circuit.

DE 2853897 A1 discloses a method for measuring the concentration of a compound in a flowing substance, the substance under investigation being passed through an enzyme bed or the like, for the purpose of converting the substance under investigation with the formation or removal of a reaction product that can be measured in a subsequent measuring unit.

The lack of a fast and as simple as possible suitable method for determining the plasma concentration of e.g. doxorubicin or related chemotherapeutic agents means that a routine determination of the plasma level of such substances is currently not available.

One object of the present application is to find a fast, simple, inexpensive method for the determination of chemotherapeutic agents in biological fluids which overcomes the disadvantages of known determination methods. For example, a plasma level measurement of a chemotherapeutic agent should be carried out during (i.e. online) or at least promptly to an i.v. Allow bolus injection or infusion of chemotherapy drugs. The process should ideally be easy to automate and miniaturize.

1. (i) das Abtrennen von in der Flüssigkeitsprobe enthaltenen Proteinen durch Ausfällen der Proteine mit einem Fällungsreagenz und Zentrifugation oder Filtration der Flüssigkeitsprobe, wobei das Fällungsreagenz ausgewählt ist aus der Gruppe bestehend aus Trichloressigsäure,Methanol, Ethanol, Acetonitril, Zinksulfat und Mischungen von zwei oder mehr dieser Substanzen, und
2. (ii) Messung der Konzentration eines Chemotherapeutikums und seiner gegebenenfalls anwesenden Matabolite in einer biologischen Flüssigkeitsprobe mittels eines Fluoreszenzsignals, das von der Flüssigkeitsprobe nach Proteinabtrennung bei Bestrahlung mit Anregungslicht ausgeht,

dadurch gekennzeichnet, dass das Verfahren keinen chromatographischen und keinen elektrophoretischen Verfahrensschritt und keine flüssig-flüssig Extraktion umfasst. Alternativ kann im beschriebenen Verfahren das Abtrennen von in der Flüssigkeitsprobe enthaltenen Proteinen (Schritt (i)) auch durch Ultrazentrifugation erfolgen anstelle oder zusätzlich zu Schritt (i).A method is disclosed which achieves this problem. The method according to the invention is a method for determining the concentration of a chemotherapeutic agent and its possibly present metabolites in a biological fluid sample of an individual at a point in time between 0 and 60 minutes after the start of administration of the chemotherapeutic agent or one of its salts to the individual, the chemotherapeutic agent being selected from the group consisting of doxorubicin, epirubicin, idarubicin, daunorubicin, etoposide, 5-fluorouracil, fludarabine and chlorambucil

1. (i) the separation of proteins contained in the liquid sample by precipitating the proteins with a precipitation reagent and centrifugation or filtration of the liquid sample, wherein the precipitation reagent is selected from the group consisting of trichloroacetic acid, methanol, ethanol, acetonitrile, zinc sulfate and mixtures of two or more of these substances, and
2. (ii) Measurement of the concentration of a chemotherapeutic agent and its possibly present metabolites in a biological fluid sample by means of a fluorescence signal emanating from the fluid sample after protein separation upon irradiation with excitation light,

characterized in that the process comprises no chromatographic and no electrophoretic process step and no liquid-liquid extraction. Alternatively, in the method described, the proteins contained in the liquid sample (step (i)) can also be separated off by ultracentrifugation instead of or in addition to step (i).

The biological fluid sample is typically a fluid sample from a person undergoing chemotherapy. In individual cases, however, it can also involve fluid samples from animals treated with chemotherapy. The method according to the invention provides a value which the doctor of the individual who is being treated with a chemotherapeutic agent or its salt can utilize in the decision about the further chemotherapeutic treatment of the individual. The method for determining the concentration of a chemotherapeutic agent and its metabolites, if any, present in a biological fluid sample of an individual relates to an application outside the body of the individual.

The chemotherapeutic agent or a salt of the chemotherapeutic agent can in particular be administered intravenously. The administration of the The chemotherapeutic agent or a salt of the chemotherapeutic agent is preferably carried out in one embodiment as an intravenous bolus injection. In another embodiment, the chemotherapeutic agent or a salt of the chemotherapeutic agent is administered by infusion, for example over 1 hour. According to the invention, an intravenous bolus injection is understood to mean an intravenous injection that lasts 30 seconds or less. If the intravenous administration lasts longer than 30 seconds, it is referred to according to the invention as an infusion. In the present invention, an infusion can last, for example, 30 minutes or 1 hour or 2 hours. In an alternative embodiment, the medicament can also be administered locally, for example by means of a syringe at a specific location, for example on the tumor.

With the method according to the invention, the concentration of the chemotherapeutic agent and its possibly present metabolites is determined in a biological fluid sample, as it is present close to the beginning of the administration (e.g. close to the beginning of the intravenous bolus injection or infusion) of the chemotherapeutic agent or a salt of the chemotherapeutic agent, ie up to for a maximum of 60 minutes after the start of administration. The start of administration can be determined by the first contact of the chemotherapeutic agent or its salt with the individual to whom it is administered, within a treatment cycle. For example, if the individual is administered a chemotherapeutic agent or its salt as an infusion over 1 hour, the start of administration can be defined as the point in time at which the individual first comes into contact with the infusion fluid. If an individual is subjected to several treatment cycles with a chemotherapeutic agent or its salt (e.g. an infusion of the chemotherapeutic agent over 1 hour every 7 days for 4 weeks), the term of the beginning of the administration refers to the respective treatment cycle with the chemotherapeutic agent or its salt and not upon the first administration of the chemotherapy drug or its salt to the individual at all.

If, for example, the concentration in a biological fluid is to be determined 10 minutes after the start of an intravenous bolus injection, a biological fluid sample can be taken from the individual at this point in time (10 minutes after the start of administration). The method according to the invention then allows the concentration to be determined within a short time, so that it is possible to quickly determine what the concentration of the chemotherapeutic agent and its possibly present metabolites was at the time 10 minutes after the start of administration. In one embodiment, the method according to the invention relates to a method for determining the concentration of the chemotherapeutic agent and its possibly present metabolites in a biological fluid of an individual at a point in time between 0 and 40 minutes, or between 0 and 30 minutes after the start of administration of the chemotherapeutic agent or its salt to the individual. In one embodiment, the method according to the invention relates to a method for determining the concentration of the chemotherapeutic agent and its possibly present metabolites in a biological fluid of an individual at a time between 0 and 15 minutes, preferably between 0 and 10 minutes or between 0 and 5 minutes after the start of the Administration of the chemotherapy drug or its salt to the individual.

In one embodiment, the method according to the invention relates to a method for determining the concentration of doxorubicin and its possibly present metabolites in a biological fluid sample of an individual at a time between 0 and 12 minutes, preferably between 0 and 6 minutes after the start of administration of the chemotherapeutic agent or one of its Salts.

In one embodiment, the method according to the invention relates to a method for determining the concentration of epirubicin and its possibly present metabolites in a biological fluid sample of an individual at a time between 0 and 12 minutes, preferably between 0 and 6 minutes after the start of administration of the chemotherapeutic agent or one of its Salts.

In one embodiment, the method according to the invention relates to a method for determining the concentration of idarubicin and its possibly present metabolites in a biological fluid sample of an individual at a time between 0 and 12 minutes, preferably between 0 and 6 minutes after the start of administration of the chemotherapeutic agent or one of its Salts.

In one embodiment, the method according to the invention relates to a method for determining the concentration of daunorubicin and its any metabolites present in a biological fluid sample of an individual at a point in time between 0 and 12 minutes, preferably between 0 and 6 minutes after the start of administration of the chemotherapeutic agent or one of its salts.

In one embodiment, the method according to the invention relates to a method for determining the concentration of etoposide and its possibly present metabolites in a biological fluid sample of an individual at a time between 0 and 60 minutes, preferably between 0 and 45 minutes after the start of administration of the chemotherapeutic agent or one of its Salts.

In one embodiment, the method according to the invention relates to a method for determining the concentration of 5-fluorouracil and its possibly present metabolites in a biological fluid sample of an individual at a time between 0 and 16 minutes, preferably between 0 and 8 minutes after the start of administration of the chemotherapeutic agent or one of its salts.

In one embodiment, the method according to the invention relates to a method for determining the concentration of fludarabine and its possibly present metabolites in a biological fluid sample of an individual at a time between 0 and 5 minutes, preferably between 0 and 2.5 minutes after the start of administration of the chemotherapeutic agent or one of its salts.

In one embodiment, the method according to the invention relates to a method for determining the concentration of chlorambucil and its possibly present metabolites in a biological fluid sample of an individual at a time between 0 and 60 minutes, preferably between 0 and 45 minutes after the start of administration of the chemotherapeutic agent or one of its Salts.

Metabolites are products that result from the conversion of drugs (chemotherapeutic agents) by enzyme systems over time in the body of the individual. Examples include products resulting from oxidation or reduction or molecular-splitting processes such as hydrolysis, or substances resulting from conversion into derivatives by binding the drug to the body's own substances (glucuronic acid conjugation, glucoside conjugation, hippuric acid synthesis, glutamine conjugation, Methylation, acetylation). The main metabolites of doxorubicin in the human body are e.g. doxorubicinol, as well as the 7-deoxy-doxorubicin and 7-deoxy-doxorubicinol aglycones.

Under the measurement conditions of the method according to the invention, in addition to the chemotherapeutic agent (which, depending on the measurement conditions, can also be present in protonated or deprotonated form, for example) in the biological fluid, metabolites that may be present in the biological fluid can contribute to the measured fluorescence signal when irradiated with excitation light, if not only the chemotherapeutic agent with irradiation Excitation light emits a fluorescence signal but also present metabolites of the chemotherapeutic agent. In the method according to the invention for determining the concentration of the chemotherapeutic agent and its metabolites which may be present, the term metabolites is therefore only to be understood as meaning those metabolites which also contribute to the fluorescence signal under the measurement conditions. However, this is not a disadvantage of the method, especially when also including active metabolites. In addition, if the metabolism rate is significantly lower than the primary breakdown rate of the chemotherapeutic agent, the metabolites, e.g. for determining the maximum plasma concentration and the primary breakdown rate of the chemotherapeutic agent, are of minor importance.

In the following, the determination of the concentration of the chemotherapeutic agent in a biological fluid sample of an individual will only be referred to in abbreviated form, instead of the longer expression of the concentration determination of the chemotherapeutic agent and its possibly present metabolites.

The biological fluid sample is preferably a fluid sample from an individual who is being treated with monochemotherapy, i.e. only one chemotherapeutic agent and possibly metabolites of the chemotherapeutic agent are present in the fluid sample. It is preferably a monotherapy with doxorubicin, epirubicin, idarubicin or daunorubicin, or a salt thereof, in particular doxorubicin or a salt of doxorubicin.

If, in one embodiment of the method according to the invention, the concentration of etoposide and its possibly present metabolites or of fludarabine and its possibly present metabolites in a biological fluid sample of an individual at a point in time between 0 and 60 minutes after the start of the administration of etoposide or fludarabine or a If the salts are determined for the individual, the etoposide or fludarabine can of course also be administered, for example, in the form of the etoposide phosphate or in the form of the fludarabine phosphate.

The method according to the invention preferably relates to a method in which the chemotherapeutic agent is doxorubicin, epirubicin, idarubicin or daunorubicin. The chemotherapeutic agent doxorubicin is preferred. In the latter case, the method according to the invention thus relates to a method for determining the concentration of doxorubicin and its possibly present metabolites in a biological fluid sample of an individual at a point in time between 0 and 60 minutes after Beginning administration of doxorubicin or a salt of doxorubicin, for example doxorubicin hydrochloride, to the individual.

In one embodiment, the method according to the invention relates to a method in which the chemotherapeutic agent or one of its salts is administered to the individual as a liposomal preparation. In this case, the chemotherapeutic agent is preferably doxorubicin, epirubicin, idarubicin or daunorubicin, in particular doxorubicin. Liposomal preparations of chemotherapeutic agents and their salts are known, for example, under the trade names Caelyx, Doxil or Myocet.

The biological fluid sample can be, for example, a blood sample, serum sample or plasma sample, preferably a plasma sample. The biological fluid sample can contain anticoagulants. The blood or plasma sample can be, for example, a sample that has been anticoagulated with EDTA. If, for example, blood is taken from an individual 5 minutes after the start of administration of a chemotherapeutic agent or its salt, a plasma sample can be obtained from it, and the concentration of the chemotherapeutic agent and its possibly present metabolites in the plasma sample at the point in time 5 minutes after the administration of the chemotherapeutic agent has started can be determined will.

According to the invention, a method is preferred in which the proteins contained in the liquid sample are separated off by precipitating the proteins with a precipitation reagent and centrifugation or filtration of the liquid sample, the precipitation reagent being selected from the group consisting of trichloroacetic acid, methanol, ethanol, acetonitrile, zinc sulfate and Mixtures of two or more of these substances. In the method according to the invention, the proteins contained in the liquid sample are particularly preferably separated off by precipitating the proteins with a precipitation reagent as described above and centrifuging the liquid sample. Zinc sulphate is preferably used as a precipitation reagent in dilute sodium hydroxide solution, e.g. 10% w / v zinc sulphate in 0.25M NaOH. Trichloroacetic acid is used as the precipitation reagent, preferably as an aqueous trichloroacetic acid solution, and is particularly preferred. For example, 3M aqueous trichloroacetic acid can be used. In one embodiment, the precipitation reagent is methanol, ethanol, acetonitrile or trichloroacetic acid (e.g. as 3M aqueous trichloroacetic acid). If mixtures are used as the precipitation reagent, mixtures of 2 substances are preferred, e.g. zinc sulfate (e.g. as 10% w / v zinc sulfate in 0.25M NaOH) / methanol.

The precipitation reagent causes the proteins to be precipitated. The centrifugation or filtration described in connection with the precipitation of proteins is then used to separate the precipitated (precipitated) proteins. Centrifugation and filtration processes have in common that they make use of the large size or high weight of the (precipitated) proteins for their separation. By separating the proteins, the background for the fluorescence measurement is minimized. The separation of proteins contained in the liquid sample is a process step that can be carried out easily and routinely with simple means, which differs, for example, from more complicated determination methods for chemotherapeutic agents in biological fluid samples, which are based on a (specific) isolation of the chemotherapeutic agents from the rest of a sample (for example mentioned isolation of anthracyclines by binding to a DNA “probe”).

The method according to the invention relates, for example, to a method in which the proteins contained in the liquid sample are separated off by precipitating the proteins with a precipitation reagent and centrifugation of the liquid sample, the precipitation reagent being trichloroacetic acid.

The centrifugation after precipitation of the proteins with a precipitation reagent in the method according to the invention for determining the concentration of a chemotherapeutic agent is preferably carried out at 12000 g or less, e.g. 8000-12000 g. It was found that centrifugation at 12,000 g is sufficient or enables excellent sample preparation for a subsequent fluorescence measurement from the supernatant with little background fluorescence and high robustness. This also enables the use of a simple table-top centrifuge in the method according to the invention, which is often not suitable for centrifugation at a much higher acceleration.

In one embodiment of the method according to the invention, the proteins contained in the liquid sample are separated off by precipitating the proteins with a precipitation reagent and centrifugation of the liquid sample, the precipitation reagent being selected from the group consisting of trichloroacetic acid, methanol, ethanol, acetonitrile, zinc sulfate and mixtures two or more of these substances are centrifuged for 2 minutes or less. In this embodiment, the precipitation reagent is preferably trichloroacetic acid. In the method according to the invention, in which the proteins contained in the liquid sample are separated off by precipitating the proteins with a previously described precipitation reagent, preferably trichloroacetic acid, centrifugation can be carried out for 2 min at 12,000 g, for example. This applies in particular to a sample volume of 1.5 ml or less.

A short centrifugation time enables the concentration of the chemotherapeutic agent to be determined very quickly in the biological fluid sample, e.g. in a blood sample or plasma sample, and is particularly advantageous for determining e.g. the blood level or the plasma level of a chemotherapeutic agent in an individual during (e.g. an infusion) or close to the administration of a To be able to determine chemotherapy drug or its salt. On the basis of the measurement results, for example, the dose can be adjusted during the infusion, or after a bolus injection of the chemotherapeutic agent or its salt, for example, additional dosing can be made if the blood or plasma levels are measured which are too low.

The method according to the invention for determining the concentration of a chemotherapeutic agent is preferably carried out at room temperature, i.e. 15-25 ° C. Cooling, e.g. to 0-4 ° C, of the biological fluid samples to be examined while the method according to the invention is being carried out is not necessary and is therefore preferably not carried out either. This in turn makes it possible to keep the method simple and uncomplicated and still achieve a reliable determination of the chemotherapeutic agent concentration in a biological fluid sample.

In one embodiment, with the separation of proteins contained in the biological fluid sample, a separation of cells contained in the fluid sample can also take place at the same time. It was surprisingly found that a separate step of cell separation from a biological fluid sample preceding the protein separation, e.g. cell separation from a blood sample to obtain a plasma sample, can be dispensed with in order to be able to determine the chemotherapeutic agent concentration. According to the invention, a blood sample is preferably understood to be a whole blood sample.

In one embodiment, the biological fluid sample, for example blood sample, can be mixed directly with the precipitation reagent, preferably trichloroacetic acid (cf. 8th ). It is also possible to dilute the biological fluid sample, for example blood sample, with a buffer, for example 10 mM sodium hydrogen phosphate / 140 mM sodium chloride, prior to the simultaneous separation of proteins and cells contained in the fluid sample. By simultaneously separating proteins and cells contained in the liquid sample, the process can be carried out even more quickly and easily. In order to be able to better convert the chemotherapeutic concentration in the individual's plasma from the determined chemotherapeutic concentration in the blood sample, the hematocrit of the individual can be determined before the administration of the chemotherapeutic agent begins and the plasma concentration of the individual can be calculated with the help of this. In one embodiment, the method according to the invention relates to a method in which the biological fluid sample is a blood sample.

1 and 2 show, by way of example, the suitability of the method according to the invention for determining the concentration of 5-fluorouracil, chlorambucil, fludarabine, etoposide, doxorubicin or epirubicin.

It has been found that the background fluorescence of the liquid sample after the separation of proteins contained in the liquid sample is low in the method according to the invention, so that a quick and easy fluorometric determination of the concentration of chemotherapeutic agents in the liquid sample can take place. 3 shows an example of the low background fluorescence of liquid samples after separation of proteins contained in the liquid sample. This is surprising and cannot be inferred from the prior art, since there, in terms of apparatus, time and / or implementation, more complex separation processes are described before a fluorometric concentration of the chemotherapeutic agents is determined.

The fact that the method according to the invention also has good robustness is also evident from this 4th . It has been found that the method according to the invention for determining the concentration of chemotherapeutic agents leads to good agreement of the results even when using plasma samples from different test subjects.

the end 5 the low variance of the method according to the invention can also be seen on different measurement days and thus the reliability of the method according to the invention.

However, it is also possible, if desired, to take into account the individual background signal in the biological fluid sample, for example plasma sample, of an individual patient. For this purpose, a liquid sample (e.g. plasma sample) can be obtained from the patient in whose biological fluid sample the concentration of a chemotherapeutic agent and its possibly present metabolites are to be determined according to the invention, e.g. immediately before administration of the chemotherapeutic agent or its salt (so-called zero sample) and its fluorescence can be determined after the blank sample has been subjected to the same procedural steps as the liquid sample (in the example: plasma sample), which is taken after administration of the chemotherapeutic agent and in which the concentration of the Chemotherapy drug should be determined. The difference in the measurement signal between the blank sample and the liquid sample after administration of the chemotherapeutic agent or its salt can then be traced back to the chemotherapeutic agent and its metabolites which may be present.

The pharmaceutical formulation with which the chemotherapeutic agent or its salt is administered preferably does not contain any substances which, in addition to the chemotherapeutic agent or possibly its metabolites, also contribute to the fluorescence signal in the biological fluid sample of the individual when the method according to the invention is carried out under measurement conditions. If substances that contribute to the measurement signal are present, such substances should only be present in such an amount that they only contribute to less than 1% of the measured fluorescence signal of the liquid sample in the method according to the invention under the selected measurement conditions after subtracting any fluorescence signal from a blank sample.

6a shows, by way of example, a comparison of the background signal of liquid samples which were obtained from biological liquid samples after separating proteins precipitated with a precipitation reagent mentioned according to the invention. A comparison with the 0.3 M HCl / ethanol 1: 1 (v / v) not according to the invention shows the surprisingly low background fluorescence of liquid samples which were treated with the precipitation reagents used in the present invention. A lower background fluorescence makes it possible, for example, to determine low chemotherapeutic agent concentrations well in biological fluid samples. Precipitation with trichloroacetic acid as the precipitation reagent with subsequent optional dilution of the liquid sample with water is particularly advantageous with regard to a low background fluorescence or an advantageously high ratio of the fluorescence intensity and background fluorescence emanating from the chemotherapeutic-containing liquid sample.

The method according to the invention is also advantageous for the determination of low chemotherapeutic agent concentrations in biological fluid samples insofar as the precipitation reagent, optionally after dilution with water or as a solution in water or dilute sodium hydroxide solution, in a volume ratio of only 1: 4 or even in a lower volume ratio to Volume of the biological fluid sample can be used. For example, it can be used in a volume ratio of 1: 5 or in an even lower volume ratio to the volume of the biological fluid sample. For example, it can also be used in a volume ratio of 1:10 to the volume of the biological fluid sample or in an even lower volume ratio.

In a preferred embodiment of the method according to the invention for determining the concentration of chemotherapeutic agents, e.g. of anthracyclines such as doxorubicin, and their metabolites which may be present in a plasma sample, the precipitation reagent is aqueous 3M trichloroacetic acid. This is preferably used in a volume ratio of 1: 4 to 1: 5 to the plasma sample, which is obtained, for example, after administration of an anthracycline derivative such as doxorubicin or its salt to the individual. In another embodiment, it is used in a volume ratio of 1: 4 or less, for example 1:10 or less, to the plasma sample. The liquid sample can optionally be diluted with water after the separation of the precipitated proteins by centrifugation or filtration before the measurement of the fluorescence signal takes place.

In one embodiment, the present invention is directed to a method for determining the concentration of doxorubicin, epirubicin, idarubicin and daunorubicin in a biological fluid sample, the proteins contained in the fluid sample being separated off by precipitation and centrifugation at 12,000 g or less for 2 minutes or less, wherein the precipitation reagent is trichloroacetic acid and wherein the biological fluid sample is not cooled during the implementation of the method, for example to 0-4 ° C, and wherein trichloroacetic acid is used as an aqueous solution in a volume ratio of 1: 4 or less to the volume of the biological fluid sample. In this embodiment, the liquid sample for measuring the fluorescence signal is preferably irradiated with excitation light which has a wavelength in the range of 470-475 nm and the measurement of the fluorescence signal can be carried out, for example, at a wavelength of 555 nm.

7th also shows that the method according to the invention can also be used to determine the concentration if the plasma sample contains a liposomally encapsulated anthracycline or its salt, as shown here in the example in which liposomally encapsulated doxorubicin HCl was added to a plasma sample before the method according to the invention the concentration determination was carried out.

The method according to the invention for determining the concentration of chemotherapeutic agents also includes a method in which no further steps for separating or separating the liquid sample into individual constituents or groups of constituents are carried out after the separation of the liquid sample contained proteins and a method in which no further steps for separating or separating the liquid sample into individual components or groups of components are carried out with the exception of the step for separating proteins contained in the liquid sample. This reflects the particular simplicity of the process, which is based on a few process steps. With the exception of the precipitation reagent and optionally a dilution of the liquid sample with a precipitation reagent or water in the method according to the invention, no further reagents are preferably added to the liquid sample. It is optionally also preferred that, with the exception of the precipitation reagent and a dilution of the liquid sample with a buffer and / or water in the method according to the invention, no further reagents are added to the liquid sample for the liquid sample. It is also possible that, with the exception of the precipitation reagent, only water or an agent for pH adjustment is added to the liquid sample and this addition takes place after the proteins contained in the liquid sample have been separated off. For example, NaOH or water can be added, but no further reagents are added.

8th shows the suitability of the method according to the invention for determining the concentration using the example of doxorubicin, the biological fluid sample being a blood sample. After taking the blood sample from the individual, it was only diluted with a buffer before a precipitation reagent was added and cells and proteins were separated off. The fluorescence was measured from the supernatant after dilution. This in turn demonstrates the particular simplicity of the method according to the invention.

Due to the simplicity of the method, this can also be carried out on a miniaturized measuring unit. Such a measuring unit requires only small amounts of a biological fluid sample from the patient, which relieves the patient. Such a measuring unit can, for example, have a lancet with a sample receiving capillary so that a small amount of blood can be obtained from the fingertip. A plasma sample can be obtained from the blood using known methods (see e.g. US2009107909 ) and the plasma sample can then be mixed on the miniaturized measuring unit with a small amount of trichloroacetic acid already deposited there, e.g. 3M aqueous trichloroacetic acid. The precipitated proteins can, for example, be separated off by centrifugation of the miniaturized measuring unit and a fluorescence signal emanating from the supernatant can be measured directly with a detection unit when irradiated with excitation light. The measuring unit can also be constructed in such a way that a blood sample is used directly as a biological fluid sample on the miniaturized measuring unit, in that a small blood sample is obtained from the individual with a lancet with a sample receiving capillary, which is then treated with a small amount of trichloroacetic acid already deposited, e.g. 3M aqueous trichloroacetic acid, is mixed on the measuring unit. By centrifuging the measuring unit, precipitated proteins and cells can then be separated from the liquid sample and the fluorescence measurement can be carried out as described above. The ratio of the volumes of the pre-deposited trichloroacetic acid to the biological liquid sample can be, for example, 1: 4 or 1: 5, whereby the total volume of the sample can be kept small, which is particularly advantageous for a miniaturized method.

In one embodiment, the method according to the invention relates to a method in which the chemotherapeutic agent is doxorubicin, epirubicin, idarubicin or daunorubicin, and in which the liquid sample is irradiated with excitation light having a wavelength in the range of 430-510 nm, and in which the measurement of the fluorescence signal is at a wavelength in the range of 530-600 nm takes place. For example, the liquid sample can be irradiated with excitation light that has a wavelength in the range of 470-475 nm and the fluorescence signal can be measured, for example, at a wavelength of 555 nm. A fluorescence spectrophotometer is preferably used for the measurement, slot widths of 10 nm or less, for example 1-10 nm, being used both for the excitation light and for the measurement of the fluorescence signal.

If the method according to the invention is a method in which the chemotherapeutic agent is 5-fluorouracil, chlorambucil, fludarabine or etoposide, excitation light with a wavelength in the range of 245-270 nm is preferred and the fluorescence signal is preferably measured at a wavelength in the range of 330-360 nm A fluorescence spectrophotometer can be used for the measurement, slot widths of 10 nm or less, for example 1-10 nm, being used both for the excitation light and for the measurement of the fluorescence signal.

In one embodiment of the method according to the invention, a fluorescence spectrophotometer is used, slot widths of 2.5 nm being used both for the excitation light and for the measurement of the fluorescence signal.

It is also possible to amplify the fluorescence signal of the liquid sample to be measured, for example by setting it to a certain pH after protein separation. This is in The method according to the invention is preferred if the chemotherapeutic agent is etoposide, 5-fluorouracil or chlorambucil, and can be carried out by adding NaOH to the liquid sample after the protein has been precipitated with trichloroacetic acid and the proteins have been separated off, whereas the concentration of doxorubicin, epirubicin, idarubabinicin or daunarubicin is preferably determined as well takes place in the trichloroacetic acid liquid sample. The fluorescence intensity of doxorubicin and the other anthracyclin derivatives as well as of fludarabine is increased or made possible by the measurement in acidic conditions. The fluorescence signal can also be amplified by choosing a specific precipitation reagent. Since the method according to the invention is to be kept simple, however, preferably no reagents are added to the liquid sample which, for example, can lead to an amplification of the fluorescence signal through complex formation or chemical reaction with the chemotherapeutic agent.

In one embodiment, the determination method according to the invention comprises that the intensity of the fluorescence signal emanating from the liquid sample of an individual in the method according to the invention is compared with the intensity of fluorescence signals of a standardized concentration curve of the chemotherapeutic agent. A standardized concentration curve of the chemotherapeutic agent is created, the concentration of which is to be determined in the liquid sample of the individual. If, for example, the doxorubicin concentration is to be determined in a plasma sample of a patient who has been administered doxorubicin (e.g. as doxorubicin hydrochloride), a standardized doxorubicin concentration curve can be made with the plasma of the individual, which was taken from the patient, for example immediately before the administration of doxorubicin, or with plasma from plasma donors that did not Herbal therapy are subject to be produced. Different known amounts of doxorubicin (e.g. as doxorubicin hydrochloride) can be added to this previously withdrawn plasma or the plasma withdrawn from donors in order to produce so-called different "standards", and the standards are then subjected to the same procedure as the actual liquid sample of the individual in which the doxorubicin concentration is determined should be determined. It is also possible to produce a standard to which no doxorubicin is added, corresponding to a blank sample for determining the background signal. The intensity of the fluorescence signal of the liquid sample is then compared with the fluorescence signals of the standards or the standardized concentration curve formed therefrom and the concentration of the liquid sample is given as the content of chemotherapeutic agent (in the example here: doxorubicin). The specification as the content of chemotherapeutic agent (in the example here: doxorubicin) is also made if the fluorescence signal emanating from the liquid sample is not caused entirely by the chemotherapeutic agent (in the example here: doxorubicin), but, for example, by metabolites that may be present in the liquid sample (im Example here: doxorubicin metabolites).

1. (i) die Bestimmung der Konzentration des Chemotherapeutikums und seiner gegebenenfalls anwesenden Metabolite nach einem Verfahren wie weiter oben beschriebenen in zwei oder mehr biologischen Flüssigkeitsproben eines Individuums, wobei die biologischen Flüssigkeitsproben zu verschiedenen Zeitpunkten nach Beginn der Verabreichung des Chemotherapeutikums oder eines seiner Salze an das Individuum bei diesem entnommen werden, und
2. (ii) die Berechnung der optimalen individuellen Dosierung aus den Daten des Schrittes (i), die zum Erreichen einer vorbestimmten Konzentration des Chemotherapeutikums und seiner gegebenenfalls anwesenden Metabolite in der biologischen Flüssigkeit des Individuums zu verabreichen ist.

The presented method allows a quick and easy measurement of the concentration of a chemotherapeutic agent and its possibly present metabolites in a biological fluid sample, for example in a plasma sample. Based on the measurement results, it is then possible to react immediately and to set a target concentration of the chemotherapeutic agent in the plasma. The present application therefore also relates to an ex-vivo method for determining an optimal individual dosage of a chemotherapeutic agent or one of its salts, the chemotherapeutic agent being selected from the group consisting of doxorubicin, epirubicin, idarubicin, daunorubicin, etoposide, 5-fluorouracil, fludarabine and Chlorambucil, including

1. (i) the determination of the concentration of the chemotherapeutic agent and its possibly present metabolites by a method as described above in two or more biological fluid samples from an individual, the biological fluid samples being taken at different times after the administration of the chemotherapeutic agent or one of its salts to the individual are taken from this, and
2. (ii) the calculation of the optimal individual dosage from the data of step (i) which is to be administered in order to achieve a predetermined concentration of the chemotherapeutic agent and its possibly present metabolites in the biological fluid of the individual.

The biological fluid sample is preferably a plasma sample. By administering a partial dose or test dose as an intravenous bolus injection, for example, and preferably promptly determining the plasma concentration, for example, the patient's individual pharmacokinetic parameters can be included in the calculation of a total dose of a chemotherapeutic agent to be administered in a treatment cycle. The initially administered partial dose or test dose should be below the expected optimal individual dose of the chemotherapeutic agent, which can also be referred to as the optimal individual total dose or “full” dose for the treatment cycle. Based on the measured values obtained, the full dosage can be calculated, which must be administered in the treatment cycle so that, for example, a predetermined plasma concentration of the chemotherapeutic agent and its possibly present metabolites is achieved in the patient. When the patient has then received the full dosage, the plasma concentration can be remeasured again for control using the method according to the invention for determining the concentration.

In the case of an infusion of the chemotherapeutic agent or its salt, for example, from the increase in the plasma concentration, for example by measuring the plasma concentration in 2 or 3 or 4 or more, or 5 or 6 or more fluid samples of the individual, each with an interval of a few minutes, e.g. 5-10 minutes after the start of the infusion, and the known infusion rate of the chemotherapeutic agent or its salt (amount of substance infused per unit of time), the peak plasma concentration at a certain point in time after the start of the infusion can be calculated or the point in time when a desired plasma concentration is reached is. The liquid samples can also be taken at intervals of 2-5 minutes, for example. The infusion can then continue until the calculated point in time, for example, in order to achieve an effective but less toxic chemotherapy. At the calculated point in time, the individual has then received his optimal individual dosage and the infusion can be terminated, and the plasma level can be measured again, if necessary, for control with the method according to the invention for determining the concentration. To calculate the individual elimination from the plasma, for example, the classic models and formulas for calculating the pharmacokinetics of the corresponding substance can be used. After determining the individual pharmacokinetic parameters, these parameters on the one hand and the infusion rate on the other hand can be used to extrapolate the plasma concentration to be achieved.

The influx kinetics results from a constant infusion rate and first-order elimination, usually according to the distribution in the tissue. The change in the plasma concentration c of the chemotherapeutic agent over the time t during an infusion can be described by the differential equation 1: $$\frac{\partial c}{\partial t}=k_{\text{inf}}-c\ast r$$

The constant k _inf is the quotient of the amount of substance infused per unit of time and the plasma volume. The constant r corresponds to the primary degradation rate, the degradation depending on the concentration. The linear differential equation 1 with the initial condition c (0) = 0 has equation 2 as a unique solution. $$c\left(t\right)=\frac{k_{\text{inf}}}{r}\left(1-e^{-rt}\right)$$

The chemotherapeutic agent concentration in the plasma at time t after the start of the infusion is given by c (t). The constants k _inf and r can be determined from the first measured values, ie, for example, from the measured plasma concentrations in 2 or 3 or 4 or more biological fluid samples of the individual, which were each taken a few minutes after the start of the infusion, via a corresponding regression analysis will. The non-linear regression analysis can, for example, take place iteratively with the aid of known algorithms, for example the Levenberg-Marquardt algorithm, in which case the adapted coefficients should have a minimum squared error deviation. Alternatively, k _inf can also be determined graphically from the first 2-3 values, which should be linear, and _{the constant r is then calculated from the later values with the known k inf} (see equation 2).

In order to determine the point in time τ at which the desired plasma concentration c _{target is} reached and at which the infusion can be interrupted, equation 2 can be solved for t (equation 3) $$\tau =\frac{\text{ln}\left(1-c_{Ziel}\ast \frac{r}{k_{\text{inf}}}\right)}{-r}$$

In one embodiment, the present invention therefore also relates to an ex vivo method for determining an optimal individual dosage of a chemotherapeutic agent or one of its salts, the chemotherapeutic agent being selected from the group consisting of doxorubicin, epirubicin, idarubicin, daunorubicin, etoposide, 5-fluorouracil , Fludarabine and Chlorambucil, comprising the determination of the concentration of the chemotherapeutic agent and its possibly present metabolites by a method as described above in at least 2, preferably at least 3 or more preferably at least 4 biological fluid samples, preferably plasma samples, of an individual, which at different times Beginning of an infusion of the chemotherapeutic agent or one of its salts can be taken from the individual, and the calculation of the optimal individual dosage from the measured concentrations necessary for reaching a predetermined concentration of the chemoth erapeuticum and its metabolites that may be present in to be administered to the biological fluid of the individual. The chemotherapeutic agent concentration can also be determined, for example, in 5 or more biological fluid samples. It can also take place in 6 or more biological fluid samples, for example. For example, the biological fluid samples can be taken from an individual at intervals of 2-5 minutes or 5-10 minutes after the start of the infusion, and the concentration of the chemotherapeutic agent and any metabolites thereof can be determined therein.

1. (i) ein Reservoir,
2. (ii) eine Dosierungseinheit,
3. (iii) eine Abgabeeinheit,
4. (iv) eine Gewinnungseinheit,
5. (v) eine Analyseneinheit, und
6. (vi) eine Steuereinheit

umfasst, wobei das Reservoir das Chemotherapeutikum oder eines seiner Salze enthält, die Dosierungseinheit die Zugabe der Menge des Chemotherapeutikums oder dessen Salz pro Zeiteinheit reguliert, die aus dem Reservoir über die Abgabeeinheit dem Individuum verabreicht wird, die Gewinnungseinheit eine oder mehrere biologische Flüssigkeitsprobe(n) des Individuums gewinnt, in der Analyseneinheit die Konzentration des Chemotherapeutikums und seiner gegebenenfalls anwesenden Metabolite in der oder in den biologischen Flüssigkeitsproben bestimmt wird, die Steuerungseinheit die Regulierung der Dosierungseinheit steuert in Abhängigkeit von der oder den in der Analyseneinheit bestimmten Konzentration(en), und wobei die Bestimmung der Konzentration(en) des Chemotherapeutikums und seiner gegebenenfalls anwesenden Metabolite in der Analyseneinheit nach einem Verfahren wie weiter oben beschrieben durchgeführt wird. Eine solche Infusionsapparatur ist beispielhaft in 9 dargestellt.The method according to the invention for determining chemotherapeutic agents in fluid samples from an individual can also be used in a device for administering chemotherapeutic agents because it can be carried out quickly and easily. The present invention therefore also relates to an infusion apparatus for administering a chemotherapeutic agent or one of its salts to an individual, the infusion apparatus as components

1. (i) a reservoir,
2. (ii) a dosage unit,
3. (iii) a dispensing unit,
4. (iv) a recovery unit,
5. (v) an analysis unit, and
6. (vi) a control unit

comprises, wherein the reservoir contains the chemotherapeutic agent or one of its salts, the dosage unit regulates the addition of the amount of the chemotherapeutic agent or its salt per unit of time, which is administered to the individual from the reservoir via the delivery unit, the collection unit one or more biological fluid samples of the individual wins, the concentration of the chemotherapeutic agent and its possibly present metabolites in the or in the biological fluid samples is determined in the analysis unit, the control unit controls the regulation of the dosage unit as a function of the concentration (s) determined in the analysis unit, and where the determination of the concentration (s) of the chemotherapeutic agent and its possibly present metabolites is carried out in the analysis unit by a method as described above. Such an infusion apparatus is exemplified in 9 shown.

With the infusion apparatus claimed according to the invention, the determination of the concentration of the chemotherapeutic agent and its possibly present metabolites can take place outside the body of the individual or inside.

In one embodiment of the infusion apparatus, the recovery unit obtains several biological fluid samples at different times up to a maximum of 60 minutes after the start of administration. Both the extraction of the biological fluid samples and the determination of the concentration in the fluid samples can be carried out discontinuously. In another embodiment, the infusion apparatus according to the invention is an infusion apparatus, the recovery unit continuously recovering the biological fluid sample and the analysis unit being designed so that the determination of the concentration of the chemotherapeutic agent and any metabolites that may be present in the fluid sample is carried out continuously.

The infusion apparatus can be calibrated individually, e.g. immediately before the start of the infusion with the chemotherapeutic solution of known concentration from the reservoir and the plasma of the individual.

In the infusion apparatus according to the invention, the regulation of the dosage unit can be controlled by the control unit as a function of a predetermined setpoint value for the concentration. After the attending physician of the individual has set a target value for the plasma concentration of the chemotherapeutic agent, for example before the start of the treatment cycle, which is to be achieved in the treatment cycle in the individual, the control unit controls the regulation of the dosage unit in the infusion apparatus according to the invention as a function of the or the concentration (s) determined in the analysis unit and as a function of the predetermined target value for the plasma concentration. This happens without the intervention of a doctor. The infusion apparatus can be fully automated. Depending on the measured values and the target value, the addition of the amount of the chemotherapeutic agent or its salt per unit of time can be regulated automatically via the dosage unit, which is administered to the individual from the reservoir via the dispensing unit and the infusion via the dosage unit is terminated, for example, as soon as in the analysis unit a concentration is determined which corresponds to a predetermined concentration (target value).

In a particularly preferred embodiment of the infusion apparatus, the control unit controls the dosage unit in such a way that the administration of the Chemotherapeutic agent or its salt is terminated as soon as a concentration is determined in the analysis unit which corresponds to a predetermined concentration.

In another embodiment of the infusion apparatus, the control unit controls the dosage unit in such a way that the administration of the chemotherapeutic agent or its salt is ended as soon as a certain dosage has been administered, which was determined according to the previously described method for determining an optimal individual dosage.

The infusion apparatus according to the invention can also be an infusion apparatus, the control unit controlling the dosage unit in such a way that the addition of the amount of the chemotherapeutic agent or its salt per unit of time that is administered to the individual from the reservoir via the dispensing unit is upregulated if in 2 successive determinations of the Concentration of the chemotherapeutic agent and its possibly present metabolites in the analysis unit, the second determination gives a lower concentration than the first.

The infusion apparatus according to the invention can, for example, also be an infusion apparatus, the biological fluid sample being a plasma sample which the extraction unit, which comprises a separation unit, wins by separating a plasma sample from the individual's blood, which is fed to the analysis unit and which Analysis unit comprises a protein separation unit and a detection unit, proteins contained in the plasma sample being separated in the protein separation unit, and the detection unit measuring the fluorescence signal that emanates from the plasma sample after protein separation when irradiated with excitation light. The proteins can be separated off after precipitation with trichloroacetic acid, for example. In one example of a continuously operating infusion apparatus, the separation unit is designed, for example, in such a way that the blood cells are continuously separated from the blood of the individual using a filter and a continuous plasma sample is obtained, and the protein separation unit also continuously separates the proteins precipitated from the plasma sample by adding a precipitation reagent Separates filter or a continuous centrifuge and the downstream detection unit continuously determines the concentration of the chemotherapeutic agent and its possibly present metabolites in the liquid sample.

In another embodiment, the infusion apparatus according to the invention is an infusion apparatus, the biological fluid sample being a blood sample which is fed to the analysis unit after it has been obtained in the recovery unit, and the analysis unit comprises a protein separation unit and a detection unit, with proteins contained in the protein separation unit in the blood sample and cells are also separated at the same time, and the detection unit measures the fluorescence signal that emanates from the liquid sample after protein and cell separation upon irradiation with excitation light. Here, too, the proteins can be separated off, e.g. after precipitation with trichloroacetic acid. The cells of the blood sample are separated when the proteins are separated off. In another example of a continuously operating infusion apparatus, the blood sample is obtained continuously, and precipitation reagent is added to the protein separation unit and precipitated proteins and cells are also continuously separated from the blood sample via a filter or a continuous centrifuge and the downstream detection unit continuously determines the concentration of the chemotherapeutic agent and its metabolites, if any, present in the liquid sample.

The present invention is further illustrated by the following example and the figures in which the results of the example are summarized:

Figure list

• 1 (a) - (d) shows the determination of the fluorescence intensity (RFU, relative fluorescent units) of (a) 5-fluorouracil, (b) chlorambucil, (c) fludarabine or (d) etoposide-containing liquid samples (various concentrations). Human plasma anticoagulated with EDTA was mixed with corresponding amounts of a solution of the corresponding chemotherapeutic agent (etoposide and fludarabine were used in the form of the etoposide phosphate or the fludarabine phosphate) and the fluorescence intensities were measured after adding trichloroacetic acid to the liquid sample as a precipitation reagent and separating the precipitated proteins by centrifugation. For the liquid samples for (a), (b) and (d), NaOH was added to each of the liquid samples before the fluorescence measurement; for the liquid samples for (c), water was added. In the measurements too 1 (a) - (c) the excitation slot width / emission slot width was 10 nm / 10 nm, in the measurement too 1 (d) 2.5 nm / 2.5 nm.
• 2 shows the determination of the fluorescence intensity of liquid samples containing doxorubicin and epirubicin (different concentrations). Doxorubicin and epirubicin hydrochloride were added to human plasma and the fluorescence was measured after precipitation of the proteins with trichloroacetic acid (TCA) and centrifugation (n = 4). The mean values and the standard deviation of the individual data points are shown.
• 3 shows the background fluorescence after precipitation of the plasma proteins. Plasma was using Trichloroacetic acid was added, the liquid samples were centrifuged and the fluorescence was measured at an excitation wavelength of 470 nm and an emission wavelength of 555 nm. The mean value and the standard deviation from 10 individual plasma samples are shown.
• 4th shows a comparison of the fluorescence intensities in different test subjects. Plasma samples from 10 different test subjects were treated with corresponding amounts of a 1 mM doxorubicin hydrochloride solution and treated as described. Fluorescence intensities were at 470/555 nm; Excitation slot width / emission slot width 10 nm / 10 nm measured.
• 5 shows the variance of the doxorubicin determination on different measurement days. The measurements were carried out on different days over a period of 1 year with different plasma mixtures. The proteins were precipitated using TCA as described. The precipitated proteins were separated by centrifugation.
• 6 (a) shows the background fluorescence of human plasma after protein precipitation with precipitation reagents according to the invention or after treatment with 0.3M HCl / ethanol 1: 1 (v / v) and centrifugation. The protein from human plasma (pooled) was precipitated using various methods, centrifuged and then the fluorescence was measured at 475/555 nm (excitation / emission wavelength) (n = 6). The precipitation reagent according to the invention or 0.3M HCl / ethanol 1: 1 (v / v) was mixed with the plasma sample in a ratio of 2: 1 (v / v) (1000 μl precipitation reagent: 500 μl liquid sample) or, when using TCA as a 3M aqueous solution, it was first precipitated with 100 μl of 3M TCA, then diluted accordingly with water or acetonitrile in order to achieve a final volume of 1.5 ml. Methanol, ethanol and acetonitrile (AcCN), 3M TCA (with subsequent dilution of the sample with water), 3M TCA (with subsequent dilution of the sample with acetonitrile), zinc sulfate (10% w / v in 0.25 M NaOH) were used mixed with methanol (1: 2 v / v) [Mix1]. 6 (b) shows the doxorubicin fluorescence after precipitation of plasma protein and centrifugation. Human plasma, which contained 50 μM doxorubicin as hydrochloride, was with the below 6 (a) treated precipitation reagents described, centrifuged and then the fluorescence was measured at 475/555 nm (excitation / emission wavelength) (n = 6).
• 7th shows the determination of the fluorescence intensity of human plasma samples to which pegylated liposomal doxorubicin hydrochloride (Caelyx) was added.
• Pegylated liposomal doxorubicin hydrochloride was added to human plasma and the fluorescence was measured after precipitation of the proteins with TCA and centrifugation as described (n = 7).
• 8th shows the determination of the concentration of doxorubicin in a blood sample. Various amounts of doxorubicin hydrochloride were added to whole blood. 3M TCA was added to the fluid sample and the precipitated proteins and cells were separated by centrifugation. The fluorescence was then measured in the diluted supernatant.
• 9 shows an infusion apparatus automatically controlled by the plasma concentration.
• 10 shows the determination of the concentration of doxorubicin and its main metabolites. Doxorubicin hydrochloride, doxorubicinol citrate and the 7-deoxy aglycones of doxorubicin and doxorubicinol were added to human plasma and the fluorescence was measured as described after precipitation of the proteins with TCA and centrifugation (n = 3).

example 1

Sample preparation and measurement

For those in the 1-5 , 7th and 10 The results shown were in each case 1 ml of human plasma (EDTA anticoagulated) to which various amounts of the respective chemotherapeutic agent or its salt were optionally added, mixed with 250 μl of a 3 M trichloroacetic acid solution. The solution was centrifuged at 12,000 g for 2 minutes. 750 μl were removed from the supernatant, diluted with 1500 μl H ₂ O dist. or with 1500 μl of 1 M NaOH, if indicated, and the background fluorescence or concentration of the chemotherapeutic agent was determined by fluorescence measurement. For 1 the respective wavelength pairs (excitation / emission) are indicated, in all other figures measurements were taken at an excitation of 470 nm and an emission of 555 nm. Unless otherwise stated, the excitation slot width / emission slot width was 10 nm / 10 nm.

For the in 6th The results presented here were pooled from human plasma (EDTA anticoagulated) from 5-7 volunteer donors. Part of the plasma was treated with various precipitation reagents according to the invention or with 0.3M HCl / ethanol 1: 1 (v / v), the other part of the plasma was treated with doxorubicin hydrochloride (final concentration in the plasma 50 μM) and then precipitated according to the invention. In each case 500 μl of plasma were mixed with 1 ml of precipitation reagent or 0.3M HCl / ethanol 1: 1 (v / v), when using TCA, 100 μl of TCA were first precipitated, then diluted accordingly with water or acetonitrile to achieve a final volume of 1.5 ml. It was then centrifuged for 2 minutes at 12,000 g. The fluorescence remaining after precipitation of the proteins was measured (475/555 nm, excitation slit width / emission slit width 10 nm / 10 nm) (n = 6). In addition to 0.3M HCl / ethanol 1: 1 (v / v), the following precipitation reagents according to the invention were investigated: methanol, ethanol and acetonitrile, 3M TCA (with subsequent dilution of the sample with water), 3M TCA (with subsequent dilution of the sample with acetonitrile ), Zinc sulfate (10% w / v in 0.25 M NaOH) mixed with methanol (1: 2 v / v) [Mix1].

When determining doxorubicin from a blood sample ( 8th ) 500 μl of human blood (whole blood anticoagulated with EDTA) were mixed with 500 μl of buffer pH 7.4 (10 mM sodium hydrogen phosphate / 140 mM sodium chloride) which contained various amounts of doxorubicin hydrochloride. 500 μl of 3 M TCA were added and mixed. It was then centrifuged for 2 minutes at 12,000 g. 1 ml was removed from the supernatant and diluted with 2 ml of water. The fluorescence was measured at 470/555 nm; Excitation slot width / emission slot width 10 nm / 10 nm.

All fluorescence measurements were carried out using the LS 50 luminescence spectrometer (PerkinElmer LAS GmbH, Rodgau, Germany).

Result

The concentrations of the various chemotherapeutic agents can be determined very well in biological fluid samples using the method described. From the 1 and 2 linear courses of the fluorescence signal can be seen in the described concentration ranges of the respective chemotherapeutic agent. For doxorubicin, for example, a measurement curve can be calibrated in the range from 0.1 to 50 µM. Liposomal doxorubicin can also be determined very well with the method described, since the liposomal shell is destroyed by precipitation with TCA ( 7th ).

The protein precipitation minimizes the background for the fluorescence measurement ( 3 ). The lowest background fluorescence and the best ratio between fluorescence intensity and background fluorescence are shown by the precipitation method with TCA and subsequent dilution with H ₂ O (dist.) ( 6th ). Due to the low background fluorescence, low concentrations of chemotherapeutic agents can also be determined. The method is also very robust and the measurement results hardly vary even when using liquid samples from different dispensers and on different days ( 4th and 5 ). As in 8th shown, a determination can be made directly from whole blood.

10 shows that metabolites of doxorubicin (doxorubicinol, 7-deoxy-doxorubicin aglycon, 7-deoxy-doxorubicinol aglycon) are in part also recorded by the method according to the invention (doxorubicinol is an active metabolite which is also recorded by the method, whereas the detection of inactive aglycones is low due to the low sensitivity). For the determination of the maximum plasma concentration and the primary breakdown rate, however, the metabolites are of subordinate importance, since the metabolism rate is significantly lower than the very rapid primary breakdown rate. The plasma concentration of doxorubicin falls within a relatively short time by 2 orders of magnitude. Presumably, however, the peak plasma concentration is of decisive importance for the observed toxicities and the effectiveness ( El-Kareh AW, Secomb TW, A Mathematical Model for Comparison of Bolus Injection, Continuous Infusion, and Liposomal Delivery of Doxorubicin to Tumor Cells. Neoplasia 2000; 2 (4): 325-338 ; Ishisaka T et al, A precise pharmacodynamic study showing the advantage of a marked reduction in cardiotoxicity in continuous infusion of doxorubicin. Leuk lymphoma. 2006; 47 (8): 1599-607 ), and the method presented allows a quick and safe adjustment of the plasma level, as it allows a very fast analysis in an automated / miniaturized manner.

Claims (13)

Method for determining the concentration of a chemotherapeutic agent and its possibly present metabolites in a biological fluid sample of an individual at a point in time between 0 and 60 minutes after the start of administration of the chemotherapeutic agent or one of its salts to the individual, the chemotherapeutic agent being selected from the group consisting of Doxorubicin, epirubicin, idarubicin, daunorubicin, etoposide, 5-fluorouracil, fludarabine and chlorambucil, comprising i) separating proteins contained in the liquid sample by precipitating the proteins with a precipitation reagent and centrifugation or filtration of the liquid sample, the precipitation reagent being selected from the precipitation reagent Group consisting of trichloroacetic acid, methanol, ethanol, acetonitrile, zinc sulfate and mixtures of two or more of these substances, and ii) measuring the concentration of a chemotherapeutic agent and its metabolites, if present, in a biological en liquid sample by means of a fluorescence signal emanating from the liquid sample after protein separation by irradiation with excitation light, characterized characterized in that the process comprises no chromatographic and no electrophoretic process step and no liquid-liquid extraction. Procedure according to Claim 1 wherein the chemotherapeutic agent or one of its salts is administered to the individual as a liposomal preparation. Method according to one of the Claims 1 - 2 wherein the biological fluid sample is a blood sample, serum sample or plasma sample. Method according to one of the Claims 1 - 3 , the proteins contained in the liquid sample being separated off by precipitating the proteins with a precipitating reagent and centrifuging the liquid sample, the precipitating reagent being trichloroacetic acid. Method according to one of the Claims 1 - 4th wherein no further steps for separating or separating the liquid sample into individual components or groups of components are carried out after or with the exception of the separation of proteins contained in the liquid sample. Method according to one of the Claims 1 - 5 , wherein the chemotherapeutic agent is doxorubicin, epirubicin, idarubicin or daunorubicin and wherein the liquid sample is irradiated with excitation light which has a wavelength in the range of 430-510 nm and the fluorescence signal is measured at a wavelength in the range of 530-600 nm. Method according to one of the Claims 1 - 6th , wherein the method comprises that the intensity of the fluorescence signal emanating from the liquid sample is compared with the intensity of fluorescence signals of a standardized concentration curve of the chemotherapeutic agent. Ex-vivo method for determining an optimal individual dosage of a chemotherapeutic agent or one of its salts, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, epirubicin, idarubicin, daunorubicin, etoposide, 5-fluorouracil, fludarabine and chlorambucil, comprising (i) the Determination of the concentration of the chemotherapeutic agent and its possibly present metabolites by a method of Claims 1 - 7th in two or more biological fluid samples of an individual, the biological fluid samples being taken from the individual at different times after the administration of the chemotherapeutic agent or one of its salts has started, and (ii) the calculation of the optimal individual dosage from the data of step ( i), which is to be administered in order to achieve a predetermined concentration of the chemotherapeutic agent and its possibly present metabolites in the biological fluid of the individual. Infusion apparatus for administering a chemotherapeutic agent or one of its salts to an individual, the infusion apparatus as components (i) a reservoir, (ii) a dosage unit, (iii) a delivery unit, (iv) a recovery unit, (v) an analysis unit, and ( vi) comprises a control unit, wherein the reservoir contains the chemotherapeutic agent or one of its salts, the dosage unit regulates the addition of the amount of the chemotherapeutic agent or its salt per unit of time, which is administered to the individual from the reservoir via the delivery unit, the recovery unit one or more biological Liquid sample (s) of the individual wins, in the analysis unit the concentration of the chemotherapeutic agent and its possibly present metabolites in the or in the biological fluid samples is determined, the control unit controls the regulation of the dosage unit depending on the concentration or concentrations determined in the analysis unit ntration (s) and as a function of a predetermined target value for the plasma concentration, the addition of the amount of the chemotherapeutic agent or its salt per unit of time being regulated via the dosage unit, and the determination of the concentration (s) of the chemotherapeutic agent and its possibly present metabolites in the Analysis unit according to a method of Claims 1 - 7th is carried out. Infusion apparatus after Claim 9 , the recovery unit continuously recovering the biological fluid sample and the analysis unit being designed in such a way that the determination of the concentration of the chemotherapeutic agent and its possibly present metabolites in the fluid sample takes place continuously. Infusion apparatus after Claim 9 or 10 , wherein the control unit controls the dosage unit in such a way that the administration of the chemotherapeutic agent or its salt is terminated as soon as a concentration corresponding to a predetermined concentration is determined in the analysis unit. Infusion apparatus according to one of the Claims 9 - 11 , wherein the control unit controls the dosage unit such that the addition of the amount of the chemotherapeutic agent or its salt per Time unit is upregulated, which is administered from the reservoir via the delivery unit to the individual if the second determination results in a lower concentration than the first in 2 successive determinations of the concentration of the chemotherapeutic agent and its possibly present metabolites in the analysis unit. Infusion apparatus according to one of the Claims 9 - 12th , wherein the biological fluid sample is a plasma sample which the collection unit, which comprises a separation unit, wins by separating a plasma sample through the separation unit from the blood of the individual, which is supplied to the analysis unit, and the analysis unit comprises a protein separation unit and a detection unit, wherein Proteins contained in the plasma sample in the protein separation unit are separated off, and the fluorescence signal that emanates from the plasma sample after protein separation upon irradiation with excitation light is measured with the detection unit.

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