Periodic Reporting for period 1 - POC-TDM (Development of a point-of-care microfluidic device for Therapeutic Drug Monitoring in cancer treatment (POC-TDM))
Okres sprawozdawczy: 2022-09-01 do 2024-08-31
This MSC Action is titled “Development of a point-of-care microfluidic device for Therapeutic Drug Monitoring in cancer treatment (POC-TDM)”. This project is aimed to personalize and improve current cancer care through a novel microfluidic technology enabling the monitoring of chemotherapeutics concentration in the patients’ blood during therapy.
As a cancer treatment, chemotherapy is prescribed in a bid to eliminate cancer cells. However, chemotherapy protocols are established as ‘one size fits all’, with no allowance for interpatient differences in drug pharmacokinetics. The Marie Skłodowska-Curie Actions project POC-TDM investigated the possibility of improving and personalising chemotherapy. For instance, missing target blood concentration will lead to drug resistance and/or unwanted side effects. As such, therapeutic drug monitoring could be the key to improve and personalise chemotherapy. In fact, the project proposes a new microfluidic chip-based approach to rapidly determine plasma concentrations of commonly used anticancer drugs. This new system could improve cancer survival rates through patient-tailored therapy.
Chemotherapy is here to stay, helping millions of patients annually. Surprisingly, research focusing on the improvement of chemotherapy is sorely missing. The rise of biological and immunotherapies seemingly put chemotherapy on the bench, despite treatment of most of cancer patients still includes conventional drugs. The POC-TDM MSCA project made an attempt to lay the foundation of a new, personalized cancer treatment via TDM and give chemotherapy a long-overdue improvement.
As a cancer treatment, chemotherapy is prescribed in a bid to eliminate cancer cells. However, chemotherapy protocols are established as ‘one size fits all’, with no allowance for interpatient differences in drug pharmacokinetics. The Marie Skłodowska-Curie Actions project POC-TDM investigated the possibility of improving and personalising chemotherapy. For instance, missing target blood concentration will lead to drug resistance and/or unwanted side effects. As such, therapeutic drug monitoring could be the key to improve and personalise chemotherapy. In fact, the project proposes a new microfluidic chip-based approach to rapidly determine plasma concentrations of commonly used anticancer drugs. This new system could improve cancer survival rates through patient-tailored therapy.
Chemotherapy is here to stay, helping millions of patients annually. Surprisingly, research focusing on the improvement of chemotherapy is sorely missing. The rise of biological and immunotherapies seemingly put chemotherapy on the bench, despite treatment of most of cancer patients still includes conventional drugs. The POC-TDM MSCA project made an attempt to lay the foundation of a new, personalized cancer treatment via TDM and give chemotherapy a long-overdue improvement.
WP1 aimed to develop the detection technology for the measurement of chemotherapy drug concentrations. First, the optical characterisation of the anthracycline compounds was done. The fluorescent spectrum of doxorubicin, pegylated liposomal doxorubicin, epirubicin and mitoxantrone was analysed using a Tecan Spark spectrofluorometer in 96-well plates. The results showed that the concentration of anthracyclines in different solvents (dimethyl sulfoxide, fetal bovine serum and cell culture media) can be followed with high specificity and precision. Then, the same measurements were performed in the microfluidic environment using a simple microfluidic cartridge. The encouraging results proved the usability of microfluidics in detecting fluorescent drugs in liquid samples. Finally, the sensitivity and limit of the proposed approach was tested. Published plasma drug concentrations found in patients was used as reference to identify the limit of detection. Comparing with real-life drug concentrations, the results showed that the POC-TDM technology can detect relevant drug levels in liquid samples.
The main goal of WP2 was to design and manufacture a prototype microfluidic cartridge able to separate plasma from whole blood and transport the sample into the measurement chamber. The Research Fellow learned how to design and fabricate microfluidics using the CleWin software and the host institute specialized infrastructure. At first, a simple, sample transport microfluidic cassette was established to test the fluorescent detection method in microvolumes. After it was proved that fluorescent compounds can be measured in a plate reader-compatible microfluidic system, plasma filtration methods were tested which could be combined with the sample transport microfluidic cartridge. The design was tested with both human and mouse blood samples, and it was able to separate the plasma and transport it to the detection chamber.
The testing of the microfluidics and the fluorescents detection method using blood samples from mice took place in WP3. First, to provide evidence that the POC-TDM approach will work using real samples. a small amount of blood (~100 µl) was drawn from the tail vein of healthy animals and spiked with various concentrations of doxorubicin and pegylated liposomal doxorubicin. The plasma separation was done by centrifugation and the supernatant plasma was removed and measured. The results were satisfactory, and limit of detection was similar to doxorubicin concentrations found in cancer patients. In the next step, two groups of healthy mice were treated with different doses (6 and 0.6 mg/kg) of pegylated liposomal doxorubicin through the tail vein and blood samples were taken before administration and after 5, 15, 30, 60, 180, 360, 1440, 2880 minutes. The plasma concentrations were measured with the POC-TDM method and, simultaneously, using mass spectrometry. The pharmacokinetics, while greatly differ based on the injected dose, were almost identical for the two methods. The same experiment was repeated on Brca1 and p53 double knock-out mouse mammary tumour-bearing animals. Surprisingly, despite the identical genetic background (inbred mice) and virtually indistinguishable tumour (genetic inheritance) the peak plasma drug concentrations were significantly varied, suggesting that personal pharmacokinetics of individuals could even more altered than we thought. The experiment was repeated again on mice inoculated with Lewis lung carcinoma (LLC) cells through the tail vein. These rapidly growing cells quickly colonize the lungs of the animal, forming lung tumours. The results were also identical, proving the POC-TDM approach viability in critical conditions.
At the final stage of the project (WP4), the POC-TDM methodology was tested in the veterinary oncology environment. By collaborating with the Veterinary Haematology and Oncology Center (VHOC) and the University of Veterinary Medicine (Budapest, Hungary), the anthracycline-based therapy of ten veterinary oncology patient (six dogs and 4 cats) was monitored. Blood samples were taken via the saphenous vein before administration and after 30, 60, 150 and 270 minutes. The drug concentrations were measured by both the POC-TDM system and mass spectrometry and found to be comparable. This experiment ultimately proved that the POC-TDM approach is working in the relevant clinical setting
The main goal of WP2 was to design and manufacture a prototype microfluidic cartridge able to separate plasma from whole blood and transport the sample into the measurement chamber. The Research Fellow learned how to design and fabricate microfluidics using the CleWin software and the host institute specialized infrastructure. At first, a simple, sample transport microfluidic cassette was established to test the fluorescent detection method in microvolumes. After it was proved that fluorescent compounds can be measured in a plate reader-compatible microfluidic system, plasma filtration methods were tested which could be combined with the sample transport microfluidic cartridge. The design was tested with both human and mouse blood samples, and it was able to separate the plasma and transport it to the detection chamber.
The testing of the microfluidics and the fluorescents detection method using blood samples from mice took place in WP3. First, to provide evidence that the POC-TDM approach will work using real samples. a small amount of blood (~100 µl) was drawn from the tail vein of healthy animals and spiked with various concentrations of doxorubicin and pegylated liposomal doxorubicin. The plasma separation was done by centrifugation and the supernatant plasma was removed and measured. The results were satisfactory, and limit of detection was similar to doxorubicin concentrations found in cancer patients. In the next step, two groups of healthy mice were treated with different doses (6 and 0.6 mg/kg) of pegylated liposomal doxorubicin through the tail vein and blood samples were taken before administration and after 5, 15, 30, 60, 180, 360, 1440, 2880 minutes. The plasma concentrations were measured with the POC-TDM method and, simultaneously, using mass spectrometry. The pharmacokinetics, while greatly differ based on the injected dose, were almost identical for the two methods. The same experiment was repeated on Brca1 and p53 double knock-out mouse mammary tumour-bearing animals. Surprisingly, despite the identical genetic background (inbred mice) and virtually indistinguishable tumour (genetic inheritance) the peak plasma drug concentrations were significantly varied, suggesting that personal pharmacokinetics of individuals could even more altered than we thought. The experiment was repeated again on mice inoculated with Lewis lung carcinoma (LLC) cells through the tail vein. These rapidly growing cells quickly colonize the lungs of the animal, forming lung tumours. The results were also identical, proving the POC-TDM approach viability in critical conditions.
At the final stage of the project (WP4), the POC-TDM methodology was tested in the veterinary oncology environment. By collaborating with the Veterinary Haematology and Oncology Center (VHOC) and the University of Veterinary Medicine (Budapest, Hungary), the anthracycline-based therapy of ten veterinary oncology patient (six dogs and 4 cats) was monitored. Blood samples were taken via the saphenous vein before administration and after 30, 60, 150 and 270 minutes. The drug concentrations were measured by both the POC-TDM system and mass spectrometry and found to be comparable. This experiment ultimately proved that the POC-TDM approach is working in the relevant clinical setting
The POC-TDM project reached its main goal: It proved that using a cheap and simple microfluidic system in combination with a commonly available fluorescent plate reader or spectrophotometer to detect the specific fluorescent fingerprint of anthracycline chemotherapeutics can be used for point-of-care Therapeutic Drug Monitoring. The project showed that TDM in oncology, at least for certain drugs, is feasible without expensive and specialized equipment and/or infrastructure while it also does not require special expertise or knowledge. It opened a door which virtually not existed before. Of course, there are unambiguous limitations of the current POC-TDM technology like the need of fluorescent compounds, but hopefully the collaboration with our industrial partner will help further advancing this approach. The industrial partner, as part of our collaboration, (1) will repurpose a benchtop fluorescent analyser, developed previously by the company for another IVD application, to increase our mobility and measurement capabilities, and (2) design and finance a clinical study to evaluate the POC-TDM method in the clinical setting. The results from this experiment will help decide whether our technology is ready for translation into oncology. Upon success, our method will reach cancer patients quickly.