preclinical measurement of PK curves of anticancer drugs using VAMS

An article by Sebastian P.A. Rosser et al at four institutions in Australia, published in the May 2022 edition of Therapeutic Drug Monitoring, investigated the use of Mitra^® devices with VAMS^® technology for the effect of tariquidar in combination with vincristine to improve the availability of anticancer drugs. The paper is entitled “Quantification of vincristine and tariquidar by liquid chromatography-tandem mass spectrometry in mouse whole blood using volumetric absorptive microsampling for pharmacokinetic applications.”

The study fully validated an LC-MS/MS to simultaneously measure both drugs and this was applied to a PK study on 21 mice. The study showed that vincristine drug exposure increased when tariquidar was added in combination. 

From Diabetes Therapies to Anticancer Drugs 

At the beginning of the twentieth century, the pharmaceutical giant Eli Lilly commercialized insulin for effective treatment of diabetes mellitus. Although some patients responded well to treatment, others developed drug resistance. The search for new therapies began and researchers looked to the biological effects of the extracts of alkaloids from plants as new drug targets.  

One such plant that came under the spotlight was the Madagascan Periwinkle. Although extracts from these plants were not effective in reducing hyperglycemia, they were however, shown to reduce proliferation of white cells. In the 1960s, two drugs developed from periwinkle extracts (vincristine and vinblastine), were approved by the US Food and Drug Administration (FDA) for the treatment of various cancers.   

Anticancer Drug Resistance      

Cancer can be classed as an uncontrolled proliferation of specific cells. For example, in certain blood cancers, cancerous white cells multiply uncontrollably. Drugs like vincristine help to halt cellular proliferation by binding to a protein called tubulin preventing chromosomal separation during a cell’s metaphase.

While these drugs are highly effective chemotherapeutics for many people, they also cause selection pressure on the cancer and as a result, drug resistant cells become dominant. For many years, scientists have been studying mechanisms of drug resistance and multi drug resistance (MDR) as it has become known.

One such mechanism is the cell’s utility of a class of proteins called adenosine triphosphate (ATP)-binding cassette (ABC) transporters which have a range of utilities. These include transporting specific classes of molecules inside and out of a cell. One such protein is called P-glycoprotein (P-gp), which has a broad specificity to drugs and acts to actively export drugs such as vancomycin out of cells, preventing cellular drug accumulation as a means to detoxify the cell.   

Using Drugs to Inhibit P-glycoprotein 

Inhibiting P-glycoprotein results in improved efficacy of anticancer medication. Scientists have been developing drugs which act to inhibit the P-gp pump, preventing drug efflux and thus improving chemotherapeutic efficacy. One such agent is tariquidar, a third generation P-gp pump inhibitor.   

Rosser et al commented that use of P-gp inhibitors could affect the pharmacokinetic (PK) profiles of drugs such as vincristine and so influence drug exposure. For this reason, the research group developed an LC-MS/MS method to simultaneously measure both drugs (vancomycin and tariquidar) from VAMS extracts and conduct preclinical PK measurement on mice. The group chose Mitra devices with VAMS due to the volumetric nature of the sampling approach, which prevents specific hematocrit effects seen with DBS. 

Furthermore, the microsampling element of the device is in line with the “3Rs” initiative in the research community to reduce, refine, and replace the use of animals where possible in drug development. VAMS impacts the reduction component of the 3Rs, as many more samples can be taken from far fewer animals while still obtaining high-quality samples and reliable results. In the paper reviewed here, the research group noted that VAMS was significantly less invasive than venipuncture, so it also fulfilled the refinement component of the 3Rs.   

Study Methods and Findings 

• Extraction of Mitra employed a “leave the tip behind” approach where the VAMS tips were cut from the Mitra microsampler bodies then extracted in a mixture of acetonitrile and water with IS using both vortexing and ultrasound. 
  
  
• The method was validated according to European Medicine Agency (EMEA) bioanalytical method validation guidelines. Below are some points to note:
  
  
  • Very high precision (≥0.9987) and accuracy (~90-95%) was seen for both drugs between human plasma and whole blood VAMS extracts allowing for human plasma to be used for standards and QCs, negating the need to use dried mouse blood. 
    
    
  • Analyte Stability: 
    • Vincristine remained stable (<±15%) for all measured parameters including 24° C and – 40° C over a period of 30 days. 
    • Tariquidar maintained stability for up to 14 days at room temperature and was stable at 30 days at – 40° C. 
      

• Extraction recoveries were all above 93% for both drugs and all three QC levels. 
  
  
• Imprecision for the Low QC of vincristine was 18.7%, which was just within the guidelines.  Also, imprecision for the intermediate QC for tariquidar was 15.7% which was just outside the guidelines of ±15%; the group concluded that this may have been a result of the stability of this molecule.
   
  
• Pharmacokinetic Results:
  • Mice (n=21) were either given both medications (group C) or just vincristine (group A) or just tariquidar (group B).  A control group (D) was just given glucose (drug vehicle). Blood collection was carried out by lancing the saphenous vein over 7 timepoints using 2 mouse groups.  
  • The vincristine clearance had halved when in combination with tariquidar; the research group suggested that this indicated vincristine drug exposure increased due to the P-P-gp inhibition. 

• They concluded that similar effects could also be seen with P-gp substrates other than just vincristine.  
   
• The group also commented that they would not have expected the PK of tariquidar to change as a result of other concomitant medications. 
  
  
• Preliminary evidence from other studies of tariquidar suggest administration does not impact subject tolerability. 

Anticancer Drug Study Authors’ Concluding Remarks 

• Developed a successful, LC-MS/MS method for simultaneous determination of vincristine and tariquidar from 10 µL dried blood samples and plasma samples. 
  
  
• All validation parameters were within acceptable parameters.
   
• Method was used in a successful PK study on mice. 
  
  
• Future work will be conducted to measure the effect of tariquidar on other concomitant medications. 

Neoteryx Remarks 

This study demonstrates the utility of capillary dried blood microsampling to achieve very high-quality PK data from a preclinical mouse model. Not only does microsampling significantly help in both reduction and refinement of the 3Rs of animal research to improve animal welfare, but it also increases the number of timepoints per mouse and enables greater data quality without reliance on combining data from many subjects.   

This article was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this blog, visit the original article in Therapeutic Drug Monitoring.

Image Credits: iStock, Neoteryx, Trajan

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