Pharmacodynamic Laboratory

pharmocodynamic-group-2014“The Pharmacodynamic Laboratory is now closed for the Christmas Holidays.  We will be back up and running in January 2015. “ The Mississippi State University College of Veterinary Medicine Pharmacodynamic Laboratory has been established to conduct basic and clinical research into the pharmacodynamic monitoring of immunosuppressive therapy, and to provide a diagnostic service for North American veterinarians. Our current research and diagnostic focus is the monitoring of cyclosporine therapy in dogs. Our laboratory analyzes blood samples submitted by practitioners from dogs receiving cyclosporine in order to determine the adequacy of drug-associated immunosuppression, and members of our team are available for consultation regarding immunosuppressive therapy in dogs and cats.

 

pharmacodynamic-1Cyclosporine:

Pharmacokinetic assays (measurement of blood drug levels) can provide evidence that cyclosporine concentrations are within an estimated target therapeutic range. For some dogs, this may predict immunosuppression while, for others, clinical efficacy may not occur despite achievement of blood cyclosporine concentrations in the ‘target’ therapeutic range. For this reason, our laboratory has developed pharmacodynamic assays to better estimate the dose of cyclosporine needed to maintain immunosuppression in individual dogs while minimizing the expense and adverse effects associated with excessive drug dosage.

Pharmacodynamic assays investigate a drug’s effect on target cells. Pharmacodynamic monitoring shows great promise for optimizing cyclosporine therapy and delivering individualized therapy. Here at Mississippi State University, we have done extensive work focused on the pharmacodynamic evaluation of cyclosporine in dogs, firstly using flow cytometry and, more recently, using molecular methods (qRT-PCR).

Cyclosporine inhibits calcineurin, an enzyme that is responsible for the production of cytokines such as IL-2 and IFN-γ by activated T-cells. We have evaluated the utility of qRT-PCR as an alternate method to flow cytometry for evaluating individual T-cell responses to cyclosporine. Our studies have demonstrated that qRT-PCR assays of activated T-cell mRNA IL-2 and IFN-γ expression provide results that are comparable to flow cytometry, and that assays are stable and robust enough to be performed on blood samples mailed in by practitioners.

pharmacodynamic-2Currently, our Pharmacodynamic Laboratory offers molecular (qRT-PCR) assays of activated T-cell mRNA IL-2 and IFN-γ expression as a measure of adequacy of immunosuppression in dogs receiving cyclosporine. Interestingly, using our assays, we have discovered that some dogs are inadequately immunosuppressed despite receiving high doses of cyclosporine, and that other dogs are markedly immunosuppressed despite receiving low drug doses that have typically been considered to not be immunosuppressive, thereby establishing the need for pharmacodynamic assays to enable accurate dose adjustments in the individual dog (individualized drug therapy).

When to Use Our Assays:

  • During the initial weeks of high dose cyclosporine therapy (typically, 10 mg/kg twice daily) in dogs with life-threatening or severe diseases, such as immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, glomerulonephritis, or immune-mediated polyarthritis, in order to rapidly and conclusively establish adequacy of immunosuppression at current drug doses.
  • During subsequent therapy in patients receiving cyclosporine that have failed to demonstrate an adequate clinical response, in order to establish that the drug is actually effectively suppressing T-cell function: poor clinical response despite documented suppression of T-cell function would suggest the need for alternative management strategies, whereas inadequate suppression of T-cell function would suggest the need for a cyclosporine dose increase.
  • During low dose cyclosporine therapy (5 mg/kg once or twice daily) for conditions such as atopy, anal furunculosis, or inflammatory bowel disease, if excessive and undesirable immunosuppression is suspected based on the development of unexpected infectious complications such as urinary tract infection, pneumonia, pyoderma, superficial or deep fungal infections, demodecosis, dermatophytosis or toxoplasmosis.
  • In dogs receiving cyclosporine, the assay can be performed even if the patient is concurrently receiving other drugs.

 

Currently, and for a limited time only until our diagnostic service is well-established,
our laboratory is offering pharmacodynamic analysis of samples submitted from practitioners at no charge

 

 

 “The Pharmacodynamic Laboratory is now closed for the Christmas Holidays.  We will be back up and running in January 2015. “

Sample Submission:

Cyclosporine Pharmacodynamic Assay (T-Cell Function via qRT-PCR IL-2 & IFN-g):



andres-caitlin-pharmo-labWe require a minimum of 3 ml of whole blood in heparinized (green top) tube. Ideally, the first sample should be submitted before commencement of cyclosporine, so we can use each dog’s baseline results as an untreated control. A second sample should then be submitted at least one week after commencing cyclosporine. Although any time within 12 hours of dosing is acceptable, the ideal time of sample collection is 2 hours after drug dosing. Samples can be submitted earlier than one week into treatment, at any time during first week of drug dosing, but please contact our clinicians first to discuss how to interpret results.

Although a pre-treatment sample is recommended, to allow for individualized comparison with post-treatment results, a single post-treatment sample is acceptable if a pre-treatment sample is unavailable. When we only obtain post-treatment samples, we use results from normal untreated dogs, and from untreated dogs with comparable diseases, to establish estimated ‘typical’ baseline results for the individual patient. A significant reduction in T-cell cytokine expression indicates that cyclosporine is effectively suppressing T-cell function at the cellular level.

The most reliable results are obtained if the sample is submitted chilled (that is, refrigerator temperature) but not frozen. The sample is therefore best submitted with an icepack in an insulated container. Samples should ideally be double bagged and shipped within a Styrofoam box. Samples should be shipped for overnight delivery (FedEx is recommended), as samples must be processed within 48 hours of blood collection. For processing, samples should be received by our laboratory Monday through Thursday. Samples should not be sent that will be received by our laboratory on Friday or be sent during holidays. Results are available 2-3 days after the sample arrives at our laboratory. We will contact the veterinarian with results.

Further shipping details are included in the above Sample submission form and details link.

 

 

Frequently Asked Questions:

  • I don’t have heparinized tubes. Can I use another tube?

Heparinized (green top) tubes are STRONGLY recommended for sample collection.

If heparinized tubes are not available, sodium heparin can be added to sterile, no additive tubes. ‘Red top’ tubes commonly have a silicone coating, and sometimes have a silica clot activator coating. There are also ‘no additive’ red top tubes. ‘No additive’ red tops are ideal, but silicone coating can be used if ‘no additive’ tubes are not available. Silica clot activator red top tubes are not acceptable for sample submission (these tubes may appear to have a hazy/cloudy surface).

For a 3-5 mL blood sample, we recommend adding 140 units of sodium heparin. For a 1000 U/mL sodium heparin, this would be 0.14 mL sodium heparin added to the tube prior to blood addition. Tubes should be mixed by inversion immediately.

Note that our assay has not been validated for this approach, and this should only be done in an emergency when green top tubes cannot be obtained.

 

  • My patient is a cat. Can you perform your assay on samples from cats?

Unfortunately, currently, the answer is no. We have not yet developed or validated the assay in cat

  • Why did the laboratory report that my sample had 'insufficient RNA for analysis'?

Uncommonly, we are unable to extract insufficient RNA from samples for qRT-PCR analysis.  Since we extract RNA from activated T-cells, the most likely reason for an 'insufficient RNA for analysis' is that the patient has very low lymphocyte numbers.  While we don't encounter this issue very often, when it occurs it is most likely to occur in patients with marked lymphopenia, as can be seen with severe illness or exposure to glucocortioids.  Insufficient RNA results could also potentially occur with suboptimal sample transportation and handling conditions such as a greater than two days transport time, or samples that become too warm.  'Insufficient RNA' could also result from laboratory error, but we have controls and measures to minimize this potential pitfall.  Unfortunately, if we can not extract sufficient RNA, we cannot perform our qRT-PCR analysis.


Laboratory Contact:
Dr. Evangel Kummari
Postdoctoral Associate
CVM Clinical Science Department
Phone: 662-325-0642
Email: This email address is being protected from spambots. You need JavaScript enabled to view it.

Please contact Dr. Kummari with any questions regarding sample submission or results.

 

Contact Clinicians:

Dr. Andrew Mackin: Phone: 662-325-6631, Email: This email address is being protected from spambots. You need JavaScript enabled to view it.
Dr.Todd Archer: Phone: 662-325-1226 Email: This email address is being protected from spambots. You need JavaScript enabled to view it.
Dr. Clarire Fellman: Phone: 662-325-3432: Email: This email address is being protected from spambots. You need JavaScript enabled to view it. 

 

 Our Team:

 archer todd1Dr. Todd Archer,
Assistant Professor
Small Animal Internal Medicine

mackin andrew 1Dr. Andrew Mackin
Professor and Ward Chair
Small Animal Medicine

fellman claire 1Dr. Claire Fellman,
Small Animal Medicine/Clinical Pharmacology
Resident and PhD student

 

thomason johnDr. John Thomason
Assistant Professor
Small Animal Internal Medicine

 

riggs caitlinCaitlin Riggs,
DVM/PhD student

kummaria evangelDr. Evangel Kummari,
Postdoctoral Associate
 

 

Commonly Used Veterinary Immunosuppressive Drugs:

Drug overviews:

 

Relevant Publications by Our Group:

Journal Articles:

Archer, T., Boothe, D., Langston, C., Fellman, C., Lunsford, K., and Mackin, A. (2014). Oral Cyclosporine Therapy in Dogs: A Critical Review of the Literature. J. Vet. Intern. Med.28: 1–20.

Haraschak, J.L., Langston, V.C., Wang, R., Riggs, C., Fellman, C., Ross, M.K., Bulla, C., Lunsford, K., Mackin, A., and Archer, T.(2014) Pharmacokinetic Evaluation of Oral Dantrolene in the Dog. Journal of Veterinary Pharmacology and Therapeutics. 37(3):286-294.

Archer, T., and Mackin, A. (2014). Management of Immune-Mediated Hemolytic Anemia. Today’s Veterinary Practice. March/April: 41-46.

Dudley, A., Thomason, J., Fritz, S., Grady, J., Stokes, J., Wills, R., Pinchuk, L., Mackin, A., and Lunsford, K. (2013). Cyclooxygenase Expression and Platelet Function in Normal Dogs Receiving Low Dose Aspirin. J. Vet. Intern. Med.27: 141-149.

Archer, T., and Mackin, A. (2013). Diagnosis of Immune-Mediated Hemolytic Anemia. Today’s Veterinary Practice. July/August: 30-34.

Riggs, C., Archer, T., Fellman, C., Figueiredo, A.S., Follows, J., Stokes, J., Mackin, A., and Bulla, C. (2013). Analytical Validation of a Quantitative Reverse Transcriptase Polymerase Chain Reaction Assay for Evaluation of T-Cell Targeted Immunosuppressive Therapy in the Dog. Vet Immunol Immunopathol. 156: 229-234.

Johnson, K., and Mackin, A. (2012). Canine Immune-Mediated Polyarthritis Part I: Pathophysiology. J. Am. Anim. Hosp. Assoc. 48: 12-17.

Johnson, K., and Mackin, A. (2012). Canine Immune-Mediated Polyarthritis Part II: Diagnosis and Treatment. J. Am. Anim. Hosp. Assoc. 48: 71-82.

Mullins, K.B., Thomason, J.M., Lunsford, K.V., Pinchuk, L.M., Langston, V.C., Wills, R.W., McLaughlin, R.M., and Mackin, A.J. (2012). Effects of Carprofen, Meloxicam and Deracoxib on Platelet Function in Dog.Veterinary Anesthesia and Analgesia. 39: 206-217.

Thomason, J., Lunsford, K., Stokes, J., Pinchuk, L., Wills, R., Langston, C., Pruett, S., and Mackin, A. (2012). The Effects of Cyclosporine on Platelet Function and Cyclooxygenase Expression in Normal Dogs. J. Vet. Intern. Med.26: 1389-1401.

Fellman, C.L., Stokes, J.V., Archer, T.M., Pinchuk, L.M., Lunsford, K.V., and Mackin, A.J. (2011). Cyclosporine A Affects the In Vitro Expression of T Cell Activation-Related Molecules and Cytokines in Dogs. Veterinary Immunology and Immunopathology. 140: 175–180.

Archer, T.M., Fellman, C.L., Stokes, J.V., Pinchuk, L.M., Lunsford, K.V., Pruett, S.B., Langston, V.C., and Mackin A.J. (2011). Pharmacodynamic Monitoring of Canine T-Cell Cytokine Responses to Oral Cyclosporine. J. Vet. Intern. Med. 25: 1391-1397.

Lunsford, K., Mackin, A., Langston, C., and Brooks, M. (2009). Pharmacokinetics of Subcutaneous Low Molecular Weight Heparin (Enoxaparin) in Dogs. J. Am. Anim. Hosp. Assoc. 45: 261-267.

Balch, A., and Mackin, A. (2007). Canine Immune-Mediated Hemolytic Anemia: Pathophysiology, Clinical Signs and Diagnosis. Compend. Contin. Educ. Pract. Vet. 29: 217.

Balch, A., and Mackin, A. (2007). Canine Immune-Mediated Hemolytic Anemia: Treatment and Prognosis. Compend. Contin. Educ. Pract. Vet. 29: 230.

Vasilopulos, R.J., Mackin, A.J., Lavergne, S.N, and Trepanier, A.T. (2005).Nephrotic Syndrome Associated with Administration of Sulfadimethoxine/Ormetoprim in a Dobermann.J. Small Anim. Pract. 46: 232.

Rodriguez, D.B., Mackin, A., Easley, R., Boyle, C.R., Hou, W., Langston, C., Walsh, A.M., Province, M.A., and McLeod, H.L. (2004). Relationship between Red Blood Cell Thiopurine Methyltransferase Activity and Myelotoxicity in Dogs Receiving Azathioprine. J. Vet. Intern. Med. 18: 339.

Mackin, A.J., Allen, D.G., and Johnstone, I.B. (1995). Effects of Vincristine and Prednisone on Platelet Numbers and Function in Clinically Normal Dogs.   Am. J. Vet. Res.56:100.

Abstracts:

Thomason, J., Archer, T., Bulla, C., and Mackin, A. (2014). Effects of In Vitro Exposure of Canine Platelets to Pentoxifylline on Platelet Aggregometry. 32nd Annual Forum of the ACVIM, Nashville, Tennessee. Abstract published in JVIM.

Haines, J., Thomason, J., Bulla, C., Seage, E., Wills, R., Lunsford, K., and Mackin, A. (2014) In Vitro and In Vivo Assessment of Platelet Function in Healthy Dogs During Exposure to Low-Dose Aspirin. 32nd Annual Forum of the ACVIM, Nashville, Tennessee. Abstract published in JVIM.

Thomason, J., Archer, T., Press, S., and Mackin, A. (2014). Effects of Cyclosporine and Aspirin on Canine Platelet Function. Late-breaking research abstract, 32nd Annual Forum of the ACVIM, Nashville, Tennessee.

Mackin, A., Weaver, K., Martin, P., Gunnoe, G., Shafer, L., Follows, J., Stokes, J., Pinchuk, L., McKenna, M., Birkenheuer, A., and Dow, S. (2013). Use of Liposomal Clodronate to Facilitate Detection of Subclinical Infection with Babesia canis in Greyhounds. 31st Annual Forum of the ACVIM, Seattle, Washington. Abstract published in JVIM.


Fellman, C., Archer, T., Stokes, J., Lunsford, K., and Mackin, A. (2012). Effects of Oral Cyclosporine on Canine T-Cell Expression of IL-2 and IFN-g Across a 12-Hour Dosing Interval. 30th Annual Forum of the ACVIM, New Orleans, Louisiana. Abstract published in JVIM.

Thomason, J., Lunsford, K., Stokes, J., Mullins, K., Pinchuk, L., Langston, C., Wills, R., McLaughlin, R., and Mackin, A. (2012). Effects of ‘COX-2 Selective’ Non-Steroidal Anti-Inflammatory Drugs on Canine Platelet Function and Cyclooxygenase Expression. 30th Annual Forum of the ACVIM, New Orleans, Louisiana. Abstract published in JVIM.

Fellman, C., Flores, R., Archer, T., Stokes, J., Pinchuk, L., Lunsford, K., and Mackin, A.(2011). Effects of Cyclosporine and Dexamethasone on Canine T-Cell Expression of IL-2 And IFN-γ as Measured by Flow Cytometry and Quantitative RT-PCR. 29th Annual Forum of the American College of Veterinary Internal Medicine, Denver, Colorado. Abstract published in JVIM.

Dudley, A., Thomason, J., Grady, J., Pinchuk, L., Mackin, A., and Lunsford, K. (2011). Effects of Low Dose Aspirin on Canine Platelet Function. 29th Annual Forum of the American College of Veterinary Internal Medicine, Denver, Colorado. Abstract published in JVIM.

Archer, T., Fellman, C., Stokes, J., Lunsford, K., Pinchuk, L., Pruett, S., Langston, C., and Mackin, A. (2010).Effects of Different Oral Doses of Cyclosporine On T-Lymphocyte Biomarkers of Immunosuppression in Normal Dogs.28th Annual Forum of the American College of Veterinary Internal Medicine, Anaheim, California. Abstract published in JVIM.

Thomason, J., Mackin, A., Stokes, J., Wallace, M., Pinchuk, L., Pruett, S., Langston, C., and Lunsford, K.(2010).Effects of Cyclosporine on Platelet Activity in Normal Dogs.28th Annual Forum of the American College of Veterinary Internal Medicine, Anaheim, California. Abstract published in JVIM.

Thomason, J., Lunsford, K.,Mackin, A., Pinchuk, L., Pruett, S., and Langston, C. (2010).Effects of Aspirin on Canine Platelet Cyclooxygenase Expression. 28th Annual Forum of the American College of Veterinary Internal Medicine, Anaheim, California. Abstract published in JVIM.

Thomason, J., Lunsford, K., Mackin, A., Pinchuk, L., Pruett, S., and Langston, C. (2009).

Effects of Cyclosporine on Canine Platelet Procoagulant Activity. 27th Annual Forum of the American College of Veterinary Internal Medicine, Montreal, Canada. Abstract published in JVIM.

Archer, T., Lunsford, K., Mackin, A., Fellman, C., Stokes, J., Pinchuk, L., Pruett, S., and Langston, C. (2009). Development of a Flow Cytometric Panel of T-Lymphocyte Biomarkers to Evaluate the Immunosuppressive Effects of Cyclosporine In Dogs. 27th Annual Forum of the American College of Veterinary Internal Medicine, Montreal, Canada. Abstract published in JVIM.