Prediction of Calcineurin inhibition using Phosphatase activity in immunosuppressing (Tacrolimus) oral drug dose regimen.

doi:10.21203/rs.3.rs-2758657/v1

This is a pilot study conducted to validate the intensity of immunosuppression that an individual with kidney transplant develops post-surgical grafting of the organ. It also entails the plausible clinical and pharmacological factors that can contribute to the rejection of the organ on a broader scale. The immunosuppression drug under research is Tacrolimus (FK506), that is assayed for its inhibition of Calcineurin Phosphatase Activity (CPA/CaN Activity) resulting in acceptance and stabilization of the organ system as a whole after transplant. This study also attempts to prescribe a dose regimen by estimating the bioavailability of the oral dosage form. The subjects participating in this trial are an all-male group and are studied for a period of 3–6 months immediately after the surgical transplantation procedure. Therefore, the initial results obtained with the procedures followed are potential for a cohort study (of all genders and age groups).

Clinical Pharmacology

Renal transplant

Tacrolimus

Calcineurin Phosphatase Activity

Inhibition Assay

Isolation of lymphocytes

For this research, two groups with ten male patients each comprising a control group with healthy individuals and another group of renal transplant individuals, treated with Tacrolimus are observed. Blood from patients is taken and processed for calcineurin phosphatase activity for both the groups. In addition, the Tac treated patients are assessed for drug bioavailability and inhibition rate.

Sterile surgical syringes are used to draw 3ml and 6ml of whole blood from control and Tac treated renal transplant patients. It was made sure that the blood from Tac given patients is extracted during the ‘second hour’ at C₀-level (trough level). From both the samples a 3ml is used for measuring cellular Calcineurin Phosphatase Activity (CPA), another 3ml from Tac patients for measuring Tac’s bioavailability. The following is the schematic representation of the study.

Blood samples are collected into EDTA vacutainers; separation of lymphocytes from the whole blood are carried out on the same day and the resultant is stored at -70°C for less than 2 days before CPA is assayed.

Isolation of lymphocytes:

The lymphocytes are separated by gradient centrifugation method, using Histoprep (procured from BAG Healthcare GmbH) and lab made Tris buffer saline (TBS). The whole blood was diluted with TBS in 1:1 ratio and the resultant was further diluted with Ficoll (kit component of Histoprep) in 1:2 ratio and gently homogenized. Centrifugation at 1600 rpm for 25min produces a lymphocyte ring at the interphase of plasma and Ficoll. It is extracted and washed twice with TBS for 10min at 1200rpm. The pellet obtained was added with 300µl of FCS (Fetal calf serum) and 10% DMSO for preservation.

Calcineurin assay (inhibition assay)

A colorimetric Calcineurin Assay kit is purchased from Merck Millipore, for assessment of the cellular CaN activity. All the kit reagents are thawed at room temperatures and Calcineurin, Calmodulin, and 2X Assay buffer are maintained on ice bath. Calmodulin was diluted with the assay buffer in the ratio of 1:50 and a volume of 25 µl was maintained per well. Substrate (RII phosphopeptide) is reconstituted with distilled water to 0.75 mM (1.64 mg/ml) as stock and 10 µl is used per well.

Preparation of test Sample

The purchased kit provides a 96well plate, into this a 25 µl of the assay buffer is added with calmodulin (Table 3). Calcineurin was diluted to 8 U/µl in 1X assay buffer and 5 µl of Calcineurin is added to each well. 10 µl of distilled water is added to control wells. 10µl of the test sample/inhibitor (dissolved in distilled water) is added to test sample wells. Test sample/inhibitor is allowed a reaction time of 10min at a temperature of 30° C. Reaction is triggered by addition of 10 µl phosphopeptide substrate (0.75 mM) and ceased by addition of 100 µl Malachite green. The assay plate is then incubated for 25-30min, until a visible green colour appears in the test sample wells. For accuracy, triplicates of the test sample are assayed at similar experimental conditions.

The assay plate is then scanned for absorbance with Multiskan Spectrophotometer, at 620nm with Xenon lamp pulsating at 300Hz for 1 to 64 times for each measurement. Before reading the sample plate, the system is initialized and a standard phosphate curve is obtained.

Bioavailability of Tacrolimus

Blood collected from the renal transplant patients is tested for Tacrolimus drug using Liquid Chromatography Tandem Mass Spectrometry method (API 4000 Q trap LCMS/MS) procured from Thermo Scientific.

In this study phosphatase activity in both the normal and renal transplant patients is estimated and initially a standard phosphate curve is obtained where absorption is measured at 620nm spectrophotometer. Accordingly, from the linear fit of the graph obtained for Phosphate absorption, the amount of the Phosphate released is also calculated per concentration and is mentioned in the table below. For preparation of the Phosphate (PO₄³⁻) standard curve, the procedure provided with the assay kit has been followed.

Table 1: Different concentrations of Phosphate (PO₄³⁻) absorbed and released.

S. No	Con in nM	Phosphate released	Avg of A620
1	2	2.050847458	0.37
2	1	1.593220339	0.289
3	0.5	1.248587571	0.228
4	0.25	0.960451977	0.177
5	0.125	0.920903955	0.17
6	0.063	0.90960452	0.168
7	0.031	0.824858757	0.153
8	0	0.514124294	0.098

From the graph (1), a concentration dependent Phosphate release can be established. It is calculated by the formula, (A₆₂₀-Yint)/Slope. This is used in the comparison of CaN activity in control group and renal transplant patients. The below table shows other parameters that are compared to establish a drug profile Tac as potential immunosuppressant.

Table 2

CaN activity from control group and transplant patients.
Transplant	Tac dose (mg/ml)	2.25	2.4	2.833	3.4	3.8	3.9	4.1	4.4	5.125	5.8
	Tac level (ng/ml)	8.493	9.732	6.4	8.956	10.64	10.08	9.856	11.65	10.74	7.626
	CaN activity (a.u)	0.76	0.147	0.142	0.11	0.33	0.66	0.44	0.26	0.11	0.42
Control (healthy)	CaN activity (a.u)	1.88	1.56	1.76	1.68	1.64	1.86	1.65	1.72	1.76	1.73

The data points mentioned in Table.2 are an average mean of the measurements that are taken from respective patients. The whole blood from the patients is diluted for endpoint calculations.

Graph.1 explains the inhibition activity of the patient’s immune system post renal transplant; establishing a generic oral dosage of the drug is critical, nevertheless at individual level an effect in the immunosuppresion becomes poignant for personalised treatment. Pharmacological profiling of the drug, considering to group the sample pool into two – control (healthy) and transplant, has a the statistical significance between the groups has a p-value of 1.045E-12 (< 0.05).

Tac level from a blood draw at C₀-trough, directs to the dependability of a drug dosage to pharmacokinetic parameters and the patients physical system that influences the absolute bioavailability of the drug. The half life of Tac is 16hrs for renal tranplant patients and it varies vividly if the grafting is with liver or lung or ocular organs; while in a healthy individual it takes upto 43hrs for the Tac to attain C_max (max.plasma concentration). Therefore, in graph.2 absence of a linear pattern with respect to the increase in oral dosage form is entirely attributed to the time of blood draw for Tac level estimation. This is further confirmed with a negative coefficient of correlation (Pearson) ^{[1], [2]}

The preliminary observations of this clinical research are in congruence with therapeutic outcomes; it provides for inhibition of immunity arising from the renal allograft of patients beyond the patients physiological characters, comorbidities, pharamcokinetic and pharmacodynamic process that an oral drug undergoes in its pharmacological pathway. However, the absolute bioavailbility of the immunosuppresant Tac is not an exceptional parameter as it is dependent on patient related factors consequently a lower therapeutic window at ‘zero hour’ (C₀-trough level). Hence, a larger sample pool with blood draws in line with the maximun plasma concentration is planned and a thorough monitoring of the patients is the future aim of this study. In a pandemic scenario, it is equally challenging to assert the drug efficacy or potency and pooling healthy individuals for the control group.

Declaration of interests

The authors declare that the subjects in this study included 10 normal control (healthy) volunteers and 10 renal transplant patients. Post renal transplant participants on regular treatment with CNI inhibitor (tacrolimus), attending the outpatient clinic were taken into this research. It started after a written approval from NIMS institutional ethics committee (EC/NIMS/1370/2013). The study was designed and conducted in accordance with good clinical practice guidelines. Written informed consent was obtained from each participant.

A. P. T. S. K. Sudhir K Sadul, "Development and Validation of Method for the Estimation of Tacrolimus Crystalline API in Tacrolimus Extended Release Capsules 0.5mg, 1mg and 5mg by Power X-ray diffractometer (PXRD)," Research Journal of Pharmacy and Technology, vol. 13, no. 11, 2020.
V. Dinnendahl and U. e. Fricke, Arzneistoff-Profile, vol. 9, Eschborn, Germany: Govi Pharmazeutischer Verlag, 2003.

Graphs 1 to 3 are available in the Supplementary Files section

The authors of this research report no conflict of interest.

Graph1.png
Graph 1: Standard Phosphate curve.
Graph2.png
Graph 1: Effect of immunosuppresion in healthy and transplant patients.
Graph3.png
Graph 2: Bioavailability of Tac for prescribed oral dose.

Prediction of Calcineurin inhibition using Phosphatase activity in immunosuppressing (Tacrolimus) oral drug dose regimen.

Status:

Version 1

Abstract

Figures

Study Design And Patient Participation

Isolation of lymphocytes:

Calcineurin assay (inhibition assay)

Preparation of test Sample

Bioavailability of Tacrolimus

Observations And Discussions

Conclusion

Declarations

References

Graphs

Additional Declarations

Supplementary Files

Status:

Version 1