STUDY ON THE RELATIONSHIP BETWEEN MYCOPHENOLIC ACID CONCENTRATION AND PERIPHERAL BLOOD T LYMPHOCYTE COUNT IN KIDNEY TRANSPLANT PATIENTS ABSTRACT The advent of immunosuppressive drug has
Trang 1STUDY ON THE RELATIONSHIP BETWEEN MYCOPHENOLIC ACID CONCENTRATION AND PERIPHERAL BLOOD T LYMPHOCYTE COUNT
IN KIDNEY TRANSPLANT PATIENTS
ABSTRACT
The advent of immunosuppressive drug has brought a new face
to the transplant profession in general and kidney transplantation inparticular However, the use of immunosuppressive drug aftertransplantation is highly complex
In addition to the current common and routineimmunosuppressive drug concentration tests such as Cyclosporin andTacrolimus, the Mycophenolic acid test is a test deployed innumerous countries worldwide but has not been performed inVietnam, while 100% of Post-transplant patients at Viet Duc hospitalwere assigned to use Mycophenolic acid (Cellcept or Myfotic).Nevertheless, this drug dosage adjustment merely follows therecommendations of manufacturers and the clinician’s experience but
is not quantified According to the Vietnam Organ TransplantationAssociation, whether the habit of using a single dose of MPA for allpatients is the target concentration, appropriate, safe, or not is still acontroversial issue Many studies have shown that MPA affects thebiosynthesis inhibition of the T lymphocyte, which is stronglyinvolved in the rejection process Thus, this study is conducted withthe following goals:
Trang 21) Investigating the Mycophenolic acid concentration and its area under the curve (AUC 0-12 ) on the 3rd day, 10th day, and 6 months after the kidney transplantation.
2) Evaluating the relationship between the Mycophenolic acid concentration and the immune response through the change in the number of TCD3, TCD4, TCD8 Lymphocyte in kidney transplant recipients.
New contributions to the thesis:
For the first time in Vietnam, the author conducted a quantitativedetermination of mycophenolic acid (MPA) concentration Theresults indicated that the AUC0-12 value of MPA had a significantdifference between patients who used the drug at 3 days, 10 days, and
6 months
The study has suggested the role and practical significance ofmonitoring MPA levels, which helps to personalize the use of anti-rejection medicines for post-transplant kidney patients
Thesis structure: Thesis with 113 pages; Abstract: 2 pages;
Overview: 38 pages; Objects and methods: 16 pages; Results: 23pages; Discussion: 30 pages; Limitations of the study: 1 page;Conclusion: 2 page; Recommendations: 1 page; 30 tables, 17
pictures, 2 appendices
Trang 3CHAPTER 1 OVERVIEW 1.2 Immunosuppressive drugs used in kidney transplantation 1.2.3 Several important parameters for drug absorption and concentration monitoring
In fact, when we give the medication to patients, not all of thedrug is taken into effect, but only a portion
The active fraction is the fraction of the drug entering thecirculation intact (not yet metabolized), which is called thebioavailability (BA), denoted by F (Fraction of dose), with the unit is
%
Bioavailability is a parameter representing the ratio of the drugentering the systemic circulation in the active form relative to thedrug's administered dose (F%), time (Tmax), and concentration(Cmax) penetrated into the systemic circulation Therefore, we musthave the number of drugs entering the circulation in order to havebioavailability
Nonetheless, it is well-nigh impossible because the penetration ofdrugs into the circulation always simultaneously occurs asdistribution and excretion, that is, the amount of drug entering thecirculation fluctuates continuously over time That's why peopleutilize a parameter called AUC to assist
AUC is the area under the curve (showing the variation of drugconcentration in the blood over time); (Cp-t) represents the amount ofdrug which enters the circulation in the active form after time t TheAUC is calculated as mg.h/L or µg.h/mL
AUC0 - ∞ = AUC0 - t + AUCt - ∞
Trang 4If we take a long enough sample, about 3 - 5 x t1/2, thenAUC0-t accounting for about 80% of the total drug intake isacceptable The rest can be ignored or extrapolated if desired
1.3 Mycophenolic Acid
1.3.1 Origin
Mycophenolic acid (also known as mycophenolate) is a kind
of immunosuppressive drug utilized to prevent post-transplantpatients from transplant rejection MPA, derived from the fungus
Penicillium brevicompactum, was discovered in the late 19th century.
Mycophenolic acid is known by the trade name Cellcept(Mycophenolate mofetil) in 250 mg and 500 mg; Myfortic isavailable in 180 mg and 360 mg MPA inhibits enzymes required forthe development of B cell and T cells It primarily acts as an IMPDH– first noted in 1969
Figure 1.8 Mechanism of lymphocyte proliferation inhibition
*Source: Antonio Perez-Aytes et al (2010) [33].
.
1.3.2 Pharmacokinetics
Trang 5Figure 1.9 Distribution and metabolism of MPA
*Source: Antonio Perez-Aytes et al (2010) [33].
1.3.4 Drug efficacy
In the mid-1990s, three large clinical trials were carried out
in renal transplant recipients with a view to demonstrating the clinicalefficacy of MMF The outcomes indicated superior efficacy of thecombination with CsA and steroids in lowering the rate of acuterejection in 6 months after kidney transplantation
According to the study of Jamali et al., the patients before andafter four months of kidney transplantation both increased theproportion of TCD4 lymphocytes cell lines Nevertheless, thepatients receiving Tac / MPA after transplantation indicated a more
Trang 6significant increase in the proportion of TCD4 lymphocytescompared with that of patients receiving TAC/Sirolimus.
Mahboob Lessan-Pezeshki et al., (2005) studied the evaluation
of rejection in 16 first-time kidney transplant recipients withoutdiabetes The results showed no significant difference among CD3,CD4, CD8 T-cell count, CD56 NK cell count, and CD20 B cellscounted before and after transplantation
1.4.3 Several large clinical trials help evaluate the state of monitoring MPA concentration
Van Gelder et al., studied 901 patients and indicated thatMPA AUC0-12 values range of 30 to 60 mg/L.h is acceptable Thepatient’s dosing is based on the individuals, immunological riskassessments, and how AUC values fall into this optimal range
Gaston et al, studied 720 patients and compared groupsusing fixed-dose MMF and calcineurin inhibitor dose adjustment.The results showed no change in the desired effects between thegroups
* Currently, in Vietnam, the use of MPA is following themanufacturer’s instructions and the clinician’s experience No studyevaluates the use of MPA and drug monitoring in renal transplantpatients
CHAPTER 2 STUDY SUBJECTS AND METHODS
2.1 Place, Time, and Object of the study
2.1.1 Place of the study
Biochemistry Department; Nephrology – DialysisDepartment, Transplant Center, Viet Duc Friendship Hospital; Thai
Ha Biomedical Center
Trang 72.1.2 Time of the study
- The study was conducted from February 19, 2014 to May 24,
2016
2.1.3 Object of the study
- The kidney transplant patient was at the transplant center ofViet Duc Hospital in the postoperative period and then wasmonitored and treated at the Nephrology - Dialysis Department for
up to 6 months
- Control group: healthy people (blood donors and organdonors)
2.1.3.1 Criteria for selecting study subjects
- Volunteer to participate in the study
- Criteria for selecting patients:
+ All patients of all ages and both sexes received kidneytransplants and were periodically monitored until the 6th month aftertransplantation at the Transplant Center and the Nephrology –Dialysis Department, Viet Duc Friendship Hospital
+ Use an immunosuppressive regimen with MPA twice a day(Cellcept or Myfortic both use 2 tablets/time)
- Criteria for selecting the control group:
+ Healthy people of all ages and both sexes are eligible todonate blood and organs
2.1.3.2 Exclusion criteria
- The patient was converted to a regimen without MPA duringfollow-up (will be excluded at the time of converting to therespective treatment protocol)
- The patient refused to participate in the study
- The blood sample taken did not give results when testing for T
Trang 82.2.3 Methods and techniques
2.2.3.1 The method of data collection
- Making the research medical record according to thedesigned medical record template
2.2.3.3 Implementation techniques
Determining biochemical parameters: Creatinine, albumin, GOT,GPT on AU680 machine at Biochemistry Department, Viet DucFriendship Hospital
Determining the number of red blood cells and white blood cells
on Unicel DxH600 machine at the Hematology Department, Viet
Duc Friendship Hospital
Trang 9g Determining the number of T-CD3, T-CD4, T-CD8 lymphocytes
By flow cytometry (Lympho T-CD3, -CD4, -CD8 enumeration byflow cytometry) at Thai Ha medical center
m Quantifying the MPA in the blood:
By CEDIA method on a Thermo Scientific Indiko - USA at theBiochemistry Department, Viet Duc Friendship Hospital
* Principle:
Based on Galactosidase Enzymes engineered into two inactivefragments (ED and EA), these fragments bind together to form anenzyme causing a color change in the medium that can be measured
In the presence of the analyte in the sample, it binds to the antibody,leaving the inactive enzyme fragment free to form an active enzyme
In the absence of the analyte in the sample, the antibody binds to theanalyte conjugated on the inactive fragment inhibiting thereassociation of inactive fragments; and no active enzyme is formed.The amount of active enzyme formed resulting in the absorbancechange is proportional to the amount of analyte present in the sample
2.2.4.2 Formula
We applied the trapezoidal rule of Hoang Thi Kim Huyen tocalculate the AUC; Nonetheless, the drug concentration wasquantified at time t0, t1, t2, t3, t6, while at t12, the drug concentrationwas equal to C0 Besides, we based on the chart of drug concentration
to calculate S1 - S12
AUC0-12 = S1 + S2 + S3 + S4 + S5 + S6 + S7 +S8 +S9 +S10 +S11 +S12The threshold value of AUC reached: 30 – 60 mg.h/L
Trang 102.4 Data processing: Using Stata program
- The study had the voluntary participation of the patients All
participants understand the purpose of the study and agree toparticipate in the study And, the patients have the right to withdrawfrom the study at any point without any coercion Explanations arealso not required
CHAPTER 3 RESULTS 3.1 General characteristics of the research subjects
Table 3.1 Distribution of subjects according to age and sex
Trang 11- All patients are of working age with an average age of 38.17 ±12.03.
- Men account for a higher proportion than women 1.7: 1 (62.9%compared to 37.1%)
Table 3.5 Several biochemical and hematological parameters in patients studied before and after transplantation
Time Points
Parameters
3 days post-transplanta
n = 35
10 dayspost-transplantb
n = 35
6 dayspost-transplantc
109,27 ±23,65
pa-b > 0,05pb-c > 0,05pa-c > 0,05Albumin 36,77 ± 1,93 38,26 ±
2,17
41,33 ±3,64
pa-b > 0,05pb-c > 0,05pa-c > 0,05
±7,62
26,09 ±10,64
pa-b > 0,05pb-c > 0,05pa-c > 0,05
cells
3,70 ± 0,59 3,67 ±0,62 4,12 ±
0,48
pa-b > 0,05pb-c > 0,05pa-c > 0,05White blood
cells
10,35 ± 3,99 9,58 ±
3,21
9,62 ±2,41
pa-b > 0,05pb-c > 0,05 pa-c > 0,05
Trang 12- The biochemical and hematological parameters at each timepoint post-transplant showed no difference with p > 0.05
3.2 Use of immunosuppressive drugs
Table 3.6 Distribution of patients according to therapeutic drug class
Therapeutic drug class Quantity (n) Rate (%)
The number of patients in the study group treated with Pred +Prograf + Cellcept accounted for the highest rate (57.2%); the lowestwas those treated with Pred + Neoral + Cellcept (11.4%)
3.2.1 Group employing Cellcept + Neoral (n=4)
Table 3.7 Adjusting the dose of CellCept + Neoral
Time
Drugs
Noadjustment (n, %)
The first
3 days (n, %)
From 3-10days (n, %)
From the
11th dayonwards(n, %)Cellcept
(n=4)
2
50 %
00%
125%
125%Neoral
(n=4)
00%
00%
4100%
00% When adjusting the dose of Cellcept, there was 1 patient (25%) atthe first 10 days and 1 patient (25%) from 11th day onwards
Within the first 10 days, all four patients (100%) were adjusted thedose of Neoral
Trang 133.2.2 Group employing Cellcept + Prograf (n=20)
Table 3.8 Adjusting the dose of Cellcept + Prograf.
Time
Drugs
Noadjustment(n, %)
The first 3days (n, %)
From 3-10days (n, %)
From the
11thoutwards(n, %)Cellcept
(n=20)
00%
15%
315%
1680%Prograf
(n=20)
00%
945%
840%
315%
On the first 3 days, 1 patient (5%) was adjusted the dose ofCellcept From 3 to 10 days, there were 3 patients (15%); and fromthe 11th day onwards, there were 16 patients (80%)
When adjusting the dose of Prograf, there were 9 patients (45%)
on the first 3 days, 8 patients (45%) from 3 to 10 days, and 3 patients(15%) from the 11th day onwards
3.2.3 Group employing Myfortic + Prograf (n = 11)
Table 3.9 Adjusting the dose of Myfortic + Prograf
Time
Drugs
No adjustment (n, %)
The first 3days (n, %)
From 3-10days (n, %)
From the
11th dayoutwards (n, %)Myfortic
(n=11)
00%
00%
218,2%
981,8%Prograf
(n=11)
00%
545,4%
327,3%
327,3%
Trang 14No patient (0%) was adjusted the dose of Cellcept on the first 3
days From 3 to 10 days, there were 2 patients (18.2%); and from the
11th outwards, there were 9 patients (81.8%)
When adjusting the dose of Prograf, there were 5 patients (45.4%)
on the first 3 days, 3 patients (27.2%) from 3 to 10 days, and 3
patients (27.3%) from the 11th day onwards
3.3 Evaluation of the variability of MPA concentration
Table 3.10 Kinetics of MPA concentration according
to the time of transplant
8,8
2,29 ± 1,40
16,9
14,37 ± 12,32
18,2
9,00 ± 6,17
18,7
7,60 ± 5,80
7,6
3,43 ± 1,82
Trang 15Table 3.14 C0 level at time points of post-transplant
0,96 ± 0,350,2 – 1,4(n=20)
1,07 ± 0,230,7 – 1,4(n=11)
1,94 ± 0,321,5 – 2,4(n=14)
1,87 ± 0,281,5 – 2,3(n=10)
pa-b= 0,679
pa-c= 0,338
pb-c= 0,571
Trang 16>2,5 mg/L
4,19 ± 1,332,6 – 6,9(n=10)
8,8(n=1)
3,75 ± 1,282,6 – 6,3(n=12)
23,88 ± 5,6410,73 – 28,9(n=9)
20,68 ± 3,0818,5 – 22,85(n=2)
pa-b= 0,305
pa-c= 0,917
pb-c= 0,46930-60mg.h/L
42,79 ± 6,0233,05 – 54,4(n=18)
45,38 ± 7,4332,45 – 57,6(n=23)
45,11 ± 8,2530,7 – 59,05(n=17)
69,02 ± 11,6660,45 – 82,3(n=3)
84,33 ± 97,9861,05 – 129,05(n=14)
Pa-b= 0.521
Pa-c= 0,109
Pb-c= 0,227The levels of AUC0 – 12 at time points had no difference
3.4 Variability in TCD3, TCD4, TCD8 lymphocytes counts and the ratio of TCD4/TCD3 in kidney transplant patients
Table 3.22 Variability in the number of T lymphocyte lines before and after transplantation.
Lymphocytes Pre-transplant Post-transplant
p-values TCD3 (cell/µL) 1690,31 ± 503,45 2069,14 ± 1374,4 0,222g