An assay for the determination of sirolimus levels in the lymphocyte of transplant patients.

UNLABELLED: Both tacrolimus (TAC) and sirolimus (SRL) bind to the same immunophilin FKBP12; however, their mechanisms of action are distinct. SRL inhibits mammalian target of rapamycin (TOR), which is an enzyme critical to the immune function. TOR inhibition blocks the signal that mediates T-cell proliferation by preventing cell-cycle progression from G1 to S phase. Moreover, TOR inhibition results in a decrease in antibody production by blocking B-cell proliferation and maturation into antibody producing cells. The use of SRL has resulted in a decrease in the number of rejection episodes. As with other immunosuppressive agents, SRL can cause dose-related side effects, the most notable of which are hypercholesterolemia, hyperlipidemia, anemia, and thrombocytopenia. Thus, therapeutic drug monitoring to assess efficacy and toxicity has became a necessity. SRL blood levels do not correlate with its bioactivity and are affected by the concomitant use of other immunosuppressive drugs. To determine the bioactivity of SRL we have developed an assay to determine the level of Sirolimus per lymphocyte of transplant patients. The levels were correlated with lymphocyte count. METHODS: Whole blood samples from patients on SRL were collected in Ethylene Diamine Tetra-acetic acid (EDTA) vacutainer tubes. Immediately the lymphocytes from 2 mL of blood were separated using 1.5 mL of Ficoll gradient, by centrifugation for 30 minutes at 2500 RPM. The lymphocytes were washed three times with phosphate-bufferd saline and the pellet suspended in 150 microL of Middle East research institute (MERI) drug extraction solution (Beirut, Lebanon), which was then added to 300 microL of IMx solublizing reagent. The cytoplasmic SRL concentrations in lymphocytes were measured using kits supplied from Abbott diagnostics or by high-performance liquid tomography. A corresponding whole blood sample from each patient was used to measure blood levels. To determine the level per lymphocyte, the value obtained was divided by the number of lymphocytes and expressed as Pg/cell. A pharmacokinetic profile for both blood and lymphocytes was constructed for each patient using data corresponding to predose C(0), 1 hour (C(1)) and 2 hours (C(2)) after the dose. The lymphocyte enumeration for C(0), C(1), and C(2) was performed using the FACS Calibur Flow Cytometer from Becton Dickinson. The average dose was 2.86 +/- 1.27 mg/d with a C(0) = 8.05 +/- 4.24, C(1) = 21.9 +/- 8.9 ng/mL, and C(2) = 23 +/- 0.03 ng/mL. Although there was a significant correlation (P=.0975) between the dose and C(0), there was no correlation between the dose and C(0) level on the lymphocyte count P=.897. However, there was a strong correlation between SRL lymphocyte levels (pg/cell) and the lymphocyte count (r(2)=.6.06). The higher the concentration of the drug the lower the lymphocyte counts. The assay is sensitive to within 0.45 pg/cell, reproducible with a coefficient of variance (CV) of 6.4% within assay and 7.5% for intraassay.

Cyclosporine lymphocyte maximum level monitoring in de novo kidney transplant patients: a prospective study.

OBJECTIVES: To determine prospectively the temporal variations of cyclosporine-A lymphocyte maximum level, whole blood maximum concentration, and total lymphocyte count in patients with de novo kidney transplantation. MATERIALS AND METHODS: Lymphocyte maximum level, whole blood maximum concentration, and total lymphocyte count were prospectively measured in 35 patients at 1, 2, and 3 months after kidney transplantation. Two groups--a biopsy-proven acute rejection group (REJ+) and a rejection-free group (REJ-)--were compared. RESULTS: Both groups had similar lymphocyte maximum levels, whole blood maximum concentrations, and total lymphocyte counts at the first month after transplantation. REJ+ patients had significantly lower lymphocyte maximum levels at 2 and 3 months (59+/-34 and 33+/-9 pg/Lc) and higher total lymphocyte counts (0.00204+/-0.00078x10(9)/L and 0.00203+/-0.00022x10(9)/L) when compared with their REJ- counterparts (87+/-56 and 63+/-30 pg/Lc, P<.05 and P<.007) and (0.00137+/-0.00074x10(9)/L and 0.0015+/-0.0006x10(9)/L, P<.02 and P<.003) respectively. Whole blood maximum concentrations were significantly higher in patients in the REJ+ group (2050+/-623 vs 1414+/-536 ng/mL, P<.02) at 2 months. At 3 months, the 2 groups were comparable (1158+/-340 vs 1365+/-525 ng/mL, P=NS). CONCLUSIONS: These results suggest that acute rejection is associated with a relatively low cyclosporine- A lymphocyte maximum level and high total lymphocyte count in the early posttransplant period. Cyclosporine-A whole blood maximum concentration failed to correlate with clinical outcome. Cyclosporine-A lymphocyte maximum level seems to offer a more reliable alternative than does whole blood maximum concentration for cyclosporine-A monitoring in patients with kidney transplantation.

A transplanted kidney surviving total vessel occlusion and anuria.

We report a case of a 46-year-old white male with renal graft artery stenosis who developed acute renal shutdown with total anuria while on the ACE inhibitor lisinopril, one week following the discontinuation of aspirin. The serum creatinine was 8.5 mg/dl. Doppler ultrasound and MAG3 scintigraphy of the grafted kidney were highly suggestive of a viable but nonfunctioning kidney. A femoro-femoral bypass for total thrombosis of the right common iliac artery was performed distal to the occlusion. Immediate diuresis was obtained after establishing the bypass. Serum creatinine dropped to 1.35 mg/dl three days later. In this case we believe that the collateral circulation played a significant role in immediate recovery of kidney function by maintaining renal perfusion pressure and preventing acute tubular necrosis (ATN). We also believe that the ACE inhibitor might have contributed to salvaging the kidney by improving medullary oxygen balance and maintaining adequate medullary blood flow.

Switchability of neoral and equoral according to Food and Drug Administration rules and regulations.

According to the US Food and Drug Administration (FDA), if a drug product contains a drug substance that is chemically identical and is delivered to the site of action at the same rate and extent as another drug product, then it is equivalent and can be substituted (switchable) for that drug product. Methods used to define bioequivalence as stated by the FDA rules (FDA 21 CFR 320, 24) are (1) pharmacokinetic (PK) studies in healthy volunteers, (2) comparative clinical trials, and (3) pharmacodynamic (PD) studies (bioactivity). We evaluated the switchability of Equoral (IVAX-USA) with Neoral (Novartis Switzerland using all FDA rules. In a single oral dose, we undertook a comparative bioavailability study of Equoral (IVAX, USA) Neoral (Novartis, USA), and Neoral (Novartis UK). The pharmacokinetics of Equoral and Neoral were determined with blood levels at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 16, 24, 30, 36, 42, and 48 hours. The area under curve (AUC), AUC extrapolated to infinity (AUC0-inf), rate of absorption (Tmax), extent of absorption (Cmax), half time (t1/2) of Equoral and Neoral were all within the 90% confidence interval of 80% to 125% boundaries. A comparative multinational multicenter clinical trial in stable renal transplant patients included 70 patients (22 women and 48 men) of mean age of 33 years (range, 26 to 43) was performed in Turkey, Lebanon, and Pakistan. In this study the ratios of LSM and the 90% confidence intervals for the Nontransformed/Parameters (AUC0-t, AUCinf, Tmax, and Cmax) of Equoral and Neoral SGC were 98% and 95%, respectively, which are within the 80% to 125% FDA acceptance range. For immunosuppressive drugs, the site of action is the lymphocyte and the measurable response is the decrease in lymphocyte count caused by the relative concentration of the drug in the lymphocyte. In a controlled switch, fixed-dose study, both Equoral and Neoral achieved the same concentration in the lymphocytes and caused the same degree of lymphocyte count reduction. The results of the testing (bioavailability-bioequivalence, clinical studies, and pharmacodynamic-bioactivity) required by FDA for interchangeability ("switchability") of immunosuppressive agents suggests that Neoral and Equoral are switchable.

Mycophenolic acid plasma trough level: correlation with clinical outcome.

OBJECTIVES: Assess the relationship between clinical diagnosis, state of immunosuppression, mycophenolic acid (MPA) plasma trough levels (MPACmin), and mycophenolate mofetil (MMF) dosage in renal transplant recipients. MATERIALS AND METHODS: MPACmin were determined in 30 kidney transplant patients, of whom 7 exhibited biopsy-proven acute rejection. The remaining 23 had normal graft function. Graft outcome, defined by clinical diagnosis and serum creatinine level, was compared according to MPACmin, MMF dosage, and total lymphocyte count (LC). RESULTS: Patients with acute rejection had similar MPACmin (2.4 +/- 1.7 microg/mL), MMF dosages (1.7 +/- 0.5 g), and LCs (0.001165 +/- 0.0040 x 10(9)/L) when compared with normal patients (2.2 +/- 0.7 microg/mL, 1.7 +/- 0.4 g and 0.001160 +/- 0.00527 x 10(9)/L) respectively. Rejection rates were comparable irrespective of MPACmin)ranges and higher in those receiving the 1-g dose (30%) when compared with those receiving 1.5-g and 2-g doses (12.5% and 11.7%). No relationship was observed between MPACmin and MMF doses, and neither parameter correlated with LC. CONCLUSIONS: These results suggest that MPACmin is a poor correlate of clinical outcome and state of immunosuppression. Although the usually recommended dosage of MMF (2 g) may be associated with acute rejection, low-dose MMF (1 g) seems to constitute a higher risk.

Consanguinity-associated kidney diseases in Lebanon: an epidemiological study.

Consanguineous marriages are common in many countries of the Middle East including Lebanon. Their impact on the repartition of kidney diseases and on the risk for familial nephritis is not known. We surveyed all of the dialysis centers in Lebanon. Nine hundred and twenty-five (925) patients and their private physicians were asked to answer a questionnaire. More than half of the hemodialysis (HD) patients had an unknown etiology of their kidney disease. Diabetes, polycystic kidney disease (PKD), chronic pyelonephritis and nephrosclerosis (NS) were the most commonly documented diagnoses. Consanguinity was present in 26% of the total HD population. More consanguineous patients with unknown renal etiology were diagnosed with their kidney diseases and initiated on dialysis before the age of 30 when compared with their non-consanguineous counter-parts (45% versus 33%, P<0.02 and 42% versus 27%, P<0.01), respectively. Similarly, consanguineous polycystic patients were diagnosed and started earlier on dialysis when compared with the non-consanguineous population (34% versus 12%, P<0.05 and 28% versus 8%, P<0.05), respectively suggesting a different disease pattern. Furthermore, the risk for family history of kidney disease was noticeable in the non-consanguineous population and significantly higher among the consanguineous patients (12% versus 18%, P<0.04). Consanguinity-associated kidney diseases affected all religious communities, in particular the Muslim and the Druze (36 and 39%), respectively versus 17% of the Christian community. Certain geographical areas were more involved than others such as the North, South and the Bekaa with the highest percentage (40%) in the latter. Socio-economical level was not a contributing factor. We conclude that the documentation of the underlying etiology in end-stage renal diseases (ESRD) seems to be deficient. Furthermore, consanguinity is prevalent in the Lebanese dialysis patients population, in particular the Muslim and the Druze communities. Consanguinity-associated kidney diseases pattern seems to differ from that of the general HD population by disease diagnosis and initiation at a younger age and a significantly higher risk for familial renal disease. It is a cultural phenomenon prevalent predominantly in the rural areas. We recommend a multi-approach including educational, informative and probably legislative strategy in order to limit and hopefully discourage consanguineous marriages.

Activation of intrinsic coagulation pathway in pre-eclampsia.

Disseminated intravascular coagulation, thrombocytopenia, consumption of factors VIII and II, and antithrombin deficiency have been previously demonstrated in pre-eclampsia. However, the precise mechanism responsible for initiation of disseminated intravascular coagulation has not been elucidated. The present study documents activation of the intrinsic coagulation pathway in a patient with severe pre-eclampsia. The studies revealed marked reductions of plasma coagulant activities of all intrinsic pathway factors, i.e., XII, XI, IX, and VIII. In addition, the ratio of plasma factor XII activity to antigen concentration was markedly abnormal, and plasma high-molecular-weight kininogen concentration was diminished. It is suggested that activation of the intrinsic coagulation pathway may be operative in the genesis of disseminated intravascular coagulation in pre-eclampsia.

Intestinal absorption of arachidonic acid in experimental azotemia.

The effect of renal failure (RF) on intestinal absorption of dietary fatty acids is not known. We studied the intestinal absorption of arachidonic acid (AA) in rats with experimental short-term (2 weeks post-subtotal nephrectomy) and long-term (5-6 weeks post-subtotal nephrectomy) RF. The results were compared with those obtained in sham-operated animals on liberal food intake (NL) and in those pair-fed (PF) with the respective RF groups. In vivo perfusion and in vitro incubation experiments were performed at a wide range of AA concentrations. The rates of AA transport determined both in vivo and in vitro were significantly lower in the short-term RF group than those found in the NL controls and the PF animals who showed comparable values. In contrast animals with long-term RF exhibited an increased rate of AA transport as compared with the respective controls. The observed changes in the transport rates appeared to parallel directional changes in mucosal mass which was reduced in animals with short-term RF and restored in those with long-term RF.

Intestinal transport of pyridoxine in experimental renal failure.

Renal failure (RF) has been shown to alter intestinal transport of a number of nutrients. We studied jejunal absorption of pyridoxine (B6) in rats rendered azotemic by subtotal nephrectomy (RF group) and compared the results with those obtained in normal rats subjected to sham operation (controls) and animals pair-fed (PF) with their RF counterparts. In vivo recirculating perfusion and in vitro everted sac techniques were employed. The in vitro experiments were repeated using sera from uremic and normal individuals to assess the possible effect of uremic chemical environment. The results showed significant reduction in B6 absorption in vivo in the RF group as compared to the control and PF groups. Paradoxically, the rate of in vitro B6 absorption determined for a wide range of concentrations was increased in the RF and PF groups as compared to the control group. The observed increase in B6 absorption in vitro suggests enhanced permeability in the RF and PF groups due probably to reduced nutrient intake which was common to both groups. The disparity between the in vivo and in vitro results is indicative of some inhibitory factor(s) present in the RF animals. Sacs containing uremic serum showed significantly suppressed B6 absorption in vitro as compared to those containing normal serum. These observations suggest that the uremic chemical environment may be, in part, responsible for the observed impairment of B6 transport in RF animals despite in vitro evidence of hyperpermeability.

The pharmacokinetics of equoral versus neoral in stable renal transplant patients: a multinational multicenter study.

We studied the pharmacokinetics (PKs) of the new generic cyclosporine formulation, Equoral capsules, after the switch from original formulation Neoral capsules in stable renal transplant patients. The study was carried out in accordance with the basic principles defined in the US 21 CFR Part 312.20 and the principles of the Declaration of Helsinki. The study included clinically stable first renal transplant patients maintained on cyclosporine with no rejection episode during the past 6 months. Hematology, biochemistry, and urine chemistry were determined on day 7, and day 21. The patients were all switched to Neoral (lot number 416MFD0601) on day 0 when the first sparse sampling PK was performed. On day 14 a 12-hour PK profile included predose, 30 minutes; 1 hour; 1 hour 30 minutes; 2 hours; 3 hours; 4 hours; 5 hours; 6 hours; 8 hours; 10-hours and 12-hour samples. Cyclosporine levels were determined using a CYA kit (Abbott TDx). On day 15 the patients were switched from Neoral capsules to Equoral capsules (lot 5T111014) at an equivalent dosage (mg/mg). The second sparse sampling PK was performed on day 21 and a 12-hour PK was performed on day 28. On the morning of day 29 patients were switched from Equoral capsules to Neoral capsules at an equivalent dosage (mg/mg). Additional concentrations were measured on days -7, 18, and 35. Safety parameters were monitored at each visit. The pharmacokinetics of both formulations were equivalent. The mean AUC for Neoral and Equoral was 2856 and 2892, respectively. The ratios of LSM and the 90% confidence intervals for the in-transformed parameters (AUC o-t, AUC inf, and Cmax) of Equoral and Neoral SGC were 98% and 95%, respectively, suggesting that Equoral and Neoral SGC are bioequivalent.

Spectrum of renal abnormalities in acquired immune-deficiency syndrome.

Data from 27 patients with acquired immunedeficiency syndrome (AIDS) and AIDS-related complex (ARC) managed at the University of California, Irvine Medical Center since 1982 were extracted from medical records. Functional renal insufficiency occurred with considerable frequency among the AIDS patients. In contrast renal function was stable in the ARC patients studied. The majority of the AIDS patients exhibited persistent or transient proteinuria. Hematuria, leukocyturia, bacteruria, and nonvenereal urinary tract infections were seen with considerable frequency among AIDS patients and much less frequently among ARC patients. Similarly various disorders of fluids, electrolytes, acid base and abnormal phosphorus and calcium levels were common among AIDS patients and uncommon among ARC patients. These observations point to the prevalence and significance of the renal and associated abnormalities in acquired immune-deficiency syndrome.

Cardiac pathology in patients with end-stage renal disease maintained on hemodialysis.

We report the spectrum of cardiovascular pathology found on autopsy examination in 106 consecutive patients with end-stage renal disease. Cardiovascular pathology was present in nearly all patients, and was the most frequent cause of death (36%), with acute myocardial infarction accounting for 15%. Particularly frequent pathologic findings were left ventricular hypertrophy, coronary and aortic atherosclerosis, pericarditis with effusion, myocardial fibrosis, and valvular dilatation. Cardiovascular death rate was higher during the first year than after the fifth year of dialysis. Nearly all patients had a history of hypertension. The nature of the underlying cause of renal failure and pre-existing cardiovascular disease, specifically diabetes mellitus and hypertension, were the principal predictors of cardiovascular mortality rather than maintenance hemodialysis therapy per se.