Mariadelfina Molinaro

Clinically Significant Drug Interactions with Cyclosporin An Update

SummarySince its approval in 1983 for immunosuppressive therapy in patients undergoing organ and bone marrow transplants, cyclosporin has had a major impact on organ transplantation. It has significantly improved 1-year and 2-year graft survival rates, and decreased morbidity in kidney, liver, heart, heart-lung and pancreas transplantation. Several studies have supported the efficacy of cyclosporin in preventing graft-versus-host disease in bone marrow transplantation. Cyclosporin is also possibly effective in treating diseases of autoimmune origin and as an antineoplastic agent.The introduction of therapeutic drug monitoring of cyclosporin was extremely useful because of the wide inter- and intraindividual variability in the pharmacokinetics of cyclosporin after oral or intravenous administration. Optimal long term use of cyclosporin requires careful monitoring of the blood (or plasma) concentrations.Sustained and clinically significant drug-drug interactions can occur during long term therapy with cyclosporin. The coadministration of multiple drugs with cyclosporin could result in graft rejection, renal dysfunction or other undesirable effects. Any interaction that leads to modified cyclosporin concentrations is of potential clinical importance.Cyclosporin itself may have significant effects on the pharmacokinetics and/or pharmacodynamics of coadministered drugs, such as digoxin, HMG-CoA reductase inhibitors and antineoplastic drugs affected by multidrug resistance.Many drugs have been shown to affect the pharmacokinetics and/or pharmacodynamics of cyclosporin. Interactions between cyclosporin and danazol, diltiazem, erythromycin, fluconazole, itraconazole, ketoconazole, metoclopramide, nicardipine, verapamil, carbamazepine, phenobarbital (phenobarbitone), phenytoin, rifampicin (rifampin) and cotrimoxazole (trimethoprim/sulfamethoxazole) are well documented in a large number of patients. Other interactions (such as those with aciclovir, estradiol and imipenem) are documented only in isolated case studies.

Clinical pharmacokinetics of tacrolimus in heart transplant recipients

We report pharmacokinetic data on tacrolimus in 14 heart transplant patients (2 women, 12 men). The median age and the median body weight were 55.5 years (range, 23-61 years) and 67.0 kg (55-79 kg), respectively. All patients were maintained on a triple-drug protocol (tacrolimus, azathioprine, and prednisone), with a 7-day antithymocyte globuline induction. The first tacrolimus dose, administered orally 1 to 5 days posttransplant, ranged from 0.03 to 0.4 mg/kg (median = 0.052 mg/kg). The maintenance dose ranged from 0.03 to 0.13 mg/kg/day (administered in two equal doses) to achieve blood levels of 5 of 20 ng/ml, as determined by a microparticle enzyme immunoassay (MEIA). Whole blood samples were drawn just before, at 0.5 hour, and at 1, 2, 3, 4, 6, 8, 10, and 12 hours after the administration of the first dose; trough levels were measured thereafter. The mean oral clearance (CL/F) and apparent volume of distribution (Vd/F) averaged 0.21+/-0.08 L/hour/kg and 2.4+/-0.8 L/kg while the half-life averaged 8.7+/-3.5 hours. Tacrolimus accumulation index during chronic therapy (Rac = Cmin(steady state)/Cmin(first dose) normalized to the same dose) averaged 1.3. Eighty-eight percent of the trough blood levels measured in our patients were within 5 and 20 ng/ml. The incidence of rejection in the study population was extremely low: a prevalence of grade 2 rejection or more, of 0.4+/-0.8 episodes/ patient was observed after a follow-up period of 8.8+/-2.2 months. Only one patient experienced severe renal toxicity, probably because of his preoperative precarious hemodynamic status. Pharmacokinetic data suggest that maintenance tacrolimus daily dose should be equal to 0.1 mg/kg/day to obtain trough blood concentrations of approximately 10 ng/ml. Inter- and intra-patient variability of tacrolimus blood concentration should be expected and justify careful monitoring.

High-performance liquid chromatography determination of pravastatin in plasma

Pravastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor that reduces plasma cholesterol levels. Some analytical methods have been described for determination of pravastatin levels in biological fluids, but these methods are rather cumbersome and involve expensive specialized equipment, usually not available in a clinical setting. A new technique, re-versed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection capability, has been developed for the analytical determination of pravastatin in plasma. Extraction and sample clean-up procedures are simple and rapid to execute, yet yield chromatograms virtually free of interference from endogenous plasma constituents and other antihypercholester-olemic agents or drugs usually taken concomitantly with pravastatin. Our detection limit for pravastatin was 2 ng/ml. Standard curves were linear between 5 and 200 ng/ml, with a coefficient of variation (CV) of <10% at the limits of quantitation. This method was used to study pravastatin plasma levels in two hypercholesterolemic heart-transplant recipients and two hypercholesterolemic nontransplanted patients. We conclude that the method reported here would be ideal for therapeutic pravastatin monitoring in patients.

Sirolimus Prevents Short-Term Renal Changes Induced by Ischemia-Reperfusion Injury in Rats

Background: Ischemia-reperfusion (I/R) is present at various degrees in kidney transplants. I/R plays a major role in early function and long-term survival of renal allograft. The purpose of our study was to determine if immunosuppressants modulate I/R in a model that separates I/R from all immune responses. Methods: Sprague-Dawley rats with monolateral renal I/R received daily cyclosporine (A), tacrolimus (B), sirolimus (C) or saline (D). Sham-operated rats received saline (E). After 30 days, glomerular filtration rate for each kidney was measured by inulin clearance. Kidney injury was examined, and TGF-β, fibronectin and metalloproteases were evaluated by real-time PCR, Western blot and zymography. Results: Sirolimus, but not cyclosporine and tacrolimus, prevented a glomerular filtration rate decrease in I/R kidneys (403 ± 303 vs. 1,006 ± 484 µl/min, p < 0.05; 126 ± 170 vs. 567 ± 374 µl/min, p < 0.05; 633 ± 293 vs. 786 ± 255; A, B and C group, respectively, I/R vs. contralateral kidneys). Sirolimus reduced ED-1+ cell infiltrate, interstitial fibrosis and intimal thickening of small vessels observed in I/R kidneys of controls and calcineurin inhibitor-treated rats. Tacrolimus and cyclosporine increased fibronectin and TGF-β expression and matrix deposition. Only sirolimus increased metalloprotease activity. Conclusions: Sirolimus but not calcineurin inhibitors prevented I/R-induced kidney injury.

Falsely Elevated Whole Blood Cyclosporine Concentrations Measured by an Immunoassay With Automated Pretreatment

To the Editor: Therapeutic drug monitoring of cyclosporine A (CyA) blood concentrations is needed to ensure therapeutic efficacy and to prevent toxicity, because it exhibits marked pharmacokinetic variability and, in the case of high concentrations, a dose reduction is required. An appropriate assay technique must be adopted for therapeutic drug monitoring to provide valid results. Automated systems based on immunologic principles are widely used for the routine monitoring of immunosuppressive drugs. Recent trials indicate that some endogenous antibodies interfere with the immunoassay of many molecules. Circulating antibodies can also be found in patients treated with immunotherapy, either as single infusions or through blood transfusions, or in those treated with monoclonal gammopathy. Recently we analyzed samples from a pediatric patient (23 months old, 8.8 kg) with familial hemophagocytic lymphohistiocytosis (FHLH) with a homozygous 284–285 mutation in the perforin gene; both parents were heterozygotic for this molecular defect. The patient underwent allogeneic hematopoietic stem cell transplantation (HSCT) from cord blood obtained from an unrelated donor. In line with the international prospective study HLH2004, the patient received oral CyA before transplant as maintenance treatment for his primary pathology and then as prevention of graft-versus-host disease (GVHD), he was switched to CyA (infusions 2.5 mg/kg per day) from Day –3 to Day +36 during hospitalization and switched back to oral administration (5 mg/kg per day) thereafter. The patient also received methylprednisolone (2 mg/kg per day) as infusion therapy from Day 0 to Day +24, successively tapered to oral prednisone administration (2.5 mg/day). As antiinfective prophylaxis, ceftazidime, fluconazole, and acyclovir were also prescribed together with trimethoprim and sulphamethoxazole against Pneumocystiis jiroveci pneumonia. The patient received nonspecific immunoglobulins twice a month up to 100 days after transplantation. During this period the patient always tested negative for reactivation of cytomegalovirus, EpsteinBarr Virus, and adenovirus as well as detection of Aspergillus galactomannan in serum. Before and after transplantation, a complete serologic panel for markers of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) was performed and no infection was detected. CyA blood concentrations were tested routinely by an affinity columnmediated immunoassay (ACMIA) using a Dimension Xpand Plus analyzer (Siemens Healthcare Diagnostics, Deerfield, IL) and values remained within the target range (80–200 ng/mL) from Day –3 to Day +58 after transplantation (Fig. 1). Two months after transplantation, a trend for increasing CyA blood concentrations was observed, which was confirmed by high values even after treatment discontinuation (Day +58; Fig. 1) until therapy was restarted orally at 5 mg/ kg per day dose on Day +68. To check whether the endogenous antibodies were reactive against the beta-galactosidase conjugate antibody used in the ACMIA assay, from Day +65 blood samples were analyzed for CyA concentration both by ACMIA and the enzyme multiplied immunoassay technique (EMIT) on the Viva analyzer (Siemens Healthcare Diagnostics). Using the ACMIA method, whole blood samples are mixed and lysed automatically with the conjugate that binds CyA molecules available in samples, whereas the EMIT assay requires a preanalytical step to guarantee appropriate lysis of the erythrocytes and precipitation of plasma proteins. The CyA concentrations obtained by ACMIA confirmed the increasing trend (from Day +65 to +71), whereas the corresponding values obtained by EMIT fell below its limit of quantification (40 ng/mL). Moreover, to understand and explain this discrepancy, the samples in which a sufficient surplus blood volume was still available were tested also for tacrolimus concentration by ACMIA, which uses the same betagalactosidase conjugate. However, no tacrolimus was detected; thus, the betagalactosidase conjugate could not have been a possible source of interference. Therefore, we inferred that the high blood CyA concentrations observed with ACMIA might be the result of an endogenous antibody to CyA. To assess this hypothesis, five of the blood samples were purified to remove IgG and analyzed again with ACMIA. CyA concentrations were found to fall below the limit of quantification (25 ng/mL). Briefly, aliquots (0.5 mL) of blood samples drawn on Days +65, +66, +68, +71, and +74 after transplantation were centrifuged at 6000 rpm for 10 minutes and IgG was purified by chromatography on a Protein-A-Sepharose column (GE Healthcare, Milan, Italy) according to the manufacturer’s recommendations. Protein-A-Sepharose chromatography is a type of affinity chromatography that relies on the specific interaction of protein A with the Fc region of immunoglobulins from a number of species. Protein A, a polypeptide originally isolated from the cell walls of Staphylococcus aureus, contains four binding sites, but only two Fc domains can be bound at any one time. Because the antibody combining site is left free, protein A, when covalently coupled to stationary supports such as agarose or Sepharose beads, provides an excellent reagent for isolating immunoglobulins from a crude solution. The collected materials not bound to the affinity column of each blood sample were kept at –20(C. In view of this discussion, all the results of this study are consistent with the production of anti-CyA antibodies as shown by the high CyA concentrations measured by ACMIA that persisted even after discontinuation of CyA therapy.

Considerable lack of agreement between S-FPIA and EMIT cyclosporine assay in therapeutic drug monitoring of heart transplant recipients

The authors performed a comparative analysis of 60 whole blood samples containing cyclosporine (CsA) from heart transplant (HTx) recipients (n = 60) by the two “specific” monoclonal immunoassays, enzyme-multiplied immunoassay technique (EMIT) and fluorescence polarization immunoassay (S-FPIA), using the Altman-Bland approach based on graphical techniques and simple calculations. The CsA blood concentrations measured by S-FPIA [mean (SD): 268.1 (108.8) ng/mL] showed a statistically significant difference (P < 0.001) from the corresponding concentrations measured by EMIT [219.6 (118.7) ng/mL]. The CsA concentrations were 27% (median) higher when determined by monoclonal S-FPIA than by EMIT. The comparison between EMIT and S-FPIA showed a good correlation (S-FPIA conc.(ng/mL) = EMIT conc.(ng/mL) · 0.88 + 76.1, r = 0.96, P < 0.001). However, a high correlation does not mean that the two methods agree, and their use as interchangeable might be misleading. The authors summarized the degree of agreement by calculating the bias estimated by the mean difference (d) and the standard deviation of the difference (SD). For CsA concentration data, the mean difference (S-FPIA minus EMIT) is +49.9 ng/mL and SD is 31.2 ng/mL. Altman-Bland analysis indicates considerable lack of agreement between EMIT and S-FPIA, with discrepancies of more than 100 ng/mL. The present studys data clearly show that there is a considerable and clinically unacceptable lack of agreement between the S-FPIA and the EMIT techniques in HTx recipients for the whole range of concentrations evaluated (25–500 ng/mL), and this is caused by the variation in the overestimation of the CsA parent compound. Even though a similar CsA reference range was reported during maintenance therapy for both methods (150–250 ng/mL), which might encourage their interchangeability in the clinical setting, this approach should be avoided. Laboratory reports should always state both the concentration of CsA and the analytical method.

Efficacy of intraventricular amikacin treatment in pan-resistant Pseudomonas aeruginosa postsurgical meningitis

Background We describe a case of pan-resistant Pseudomonas aeruginosa postsurgical meningitis associated with the presence of an external ventricular device. We changed therapy twice; finally, by using amikacin and a continuous infusion of cefepime, we obtained clinical improvement. Case presentation A female patient, who underwent surgery for a cavernous angioma, presented with meningitis. Cerebrospinal fluid culture revealed a multidrug-resistant Pseudomonas aeruginosa, initially sensitive only to colistin. We successfully used intrathecal amikacin and intravenous cefepime continuous infusion plus intravenous amikacin after two previous ineffective therapeutic approaches. Conclusion The evaluation of the antibiotic concentration and the bactericidal activity in cerebrospinal fluid may contribute to the choice of the drug in cases of multidrug-resistant meningitis.

Evaluation of two buflomedil tablet formulations in patients with atherosclerotic disease

The bioequivalence of a 600‐mg methocel tablet containing buflomedil hydrochloride in sustained‐release form was determined relative to a 300‐mg CAP/carbovax‐coated tablet of buflomedil hydrochloride in immediate‐release form. The tablets were given to 20 patients in a double‐blind placebo‐controlled clinical study with cross‐over between the administration plans. The 300‐mg tablets were given b.i.d., at 8 a.m. and 8 p.m. while the 600‐mg tablets were taken once a day at 8 a.m. (+placebo at 8 p.m.). Plasma samples were collected at appropriate times up to 24 h after administration and were analysed for buflomedil with a validated high‐performance liquid chromatographic procedure. Results showed an overall significant mean difference in absorption rate between the two formulations. The mean tmax (5‐5 Ω 3–5 h) for the 600‐mg tablet was longer (P>0–001) than the tmax value (1–8 ± 0–8 h) seen after administration of the first 300‐mg tablet. Analysis of AUC(0‐∞) values indicated that the sustained‐release preparation (32.1 ± 20‐7 μg/ml h) was not significantly different from the 300‐mg tablet b.i.d. (28‐7 ± 16‐0 μg/ml h).