Toxicology

Cyclosporine is a cyclic undecapeptide of fungal origin and a potent immunosuppressant.

Cyclosporine is used as a primary agent during immunosuppressive therapy for solid organ transplants.

Immunosuppression is thought to be the result of impairment of T-cell receptor transcription of the IL-2 gene.

Cyclosporine therapy has greatly improved organ transplant survival of heart, liver, and kidney transplants.

Cyclosporine may be administered by IV or orally.

Absorption from the gastrointestinal tract is variable, unpredictable, and incomplete.

Bioavailability increases during treatment so oral doses must be gradually reduced in order to maintain a constant cyclosporine concentration in the blood.

Assessment of cyclosporine concentrations in blood aids in adjusting patients’ dosage and avoids cyclosporine underdosage inefficacy or overdosage toxicity.

Cyclosporine is eliminated almost completely by hepatic metabolism.

Cytochrome P-450 enzymes are responsible for the biotransformation of cyclosporine and its metabolites.

More than thirty metabolites have been identified.

Preliminary data indicate cyclosporine metabolites are less immunosuppressive and less toxic than their parent compound.

Many drugs affect cyclosporine blood concentrations. These drugs alter cyclosporine blood concentration by inducing drug metabolism, interfering with drug metabolism, or affecting drug absorption.

Interactions between cyclosporine and danazol, diltiazem, erythromycin, fluconazole, itraconazole, ketoconazole, metoclopramide, nicardipine, verapamil, carbamazepine, phenobarbital, phenytoin, rifampicin, and cotrimoxazole are well documented.

The use of cyclosporine is associated with serious toxic side effects, primarily nephrotoxicity and hepatotoxicity.

Other adverse effects include diarrhea, gum hyperplasia, nausea, vomiting, hirsutism, tremor, and hypertension.

Monitoring cyclosporine concentrations reduces the incidence of biopsy proven acute rejection.

The ARCHITECT Cyclosporine assay is a two-step immunoassay for the quantitative determination of cyclosporine in human whole blood using CMIA technology with flexible assay protocols, referred to as Chemiflex.

The whole blood sample is lysed with solubilization reagent, extracted with precipitation reagent, and centrifuged; the supernatant is decanted into a Transplant Pretreatment Tube.

Sample, assay diluent, and anti-cyclosporine coated paramagnetic microparticles are combined; the cyclosporine present in the sample binds to the anti-cyclosporine coated microparticles.

After washing, cyclosporine acridinium-labeled conjugate is added to create a reaction mixture.

Following another wash cycle, Pre-Trigger and Trigger Solutions are added to the reaction mixture.

The resulting chemiluminescent reaction is measured as relative light units (RLUs).

1. Sample, assay diluent, and anti-cyclosporine coated paramagnetic microparticles are combined. The cyclosporine present in the sample binds to the anti-cyclosporine coated microparticles.
2. After washing, cyclosporine acridinium-labeled conjugate is added to create a reaction mixture.
3. Following another wash cycle, Pre-Trigger and Trigger Solutions are added to the reaction mixture.
4. The resulting chemiluminescent reaction is measured as relative light units (RLUs). There is an indirect relationship between the amount of cyclosporine in the sample and the RLUs detected by the ARCHITECT iSystem optics.

There is an indirect relationship between the amount of cyclosporine in the sample and the RLUs detected by the ARCHITECT iSystem optics.

To prevent reagent evaporation, contamination, and ensure reagent integrity. To avoid contamination, wear clean gloves when placing a septum on an uncapped reagent bottle. Do not invert the bottle as this will result in reagent leakage and may compromise assay results.

Tacrolimus is an immunosuppressive drug discovered in 1984 by the Fujisawa Pharmaceutical Co., Ltd.

Tacrolimus has been shown to be effective for the treatment of rejection following transplantation.

Clinical trials with liver and kidney have been published. Clinical studies are continuing for a variety of indications.

Tacrolimus binds to a family of proteins termed FK506 (tacrolimus) binding proteins (FKBPs).

It forms a large pentameric complex of FKBP, tacrolimus, calmodulin, and calcineurins A and B results in the inhibition of the phosphatase activity of calcineurin.

The phosphatase activity of calcineurin

It blocks the action of transcription factors requiring dephosphorylation for transport to cell nucleus, leading to T-cell proliferation and function blockage.

Tacrolimus may be administered IV or orally.

Absorption from the gastrointestinal tract is variable and irregular.

They show large inter- and intra-individual differences in its kinetics in organ transplant patients.

Whole blood rather than plasma may serve as the more appropriate medium to describe the pharmacokinetic characteristics of tacrolimus.

Tacrolimus is bound to albumin and alpha-1-acid glycoprotein.

Erythrocytes.

Hematocrit, temperature of plasma separation, drug concentration, and plasma protein concentration.

The ratio ranged from 12 to 67 with a mean of 35.

Tacrolimus is extensively metabolized in the liver and small intestine microsomes utilizing the cytochrome P-450 enzymes.

Nine different metabolites of tacrolimus have been identified; several of the metabolites have been found and tested in whole blood.

Nephrotoxicity is the primary side effect. At the present time it is not clear whether the nephrotoxicity of tacrolimus is the result of parent drug, metabolites, or a combination of both.

Neurotoxicity, hypertension, insomnia, and nausea.

A delayed one-step immunoassay for the quantitative determination of tacrolimus in human whole blood using CMIA (chemiluminescent microparticle immunoassay) technology with flexible assay protocols, referred to as Chemiflex.).

Whole blood is extracted with a precipitation reagent and centrifuged; the supernatant is decanted into a Transplant Pretreatment Tube.

Sample, assay diluent, and anti-tacrolimus coated paramagnetic microparticles are combined to create a reaction mixture. Tacrolimus present in the sample binds to the anti-tacrolimus coated microparticles.

Tacrolimus acridinium-labeled conjugate is added to the reaction mixture.

The tacrolimus on the acridinium-labeled conjugate competes for the available binding sites on the microparticles.

Following incubation, the microparticles are washed, and pre-trigger and trigger solutions are added to the reaction mixture.

Chemiluminescent reaction is measured as relative light units (RLUs).

Sample, assay diluent, and anti-tacrolimus coated paramagnetic microparticles are combined to create a reaction mixture. Tacrolimus present in the sample binds to the anti-tacrolimus coated microparticles. After a delay, tacrolimus acridinium-labeled conjugate is added to the reaction mixture. The tacrolimus on the acridinium-labeled conjugate competes for the available binding sites on the microparticles. Following incubation, the microparticles are washed, and pre-trigger and trigger solutions are added to the reaction mixture. The resulting chemiluminescent reaction is measured as relative light units (RLUs).

An indirect relationship exists between tacrolimus concentration and RLUs detected by the ARCHITECT optics.

Sirolimus is also known as rapamycin or Rapamune.

Sirolimus, an immunosuppressant, inhibits cytokine-stimulated T-cell proliferation. Sirolimus initially binds to FK-binding protein-12 (FKBP-12). This sirolimus-FKBP-12 complex in turn binds to a specific cell cycle regulatory protein, the mammalian target of rapamycin (mTOR) kinase, thereby inhibiting mTOR action.

mTOR is a mammalian target of rapamycin kinase involved in cell cycle progression.

It prevents progression from G1 to S phase in T-cells, stopping T-cell proliferation.

Sirolimus inhibits mTOR, a different mechanism of action than that of calcineurin-inhibiting agents such as cyclosporine. A fact that may account for the synergistic effect of sirolimus/cyclosporine combined therapy following renal transplant.

Sirolimus is currently recommended for use in conjunction with cyclosporine (and corticosteroids) to reduce or prevent graft rejection by the host.

Increased serum levels of cholesterol, triglycerides, creatinine, and decreased glomerular filtration rate.

Hypertension, rash, anemia, arthralgia, diarrhea, hypokalemia, leucopenia, and thrombocytopenia.

Sirolimus bioavailability and clearance are dependent on intestinal and hepatic metabolism by cytochrome P-450 (CYP) 3A4 enzyme and on countertransport by the multidrug efflux pump p-glycoprotein in the intestine.

Pharmacokinetic studies reveal an approximate 4–5 fold range in interindividual behavior.

Pharmacokinetics studies reveal a correlation between trough blood concentrations and both efficacy and toxicity.

The pharmacokinetics are altered during drug co-administration. For example, when sirolimus is administered concomitantly with the microemulsion formulation of cyclosporine rather than administered 4 hours apart, sirolimus trough levels increase.

Diltiazem and ketoconazole increase it, while rifampin decreases it.

A therapeutic range has not been establish. Trough levels between 4-20 ng/mL are recommended to maximize efficacy and minimize adverse effects. Results are reported to one decimal.

A delayed one-step immunoassay for the quantitative determination of sirolimus in human whole blood using CMIA (chemiluminescent microparticle immunoassay) technology with flexible assay protocols, referred to as Chemiflex.

Whole blood is extracted with a precipitation reagent, heated, centrifuged, and the supernatant is decanted into a Transplant Pretreatment Tube.

Sample, assay diluent, and anti-sirolimus coated paramagnetic microparticles are combined to create a reaction mixture. Sirolimus present in the sample binds to the anti-sirolimus coated microparticles.

After a delay, sirolimus acridinium-labeled conjugate is added to the reaction mixture.

The sirolimus on the acridinium-labeled conjugate competes for the available binding sites on the microparticles.

Following incubation, the microparticles are washed and pre-trigger and trigger solutions are added to the reaction mixture.

By chemiluminescent reaction as relative light units (RLUs).

An indirect relationship exists between sirolimus concentration and RLUs detected by the ARCHITECT iSystem optics.

Human prolactin (hPRL) is a single chain polypeptide of 199 amino acids with a molecular weight of approximately 23,000 daltons. Its existence as a distinct chemical entity, separate from growth hormone, was established through a series of studies between 1965 and 1971.

Prolactin is produced by the anterior pituitary.

It’s secretion is regulated physiologically by inhibitory and releasing factors of the hypothalamus.

Prolactin appears in blood promptly after administration of thyrotropin-releasing hormone (TRH).

Initiation and maintenance of lactation in women.

Infertility and gonadal disorders in men and women.

It inhibits secretion of ovarian steroids and interferes with follicle maturation and LH/FSH secretion in the human female.

Measurement of elevated serum prolactin levels may provide the first quantitative evidence of pituitary dysfunction.

Quantitation of prolactin levels is also of interest in the evaluation and management of patients with amenorrhea and galactorrhea.

Pregnancy, breast stimulation, stress, coitus, administration of estrogens, progesterone, androgens, some psychotropic and antihypertensive drugs, and TRH.

Administration of L-dopa and bromocriptine.

To aid in diagnosis of male and female infertility and pituitary dysfunction, monitoring of male and female gonadal disorders, and management of amenorrhea, and galactorrhea.

A two-step CMIA to determine prolactin in human serum and plasma using using CMIA technology with flexible assay protocols, referred to as Chemiflex.

Sample and anti-prolactin coated paramagnetic microparticles are combined; prolactin binds to the anti-prolactin coated microparticles.

Anti-prolactin acridinium-labeled conjugate is added to create a reaction mixture. Following another wash cycle, Pre-Trigger and Trigger Solutions are added to the reaction mixture.

By chemiluminescent reaction measured as relative light units (RLUs).

There is a direct relationship between the amount of prolactin in the sample and the RLUs detected by the ARCHITECT iSystem.

Everolimus is a macrolide immunosuppressant derived by chemical modification of the natural product rapamycin.

Rapamycin is produced by certain strains of Streptomyces hygroscopicus.

Immunosuppressive treatment strategies are aimed at prevention of T cell activation and/or proliferation. Everolimus is classified as a mechanistic target of rapamycin inhibitor. On a cellular level everolimus inhibits, in general, growth factor-stimulated cell proliferation irrespective of the cell lineage or growth factor involved.

Inhibition is reversible since everolimus is not a cytotoxic compound.

Everolimus inhibits the T cell response to growth factors arresting clonal expansion of activated T cells by inhibiting G1 to S phase.

To aid in patient management during clinical use of everolimus.

The preferred matrix is whole blood because, at therapeutic concentrations the compound is predominately partitioned into erythrocytes.

Liquid chromatography coupled to mass spectrometry (LC-MS/MS) has been used to measure the concentration of everolimus in blood.

A homogeneous particle-enhanced turbidimetric immunoassay.

The assay is based on competition between drug in the sample and drug coated onto a microparticle for antibody binding sites of the everolimus antibody reagent.

The everolimus coated microparticle reagent is rapidly agglutinated in the presence of the anti-everolimus antibody reagent and in the absence of any competing drug in the sample.

By rate of absorbance change measured photometrically.

When a sample containing everolimus is added, the agglutination reaction is partially inhibited, slowing down the rate of absorbance change.

A concentration-dependent classic agglutination inhibition curve can be obtained with the maximum rate of agglutination at the lowest everolimus concentration and the lowest agglutination rate at the highest everolimus concentration.

The sweat glands of healthy people produce isotonic sweat that is made hypotonic in the sweat duct by active electrolyte re-absorption.

Patients with cystic fibrosis have a defect in a transport mechanism, resulting in increased levels of sodium or chloride concentrations in sweat.

The definitive test for cystic fibrosis is the sweat test.

Low total protein, prolongation of prothrombin time, and abnormalities related to evolving hepatic cirrhosis.

The ChloroChek Chloridometer operates according to the principle of coulometric titration.

An accurately measured sample volume (10 μL) is added to an acid buffer.

The system automatically titrates chloride ions by passing a known constant current between two silver electrodes, which provides a constant generation of silver chloride, which is held in suspension by the colloid stabilizer. By adding a chloride sample, the free silver ions form a non-soluble silver chloride precipitate together with the free chloride ions in the sample.

Ag⁺ + Cl⁻ → AgCl. The number of silver ions introduced into the sample combines with one unit measurement of chloride.

A non-soluble silver chloride precipitate.

When all the chloride has been precipitated as silver chloride, free silver ions begin to appear and the solution conductivity changes.

This change is detected by the sensing electrodes.

Once the change is detected by the sensing electrode, the readout stops, displaying results directly in mg of chloride per liter (mg/mL) or milligrams % salt.

Yes, another sample may be added to the same acid buffer and the cycle repeated.

Sample volume requirement is not an analytical concern but a physiological one. Measurement on samples from less than maximally stimulated sweat glands may lead to false-negative results.

Remove them carefully with a lint-free tissue without drawing solution from the pipette tip; if necessary, use a new pipette tip.

The chloride concentration in the sample is less than 10 mmol/L.

If the chlorochek fails to automatically recognize the sample, then the chloride concentration in the sample is less than 10 mmol/L. Verify this by adding 10uL of the 100 mmol/L NaCl/H2O standard solution. The reading will likely be above 100 mmol/L. Subtract 100 mmol/L from this final reading for the approximate final result of the sample. Record the values as <10 mmol/L.

Fluoride, bromine, and iodide.

They can falsely elevate the sweat chloride result.

Skin covered by rashes or lesions.

Adrenal insufficiency, ectodermal dysplasia, hereditary nephrogenic diabetes insipidus, hypothyroidism, malnutrition, fucosidosis, Addison’s disease, glucose-6-phosphatase deficiency, mucopolysaccharidosis, and hypoproteinemia.

Edema and hypoproteinemia.

Borderline tests must be repeated.

Yes, even negative tests should be repeated if the clinical picture suggests cystic fibrosis.

One to two percent of cystic fibrosis patients do not have diagnostic sweat chloride patterns.