Clinical pharmacokinetics in heart failure. An updated review.

Pharmacokinetics is the study of the effect that the body has on drug absorption, distribution, metabolism and excretion. The pharmacokinetics of a specific drug are assessed by the volume of distribution, bioavailability, clearance and elimination half-life. Elimination half-life is directly related to the volume of distribution and inversely related to clearance. Any 1 or more of these parameters may be altered by physiological changes such as ageing, or disease states such as congestive heart failure. Congestive heart failure is associated with hypoperfusion to various organs including the sites of drug clearance, i.e. the liver and kidneys. It also leads to organ congestion as seen in the liver and gut. The main changes in drug pharmacokinetics seen in congestive heart failure are a reduction in the volume of distribution and impairment of clearance. The change in elimination half-life consequently depends on whether both clearance and the apparent volume of distribution change, and the extent of that change. Pharmacokinetic changes are not always predictable in congestive heart failure, but it seems that the net effect of reduction in the volume of distribution and impairment of clearance is that plasma concentrations of drugs are usually higher in patients with congestive heart failure than in healthy subjects. The changes in pharmacokinetics assume importance only in the case of drugs with a narrow therapeutic ratio (e.g. digoxin) and some of the antiarrhythmics such as lignocaine (lidocaine), procainamide and disopyramide. This necessitates reduction in both the loading and maintenance doses. Prolongation of the elimination half-life leads to delay in reaching steady-state, and therefore dose increments must be made more gradually. Plasma concentration measurements of the drugs concerned are a good guide to therapy and help to avoid toxicity. Pharmacokinetic changes are of less importance in the case of drugs with immediate clinical response, e.g. diuretics and intravenous vasodilators such as nitrates and phosphodiesterase inhibitors. The dose in the latter group can be titrated to the desired effect. Not all adverse reactions to drugs that may occur in heart failure are the result of alterations in pharmacokinetics; rather, some may be due to important drug interactions. An interaction may occur directly e.g. reduction of renal clearance of digoxin by captopril and quinidine; or indirectly, e.g. through diuretic-induced hypokalaemia, which exacerbate arrhythmias associated with digoxin and antiarrhythmics such as quinidine and procainamide.

Serum concentrations of tumour necrosis factor-alpha during chemotherapy-induced leukopenia in patients with acute leukaemia and bacterial infections.

Serum concentrations of tumour necrosis factor-alpha (TNF-alpha) increase during septicaemia in previously healthy individuals. To investigate whether a similar increase in TNF-alpha can be seen in severely immunocompromised patients with acute leukaemia and chemotherapy-induced leukopenia, serum TNF-alpha was analysed in leukopenic patients with bacterial infections. Pretherapy serum levels of TNF-alpha were decreased in leukaemia patients compared with healthy controls, and serum TNF-alpha levels showed a further decrease when patients developed chemotherapy-induced leukopenia. When leukopenic patients developed bacterial infections, serum concentrations of TNF-alpha increased. Serum levels of TNF-alpha decreased when clinical signs of infection resolved during antibiotic therapy, but an increase occurred later in parallel with haematopoietic reconstitution.

Chronic cold agglutinin disease of the "idiopathic" type is a premalignant or low-grade malignant lymphoproliferative disease.

We investigated the clinical, pathological, and immunological features of "idiopathic" cold agglutinin disease (CAD) in a population-based study. Fourteen patients were studied, giving a prevalence of about 14 per million with a mean age of 75 years. Haemolysis was present in all cases, but only eight patients had clinical symptoms of peripheral haemagglutination. Serum electrophoresis, immunofixation, morphological bone marrow evaluation, and flow cytometric immunophenotyping were used to detect any monoclonal lymphoproliferative disorder. Flow cytometry seemed to be a sensitive way to demonstrate a clonal B-cell proliferation. Some evidence of clonality was found in 13 patients, and a clonal lymphoproliferative disease was documented by flow cytometry or biopsy in 10 out of 11 patients. We conclude that CAD is a symptom-producing monoclonal lymphoproliferative disorder in nearly all patients.

Quantitative analysis of cytokeratin 20 gene expression using RT-PCR and capillary electrophoresis with fluorescent DNA detection.

OBJECTIVE: We developed a quantitative reverse-transcription polymerase chain reaction (RT-PCR) to determine CK20 expression in colorectal tumor and hematopoietic tissue. DESIGN AND METHODS: Our method incorporates a calibrated PCR with an internal competitor and an external standard. RESULTS: The RT-PCR assay is sensitive detecting 10 target molecules of CK20 in solution with one round of 38 amplification cycles. Genomic DNA contamination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high throughput of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow from 20 healthy volunteers revealed a low level of CK20 expression in all samples. CONCLUSION: To study the clinical significance of CK20 expression as a marker of systemic metastatic disease it is essential to measure CK20 mRNA levels in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described.

Fluorescence-based method for measuring and determining the mechanisms of recombination in quantitative PCR.

We quantitatively measured the amount of recombinant molecules formed during PCR when the break point cluster region (BCR) cDNA was coamplified with a homologous internal standard using Taq polymerase. The products were analysed under denaturing conditions using capillary electrophoresis followed by detection of the fluorescently labelled products and the recombinant molecules were differentiated by their size. Early termination of chain synthesis and reannealing of incomplete fragments, to each other as well as to BCR and internal standard, is one mechanism for generating recombinants during PCR since prolonging extension time reduced, but did not totally suppress recombinant molecule formation. Template switching by the extending chain is another mechanism since recombinant molecules could be detected even after only one round of primer extension. The latter mechanism is probably facilitated by increasing number of templates. Thus, the large increase of recombinant molecules formed in plateau phase is mediated by direct amplification of the recombinants and de novo synthesis by template switching. The effect of additives on recombination could be quantitatively measured and both betaine and DMSO were effective in suppressing recombination. Thus, prolonging extension time, reducing the number of amplification cycles and incorporating additives in the PCR reaction, reduced recombinant molecule formation.

Serial quantitative PCR analysis of bone marrow samples from breast cancer patients to monitor systemic micrometastases.

BACKGROUND: We have previously developed a quantitative calibrated PCR assay to measure cytokeratin 19 (CK19) expression in haematopoietic tissue in order to detect systemic micrometastases. PATIENTS AND METHODS: Serial measurements of CK19 expression in bone marrow of patients with primary breast cancer were performed at operation, at 3 weeks and 6 months postoperatively. RESULTS: Reference values for CK19 expression were established by analysing bone marrow samples from 48 healthy female volunteers or patients without epithelial cancer. Samples from breast cancer patients with CK19 values above the upper reference limit were considered positive. Bone marrow samples taken at operation were positive in 29 out of 141 patients (20.6%) and remained positive in 12, turned negative in 4 and were unavailable in 13 at 6 months postoperatively. CONCLUSION: Serial measurements increase the reliability of detecting micrometastases perioperatively. Further studies are in progress to evaluate the relationship between elevated CK19 values and clinical outcome.

Glycogen content and PAS staining pattern of human megakaryocytes.

The glycogen content of human megakaryocytes was studied using a quantitative method. Smears of bone marrow from 13 individuals were stained with the modified PAS reaction with and without prior treatment with alpha-amylase. The intensity of the reaction was determined by microspectrophotometry in 50 megakaryocytes from each individual. It was found that megakaryocytes are rich in glycogen which is not only confined to the intensely PAS-positive granules and inclusion bodies, but also makes up a good part of the diffuse cytoplasmic staining. In the diffusely stained megakaryocytes, glycogen makes up 32% of the intensity of the PAS reaction, while it reaches 47% in those with granules and up to 60% in those with inclusion bodies. The total extinction of the PAS-stained megakaryocytes is not only dependent on the morphological appearances of the cells, but is also positively correlated with their size.

[Secondary myelodysplastic syndrome]. / Sekundaert myelodysplastisk syndrom.

Secondary or therapy related myelodysplastic syndrome may develop following treatment with alkylating agents and radiotherapy. The condition may also follow high dose therapy for malignant lymphomas. We describe two patients who developed secondary myelodysplasia. The first is a 76-year old female with a low grade lymphoma who developed pancytopenia with monosomy 7. Secondary myelodysplasia was diagnosed 24 months after treatment with chlorambucil (total dose of 900 mg) and 12 months after treatment with trophosphamide (total dose of 24 g). The second patient was a 48-year old male who underwent autologous bone marrow transplantation for a relapsed low grade lymphoma. Following autografting he had persistent anemia and trombocytopenia. Secondary myelodysplastic syndrome with complex karyotype was diagnosed 54 months after high dose therapy. He died nine months later of bone marrow failure.

Cisplatin and 5-fluorouracil in advanced cancer of the penis.

A total of 8 patients with advanced squamous cell carcinoma of the penis (Jackson stages III and IV) received chemotherapy with 100 mg./m2. cisplatin intravenously on day 1 and a 24-hour infusion of 1,000 mg./m.2 5-fluorouracil on days 1 to 5. Of the patients 2 (25%) achieved a partial response: 1 required a further operation and 1 required surgery with radiotherapy to achieve a complete response. These 2 patients were disease-free at 32+ and 57+ months. Nonresponders had a survival range of 2+ to 28 months after chemotherapy. Nausea and vomiting were the most frequent side effects of chemotherapy. Chemotherapy-related increase in serum creatinine occurred in 3 patients. Two patients had septicemia and 1 complained of tinnitus. Poor tolerability especially in the elderly was the main reason for discontinuing chemotherapy. The combination of cisplatin and 5-fluorouracil may have a role in the management of advanced penile cancer together with surgery and radiotherapy.

[Diagnosis and follow-up in chronic myeloid leukemia. Detection and quantification of specific transcripts with the help of reverse transcriptase-polymerase chain reaction]. / Diagnostikk og oppfølging ved kronisk myelogen leukemi. Påvisning og kvantitering av spesifikke transkripter ved hjelp av revers transkriptase-polymerasekjedereaksjon.

The Philadelphia chromosome in cells of patients with chronic myeloid leukemia and acute lymphoblastic leukemia can be detected by reverse transcriptase-polymerase chain reaction (RT-PCR). We have tested two new methods for this purpose. For diagnostic purposes, three different BCR-ABL translocations (b3a2, b2a2 and ela2) can be detected in a multiprimed, one step PCR reaction. By using a competitor DNA construct and a two-step, nested PCR reaction, a quantitative measure of the number of specific BCR-ABL transcripts can be estimated. We tested five patients with chronic myeloid leukemia. All of them showed positive BCR-ABL translocations in the diagnostic test. Patients with other myeloproliferative disorders, used as controls, were all negative. Quantitative measurements of specific BCR-ABL mRNA showed that as few as ten transcripts could be quantified in the assay. The analysis showed that coefficients of variation between 15% and 30% were obtained for specific transcripts per micrograms RNA, whereas specific BCR-ABL per normal ABL showed a coefficient of variation of 10%. These new methods to detect BCR-ABL translocation by RT-PCR should provide easy and sensitive diagnosis, and possibilities of monitoring residual disease or relapse.

Clinical immunology of chronic cold agglutinin disease.

We studied clinical and immunological characteristics of 15 patients with chronic cold agglutinin disease (CAD). Mean age at disease debut was 68 years for female and 67 years for male patients. The patients had no signs of other autoimmune diseases. All patients had V(H)4-34 encoded IgM kappa cold agglutinins (CA) in high titre. In five patients IgM increased significantly with advancing disease. Seven patients had reduced concentrations of lymphocytes, largely of CD4 and CD8 T cells. Percentages of NK cells (CD56) and B cells (CD19) were increased in seven and three patients, respectively. In six out of nine patients a clonal expansion of kappa positive B cells was found. Serum C3 was decreased in nine patients and C4 was decreased in 11 patients, six of whom had reduced CH50. Such data indicate that patients with CAD experience a continuous low-grade complement consumption. Five patients had experienced increased haemolysis during infections. After addition of active complement to patient sera in vitro, six sera showed increased haemolytic activity. Our results indicate that some patients with CAD have a relative deficit of complement in their serum and that an increase of complement production occurs during an acute phase reaction which enhances haemolysis. Our data also indicate that both CA titre and thermal amplitude are important characteristics when predicting complement activation and clinical course in CAD.

Sensitive and quantitative one-step polymerase chain reaction using capillary electrophoresis and fluorescence detection for measuring cytokeratin 19 expression.

An improved quantitative assay to measure cytokeratin 19 (CK19) expression has been developed. The assay utilizes reverse transcription and a one-step polymerase chain reaction (PCR), with capillary electrophoresis and fluorescent labelling, to separate and detect the PCR products. Calibration curves were constructed from a serial dilution of CK19 cDNA coamplified with a fixed amount of CK19 internal standard, which was found to be linear between 10 and 500 molecules. Quantitative measurement of CK19 in samples was carried out by coamplifying the cDNA with a fixed amount of internal standard. The values were calculated from the calibration curve. The integrity of RNA and cDNA synthesis was checked by quantitative measurement of the breakpoint cluster region (BCR) gene expression. The assay is sensitive, detecting < 10 CK19 transcripts, and reproducible with a coefficient of variation of approximately 10%. CK19 expression showed overlapping values when measured in samples from peripheral blood and bone marrow in operable breast cancer patients, in healthy volunteers or patients without epithelial cancer and in blood samples from patients with metastatic breast cancer. As the assay is easier to perform than traditional quantitative competitive PCR assays, it might be useful for quantitative measurement of other specific transcripts.

[Chronic myeloid leukemia in health regions 1, 3, 4 and 5 during the period 1990-96]. / Kronisk myelogen leukemi i helseregion 1, 3, 4 og 5 i perioden 1990-96.

The aim of the present investigation was to obtain information about treatment, clinical course and outcome for all patients with chronic myeloid leukaemia through a six-year period in a defined part of Norway. A total number of 141 patients fulfilled the diagnostic criteria. This is equivalent to 0.9 patients per 100,000 per year. The median age was 62 years. More than 70% of the patients were primarily treated with hydroxyurea, either alone or combined with interferon. 40 out of 57 patients younger than 55 years underwent allogeneic stem cell transplantation. Median survival for all patients was 36 months with an estimated five-year survival rate of 33%. Patients older than 55 years had a median survival of 30 months with 16% alive after five years. The five-year survival rate for patients younger than 55 years was 56%, for transplanted patients 72%. 60 of 84 patients older than 55 years have died after 4 1/2 years median observation time. Two thirds of those died of leukaemia; one third of other causes. 23 of 57 patients younger than 55 years have died. 11 of them had had transplantations and most of them died from transplantation-related causes, while leukaemia was the dominating cause of death in the others.