Non-linear pharmacokinetics of high-dose intravenous verapamil

Aims In an attempt to reverse multidrug resistance, in a recent trial of verapamil in association with doxorubicin, we used escalating doses of continuous intravenous (i.v.) verapamil under close haemodynamic monitoring. We report the pharmacokinetics of escalating doses of verapamil. Methods We studied nine patients [ seven males, two females; median age 46 years (range, 31–57)] with advanced adenocarcinoma of the colon and normal renal, hepatic, and cardiac functions. After a loading dose (0.15 mg kg⁻¹ followed by 12 h continuous i.v. infusion at 0.20 mg kg⁻¹ h⁻¹ ), the infusion rate (ko) of verapamil was increased every 24 h (0.25, 0.30, 0.35, and 0.40 mg kg⁻¹ h⁻¹ ). The highest rate was maintained for 48 h. Doxorubicin was given as a continuous i.v. infusion from 12 to 108 h (n=4) or 60 to 108 h (n=5). Blood samples and urine collections were taken every 12 h. Verapamil and nor-verapamil were assayed by high performance liquid chromatography. We calculated systemic clearance of verapamil (CL=ko/C_ss ) and renal clearance (CLr) of verapamil and nor-verapamil. The C_ssvs rate relationship was fitted to a Michaelis-Menten equation: C_ss=ko(K_m+C_ss )/(V V_m ). Results CL was dose-dependent and in all nine patients a significant reduction in CL was observed over the dose range (mean CL±s.d. were 0.51±0.31, 0.38±0.16, 0.32±0.18, and 0.27±0.11 l h⁻¹ kg⁻¹, respectively, at 0.25, 0.30, 0.35, and 0.40 mg kg⁻¹ h⁻¹; P=0.0001). C_ss increased more than proportionally to the dose rate and the C_ssvs rate relationship was best defined by a Michaelis-Menten equation (K_m=730 μg l⁻¹; V V_m=0.55 mg kg⁻¹ h⁻¹ ), (r=0.994; P=0.006). CLr of verapamil and nor-verapamil was not saturable but the contribution to the elimination was only 2 to 4% of the dose. Conclusions These findings suggest a non-linear, capacity-limited metabolic clearance of high-dose verapamil. Using escalating infusion rates, high verapamil concentrations (1500–2500 ng ml⁻¹ ) were achieved without major toxicity. Saturable clearance may cause higher bioavailability and slower elimination of verapamil after acute oral overdoses.     

Autologous implantation of chondrocytes on a solid collagen scaffold: clinical and histological outcomes after two years of follow-up

Abstract From our overall experience in 56 patients, we here report the treatment with matrix-induced autologous chondrocyte implantation (MACI) of 35 patients suffering from knee cartilage defects measuring about 4 cm², and followed for a minimum of 6 months. A total of 36 knees were treated (1 patient on both knees) and clinically observed for 22 months (in some cases for over 39 months), in accordance with a standardised protocol. Subjective parameters (pain, well-being, functional state, symptoms during specific activity) and objective outcomes (IKDC score and Lysholm and Tegner scores) were recorded. One or 2 years after implantation, some biopsies of the regenerated cartilage were histologically evaluated. The subjective parameters (VAS pain score, 2.80±1.49, p<0.0001; change vs. basal score, 2.72) promptly normalized after 1 month, as did the objective ones (IKDC score after 6 months, 1.53±0.59, p<0.0001; change vs. basal score, 1.78). Similar results were observed after the treatment of a femoropatellar kissing lesion. The three cartilage biopsies that were analysed from different patients showed a tissue positivity to immunohistochemical markers of hyaline cartilage. The conclusions of this preliminary analysis are that the clinical outcome and histological evaluation suggest that MACI is able to relieve pain and restore the functionality of the knee, and that the treatment appears capable of regenerating hyaline cartilage.     

Increased external tibial torsion in Osgood-Schlatter disease

We studied the relationship between Osgood-Schlatter disease and torsional abnormalities of the lower limb in 21 boys with this condition and 20 age- and sex-matched controls. 3 groups of knees (20 control knees, 21 symptomatic and 21 asymptomatic or less symptomatic knees) were subjected to clinical, radiographic and CT evaluation. We found no statistically significant differences between patients and controls, as regards femoral anteversion, patellar congruence angle, patellar tilt angle and anterior tibial tuberosity-trochlear groove distance, but the condylomalleolar angle and tibial torsion angle were greater in patients. We found no differences between symptomatic and asymptomatic or less symptomatic knees in any of the parameters. All the symptomatic knees were on the side preferentially involved in jumping and sprinting. This increase in external tibial torsion may play a role as a predisposing mechanical factor in the onset of Osgood-Schlatter disease in male athletes.     

Identification of a Promiscuous T-Cell Epitope in Mycobacterium tuberculosis Mce Proteins

The characterization of Mycobacterium tuberculosis antigens inducing CD4(+) T-cell responses could critically contribute to the development of subunit vaccines for M. tuberculosis. Here we performed computational analysis by using T-cell epitope prediction software (known as TEPITOPE) to predict promiscuous HLA-DR ligands in the products of the mce genes of M. tuberculosis. The analysis of the proliferative responses of CD4(+) T cells from patients with pulmonary tuberculosis to selected peptides displaying promiscuous binding to HLA-DR in vitro led us to the identification of a peptide that induced proliferation of CD4(+) cells from 50% of the tested subjects. This study demonstrates that a systematic computational approach can be used to identify T-cell epitopes in proteins expressed by an intracellular pathogen.     

A PCR-colorimetric microwell plate hybridization assay for detection of Mycobacterium tuberculosis and M. avium from culture samples and Ziehl-Neelsen-positive smears

Differentiation between Mycobacterium tuberculosis and M. avium is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-Mycobacterium primers followed by hybridization of the amplification products to biotinylated M. tuberculosis and M. avium-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of M. tuberculosis and M. avium. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.