An investigation of in vitro/in vivo correlations for salbutamol nebulized by eight systems.

The choice of a nebulized system for a patient usually depends on the equipment available. To date, there is limited guidance on the selection and use of a nebulizer. We have conducted in vitro tests described within the draft European Standard for Nebulisers (BSEN13544-1; CEN) and correlated these to in vivo pharmacokinetic performance of relative lung and systemic deposition in healthy volunteers. Eight nebulizer systems have been tested. The in vitro analysis used the recommended CEN methods to quantify the total dose available for inhalation as well as the size distribution from which the respirable dose was determined. The draft European Standard methods were slightly modified to use salbutamol rather than a fluoride tracer. For the in vivo study, nine volunteers provided urine samples after 30 min and then pooled for 24 h after the start of each nebulized dose (2.5 mg of salbutamol). Amounts of salbutamol and its metabolite excreted in the urine samples were determined. There was a significant (p = 0.02) correlation between the in vitro respirable dose and the amount of salbutamol excreted in the urine 30 min after the start of nebulization (relative bioavailability of salbutamol to the lung). Also, there was a significant (p < 0.001) correlation between the in vitro dose available for inhalation and the total amount of salbutamol and its metabolites excreted over the 24 h after the start of nebulization (relative bioavailability of salbutamol to the body). The results demonstrate that in vivo/in vitro correlations exist and warrant further investigations. The in vitro methods require further validation with in vivo correlations using patients with different severities of airway obstruction.

Inhaled antibiotic therapy in evidence: what delivery device?

This paper evaluates the suitability of various compressors available in Europe to generate and deliver tobramycin nebulizer solution to cystic fibrosis patients from the PARI LC PLUS jet nebulizer. This evaluation has been undertaken (i) by establishing an in vitro equivalence to the DeVilbiss PulmoAide compressor (operating at 4.6 l/min) proven effective in US clinical trials, and (ii) by determining equivalent in vitro performance of the LC PLUS nebulizer driven by alternative airflows. Equivalent performance is judged as having both an aerosol output and aerosol size within +/-10% of that obtained with the LC PLUS/PulmoAide combination. The two different in vitro methodologies applied to this investigation were based on the British Standard and a European Standard to assess nebulizer output. The results demonstrate that a wide range of compressed airflow rates generate aerosol output from the PARI LC PLUS equivalent to that obtained from the PulmoAide compressor. This range of airflows encompasses many compressors commonly available in Europe.

A Dutch population study of the STR Loci HUMTHO1, HUMFES/FPS, HUMVWA31/1 and HUMF13A1, conducted for forensic purposes.

We report on a Dutch population study of the STR loci HUMTHO1, HUMFES/FPS, HUMVWA31/1, and HUMF13A1, in which we used multiplex amplification and automated fragment detection. Genotype and allele frequencies showed no deviation from Hardy-Weinberg and linkage equilibrium. The improved Bonferroni procedure was used to combine the results of several tests. The power of discrimination of a complete profile exceeded 0.9998. We compared the allele frequencies in the Dutch sample to the frequencies in other populations using a bipilot to visualize alleles and populations simultaneously. The Dutch sample was similar to most other Caucasian samples. The data demonstrate that the genetic systems in this report are a valuable tool for forensic identity testing in The Netherlands.

MSH receptors and function in amelanotic B16 melanoma cells.

The possible mechanisms for the reduced melanin content and poor melanogenic response to MSH was investigated in B16-F10DD differentiation deficient melanoma cells. In particular, the MSH receptor status and associated signal transduction pathway linking to tyrosinase activity in these cells was studied for evidence of any defects. F10DD cells contained high-affinity binding sites for alpha-MSH, with KD values similar to those previously reported for other variants of the B16 melanoma. SDS-PAGE analysis after radioactive ligand cross-linking showed no evidence of gross structural alterations of the receptor. The F10DD cells expressed approximately twice as many receptors as the F10 parent cell line, suggesting a possible feedback response attempting to compensate for the amelanotic condition. The functional integrity of the MSH receptors in F10DD cells was confirmed by the presence of increased levels of cAMP in response to MSH stimulation. These results, coupled with the observation that F10 and F10DD cells express similar levels of tyrosinase mRNA and protein, point to a structural defect in tyrosinase or in the post-translational control mechanisms by which the activity of this enzyme is regulated.

MSH receptor expression and the relationship to melanogenesis and metastatic activity in B16 melanoma.

In this study we have compared the effects of different pro-opiomelanocortin (POMC) peptides on melanogenesis and metastasis and their relationship to MSH receptor expression in B16F1 melanoma cells. All peptides, apart from beta-endorphin, increased melanogenesis and the order of potency was Nle4DPhe7-alpha-MSH greater than alpha-MSH greater than ACTH[1-39] greater than des-acetyl alpha-MSH greater than ACTH[1-24]. A similar order of potency was found for metastasis, except for ACTH [1-24], which had a relatively greater effect on metastasis. These findings suggest that the effects on melanogenesis and metastasis are mediated via the same receptor. The results of ligand binding studies also indicated the presence of a single receptor with a KD value for Nle4DPhe7-alpha-MSH of 62 +/- 16pM. This was consistent with crosslinking studies using [125I] Nle4DPhe7-alpha-MSH which produced a single 50-55 kD band on analysis by SDS-PAGE. However, the relative binding affinities of the different peptides, measured by displacement of [125I]-Nle4DPhe7-alpha-MSH, did not closely correlate with the relative potencies in stimulating melanogenesis and metastasis. This suggests that receptor activation and the subsequent biological response is not determined solely by binding affinity.

The expression of functional MSH receptors on cultured human melanocytes.

Although alpha-MSH increases skin darkening in humans, there are several reports that it fails to have melanogenic effects on human melanocytes in vitro. The purpose of this study was to see whether cultured human melanocytes express MSH receptors. Human melanocytes were grown in the absence of artificial mitogens such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin (CT) and incubated for 2 h at room temperature with increasing amounts of 125I-labelled Nle4DPhe7-alpha-MSH with and without excess cold peptide. Binding was saturable and specific: Scatchard analysis gave a Kd of 4.9 x 10(-11) M and approximately 700 binding sites/cell. Human keratinocytes and fibroblasts showed no specific binding. The addition of 1 mM dibutyryl cAMP to the culture medium caused a 62% increase in MSH binding to human melanocytes. A smaller increase (25%) was seen with 10(-9) M CT while 25 mM TPA caused a 24% decrease. These results show that human melanocytes in culture express MSH receptors and that this expression can be modulated by mitogens.

Alpha-melanocyte-stimulating hormone immunoreactivity in melanoma cells.

Using a radioimmunoassay specific for alpha-melanocyte-stimulating hormone (alpha-MSH), significant levels of immunoreactivity were detected in a range of murine and human melanoma cell lines, including a series of ras-transfected melanocytes. The levels found in the melanoma cell lines tested varied, and overall were higher than in non-melanoma cell lines assayed for comparison. Furthermore the highest levels of immunoreactivity measured tended to be in the least differentiated and most metastatic melanoma lines. High performance liquid chromatography showed a peak of immunoreactivity which co-migrated with a desacetyl alpha-MSH standard. Additional unidentified components of immunoreactivity were found, including a high molecular weight form revealed by Sephadex-G50 gel exclusion. These may represent bound alpha-MSH or fragments of the proopiomelanocortin precursor having in common the C-terminus epitope recognised by the antibody. In view of the known effects of alpha-MSH on anchorage independent growth and metastasis of melanoma cells, our findings raise the possibility that MSH peptides may have an autocrine role in the growth and progression of melanoma. However, further characterisation of the immunoreactive species is required to determine whether these represent biologically active forms.