The bioavailability of intranasal salmon calcitonin in healthy volunteers with and without a permeation enhancer.

Serum levels of radioimmunoactive salmon calcitonin (sCT) were determined in 10 healthy volunteers after intranasal administration (IN) of 100-, 205-, and 450-IU of sCT with 0.5% sodium tauro-24,25-dihydrofusidate (STDHF), a 200-IU commercial IN formulation, and a 100-IU intramuscular (IM) formulation. Relative to the IM dose, the bioavailabilities of the IN formulations containing 0.5% STDHF were 3.9, 7.9, and 7.4%, respectively. The 200-IU commercial formulation resulted in serum levels above the limit of detection in only 5 of 10 patients, with an average bioavailability of 1.6%.

The effect of sodium tauro-24,25-dihydrofusidate on the nasal absorption of human growth hormone in three animal models.

The ability of a novel permeation enhancer, sodium tauro-24,25-dihydrofusidate (STDHF), to increase the systemic delivery of human growth hormone (hGH) after intranasal administration was investigated in rat, rabbit, and sheep. Formulations of hGH with STDHF exhibited greatly enhanced nasal absorption at concentrations of STDHF above its critical micelle concentration. The increase in bioavailability was 11-fold in rats and in rabbits and 21-fold in sheep for formulations containing 0.5% STDHF as compared to those without STDHF. Glycocholate or taurocholate at 0.5% was three to five times less effective than STDHF at enhancing hGH absorption in rats. Additionally, the pulsatile absorption kinetics observed after intranasal delivery more closely resemble the endogenous secretory pattern of hGH than those obtained following subcutaneous administration.

Alternative delivery systems for peptides and proteins as drugs.

The emergence of recombinant DNA technology has resulted in the large-scale production of a myriad of genetically engineered proteins and peptides, making many of them available for the first time for potential use as therapeutic entities. In addition, increased knowledge in the area of peptide/polypeptide hormones has resulted in an expansion of research efforts utilizing peptide synthetic chemistry, aimed toward developing novel therapeutic peptide drugs. Proteins and peptides cannot readily be administered by the conventional oral route, and thus alternative delivery methods to circumvent the necessity of frequent injections are being explored. This article reviews the current state-of-the-art technology of such methodologies, encompassing localized administration, administration to various body cavities (i.e., nasal, rectal, etc.), as well as controlled-release injectable or implantable systems. These different approaches result in quite different pharmacokinetics that may in part dictate which approach is best suited for a particular protein or peptide.

Effects of sodium taurodihydrofusidate on nasal absorption of insulin in sheep.

To investigate the utility of a novel adjuvant, sodium taurodihydrofusidate (STDHF), as an enhancer of mucosal permeation of drugs, experiments involving intranasal insulin:STDHF administration in sheep were performed. Rabbit erythrocyte lysis assays were employed to assess the relative membrane lytic activity of STDHF, as well as that of its glycine-conjugated analogue, compared with a nonionic detergent and a common bile salt. Equivalent weight concentrations of the fusidates were found to be 5- to 10-fold less lytic than the bile salt and at least 100-fold less lytic than the nonionic detergent laureth-9. Provided the concentration of STDHF was greater than its critical micellar concentration, formulations of insulin with STDHF greatly enhanced intranasal insulin absorption. Optimal nasal insulin absorption was attained at a molar ratio of STDHF to insulin of 5:1. In addition, intranasal absorption was linearly related to insulin dose. Compared with intravenous administration, the mean bioavailability of intranasal insulin was 16.4%. Interovine variability was low, with a coefficient of variation of 14% for 12 animals. It was found that intranasal absorption of sodium insulin was not significantly different from that of zinc insulin. However, formulations of both crystalline insulin preparations were absorbed more efficiently than a formulation prepared using commercially available solutions of U-500 insulin. The results taken together indicate that STDHF is an excellent enhancer of insulin absorption from the nasal mucosa.

Mechanism and quantitative contribution of the pentose pathway to the glucose metabolism of Morris hepatoma 5123C.

An investigation of the mechanism and quantitative contribution of the pentose phosphate pathway in the glucose metabolism of Morris Hepatoma 5123C is reported. Morris Hepatoma 5123C has an active non-oxidative segment of pentose pathway as judged by its ability to convert ribose 5-P to hexose 6-P in a standard assay. Based on compliance with qualitative and quantitative criteria, the cells exhibit the L-type pentose pathway reaction sequence rather than the F-type pathway. This compliance included the formation of intermediates characteristic of the L-type pathway, namely arabinose 5-P, octulose mono- and bisphosphates and sedoheptulose 1,7-bisphosphate, during the dissimilation of ribose 5-P to hexose 6-P. The intermediary role of arabinose 5-P was suggested by the incorporation of its carbon into various intermediates and products of the pentose pathway. Intermediary roles for ido octulose mono- and bisphosphates were supported by their participation in the reaction catalyzed by the phosphotransferase enzyme of the L-type pentose pathway. Presence of L-type PP reactions was further affirmed by 14C-prediction labelling experiments using [5-14C]- and [2-14C]glucose as specifically labelled substrates. Using two methods of measurement, the F-type pentose cycle made a negligibly small contribution to glucose metabolism, while the measured value of the L-type pentose pathway accounted for 30% (approx.) of the total glucose metabolism of these cells, a value consistent with the high activity of the enzymes of the L-type pentose pathway in Morris Hepatoma 5123C cells and the very high activity of the non-oxidative segment of the pathway in vitro. The findings validate the proposal that the L-type pentose pathway reactions constitute the non-oxidative segment of the pathway in Morris Hepatoma 5123C cells. Reasons involving pyruvate recycling reactions show why there is low incorporation of 14C-isotope in C-1 of glucose 6-P, when [4,5,6-14C]glucose and [6-14C]glucose are L-type PP test substrates in intact cells.

Glucocorticoid inhibition of vascular smooth muscle cell proliferation: influence of homologous extracellular matrix and serum mitogens.

We examined the influence of glucocorticoid hormones on the proliferation of cultured adult bovine aortic smooth muscle cells (BASM) using both primary mass cultures and a cloned strain. Cloned BASM cells maintained on plastic culture dishes were inhibited by approximately 40% by dexamethasone treatment but showed no inhibition when grown of homologous extracellular matrix (ECM) coated dishes. Dexamethasone inhibited growth of primary cultures by 73% on plastic and by 45% on ECM. The inhibitory effect was specific for the glucocorticoids, dexamethasone, corticosterone, and cortisol and was not observed with progesterone, aldosterone, estradiol or 17-alpha OH progesterone. In cloned cells, the abolition of glucocorticoid inhibition by ECM was independent of seeding density and serum concentration. The inhibition on plastic was dependent on serum concentrations greater than 1% and resulted in both a slow rate of proliferation and a lower saturation density. A specific subset of peptides detected on two-dimensional gels was induced by glucocorticoids under growth inhibitory conditions but was not induced when the cells were grown on ECM. Primary cultures grown on ECM and exposed to Dulbecco's modified Eagle's Medium (DME) containing high density lipoprotein and transferrin grew at 40% of the rate observed for cultures exposed to DME with 10% serum. Both conditions showed growth inhibition of 70% in the presence of dexamethasone. The addition of epidermal and platelet-derived growth factors in DME containing high density lipoprotein and transferrin to cells grown on ECM resulted in growth rates comparable to that observed with cultures exposed to 10% serum and were inhibited 45% by dexamethasone. These results suggest that glucocorticoids inhibit smooth muscle proliferation by decreasing the sensitivity of the cells to mitogenic stimulation by high density lipoprotein when the cells are maintained on a homologous substrate.

Proliferative variability of endothelial clones derived from adult bovine aorta: influence of fibroblast growth factor and smooth muscle cell extracellular matrix.

Four endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristics in vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non-FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10-50 ng/ml) and inhibited by higher doses (100-250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose-dependent manner (10-250 ng/ml) and was not growth inhibited at high FGF concentrations (250-1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM-coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM-coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM-coated dishes derived from vascular smooth muscle cells.

Glucocorticoid influence on growth of vascular wall cells in culture.

Primary mass cultures and cloned strains of bovine aortic endothelial and smooth muscle cells were investigated with respect to their growth responses to glucocorticoid hormones. The growth of primary endothelial cells was not influenced by glucocorticoid treatment in the absence of fibroblast growth factor (FGF) but was inhibited by about 30% in the presence of FGF; with cloned endothelial cells, glucocorticoids were also growth inhibitory only in the presence of FGF. In contrast, smooth muscle cell growth was inhibited 30%-70% by glucocorticoid treatment in both primary cultures and in the cloned strains in the absence of FGF, and this inhibition was totally abolished by the addition of FGF for both cultures. The corticosteroid influences on cell growth were glucocorticoid specific, concentration dependent, and were observed to be independent of the serum concentration. The results indicate that glucocorticoid hormones have direct and pronounced growth inhibiting effects on aortic smooth muscle cells but only minimal effects on endothelial cells when these components of the vascular wall are analyzed under identical conditions in vitro.

Differential radiation response of cultured endothelial cells and smooth myocytes.

In vivo observations have suggested that endothelial cells are the most radiosensitive elements of the vascular wall. To test whether this represents an intrinsic differential sensitivity, the response of bovine aortic endothelial cells and smooth myocytes was investigated in confluent cell cultures exposed to single doses of gamma radiation (250, 500, 1,000 or 2,000 rad). Both cell types showed a dose-dependent decrease in attachment efficiency when dissociated and replated at six hours after radiation. However, the attachment efficiency in both cell types was similar when a 72-hour postirradiation incubation period was used prior to dissociation of the cells. Growth inhibition was significantly greater (7- to 10-fold) in endothelial cells than in myocytes when examined four days after attachment. Confluent endothelial monolayers showed a dose-dependent, progressive cell loss during the 72-hour postirradiation period (70% after 1,000 rad); the myocyte cultures showed no radiation effect on the cell numbers. In spite of the reduction in number, the endothelial cells maintained the continuity of their monolayer by compensation with an increase in mean cell size. Endothelial cells developed multiple structural lesions, including an increase in the number and size of residual and lysosomal bodies, electron-lucent cytoplasmic defects, interruptions in the plasma membrane and irregular aggregation of chromatin, causing electron-lucent nuclei. These changes increased in severity with time and dose and were most pronounced 24 to 72 hours after 1,000 rad. No significant ultrastructural alterations were detected in myocytes four days after 2,000 rad.

Senescence of aortic endothelial cells in vitro: influence of culture conditions and preliminary characterization of the senescent phenotype.

The replicative lifespan of a cloned strain of adult bovine aortic endothelial cells has been examined by serial passage in culture using conditions where the cells were either dependent on fibroblast growth factor (FGF) for rapid growth or relatively independent on FGF for growth. In FGF-dependent cultures a replicative lifespan of 130 generations was attained with a phase III period which spanned approximately 20 generations. Withdrawal of FGF at generation 55 and repeated passage of such cultures in the absence of the growth factor resulted in the loss of proliferative potential within 15 generations. The morphological changes occurring upon FGF withdrawal were different than those that occurred when cultures senesced in the presence of FGF, FGF withdrawn cells showed a homogeneous increase in cell size and after several passages overgrew one another at confluency. Endothelial cells which senesced in the presence of FGF showed a very heterogeneous distribution of enlarged cells, many of which were binucleated but continued to form a confluent monolayer at high cell densities. Under FGF-independent conditions (begun at generation 48) a replicative lifespan of 105 generations was attained in the presence of the growth factor. FGF withdrawal under these conditions only decreased the replicative lifespan to 95 generations. Under these conditions the morphological changes occurring during phase III were identical in the presence and absence of FGF. Examination of the sensitivity of endothelial cells to FGF as they entered phase III showed that their dose-response characteristics were not qualitatively altered after the onset of phase III, although the number of cells responding to FGF progressively dropped. Comparison of the patterns of proteins synthesized in phase II and phase III cultures showed that phase III cultures even when plated at sparse densities continued to synthesize proteins which were normally observed in phase II confluent cultures. The results indicate that onset of the phase III period in aortic endothelial cells cloned and maintained in the presence of fibroblast growth factor can be delayed to a greated extent if the cells are maintained under conditions where they are made dependent on FGF for rapid growth in culture.

Use of [2-14C]glucose and [5-14C]glucose for evaluating the mechanism and quantitative significance of the 'liver-cell' pentose cycle.

1. Expressions are derived for the steady-state measurement of the quantitative contribution of the liver-type pentose phosphate cycle to glucose metabolism by tissues. One method requires the metabolism of [5-14C]glucose followed by the isolation and degradation of glucose 6-phosphate. The second procedure involves the metabolism of [2-14C]glucose and the isolation and degradation of a triose phosphate derivative, usually lactate or glycerol. 2. Measurements of 14C in C-2 and C-5 of glucose 6-phosphate are required and the values of the C-2/C-5 ratios can be used to calculate the quantitative contribution of the L-type pentose cycle in all tissues. 3. The measurement of 14C in C-1, C-2 and C-3 of triose phosphate derivatives can be used to calculate the quantitative contribution of the L-type pentose cycle relative to glycolysis. 4. The effect of transaldolase and transketolase exchange reactions, reactions of gluconeogenesis and non-oxidative formation of pentose 5-phosphate, isotopic equilibration of triose phosphate pools and isotopic equilibration of fructose 6-phosphate and glucose 6-phosphate, which could interfere with a clear interpretation of the data using [2-14C]- and [5-14C]glucose are discussed.

Quantitative measurement of the L-type pentose phosphate cycle with [2-14C]glucose and [5-14C]glucose in isolated hepatocytes.

1. Investigations of the mechanism of the non-oxidative segment of the pentose phosphate cycle in isolatd hepatocytes by prediction-labelling studies following the metabolism of [2-14C]-, [5-14C]- and [4,5,6-14C]glucose are reported. The 14C distribution patterns in glucose 6-phosphate show that the reactions of the L-type pentose pathway in hepatocytes. 2. Estimates of the quantitative contribution of the L-type pentose cycle are the exclusive form of the pentose cycle to glucose metabolism have been made. The contribution of the L-type pentose cycle to the metabolism of glucose lies between 22 and 30% in isolated hepatocytes. 3. The distribution of 14C in the carbon atoms of glucose 6-phosphate following the metabolism of [4,5,6-14C]- and [2-14C]glucose indicate that gluconeogenesis from triose phosphate and non-oxidative formation of pentose 5-phosphate do not contribute significantly to randomization of 14C in isolated hepatocytes. The transaldolase exchange reaction between fructose 6-phosphate and glyceraldehyde 3-phosphate is very active in these cells.

Globin messenger RNA content in hepatomas: a test of retrogenesis.

The globin mRNA content of foetal, neonatal and normal adult liver and of the transplantable Morris hepatoma 5123C and host liver of animals carrying the tumour was measured using molecular hybridization of total nucleic acid extracts from these tissues with a complementary DNA copy of globin mRNA from rabbit reticulocytes. The purpose of these experiments was to determine, as a test at the molecular level of the retrogenesis hypothesis, if the gene for foetal globin is activated in neoplastic hepatic tissue or induced in host liver. These experiments did not detect any foetal globin mRNA in the hepatoma or host liver nucleic acid extracts in spite of the increase in haemopoietic activity induced in host animals as a consequence of tumour bearing.

A test for foetal gene expression at the level of transcription in hepatoms.

The presence of a variety of embryonic and foetal gene products in neoplasms is well documented. Two such products, i.e. carcinoembryonic antigen and alpha foetoprotein, are currently being used for clinical diagnosis and the assessment of prognosis. The purpose of this study has been to examine the possibility of the reactivation of a foetal gene associated with foetal liver in the Morris 5123C hepatoma and host liver after prolonged tumour bearing. The foetal gene for globin was chosen for study as production of foetal globin in cancer patients has been observed and the technique for quantiation of globin messenger RNA is available. The quantitation of globin mRNA permits the identification of a gene product which is not related to the tissue of origin of the tumor being studied and which is influenced by pre-translational control mechanisms only. The influence of tumour bearing on foetal globin gene expression by the host liver is also reported. We report molecular hybridization studies of total nucleic acid extracts from foetal, 2-day neonatal, adult and host liver and the Morris 5123C transplantable hepatoma with a complementary DNA copy of globin messenger RNA. The results indicate that there is no activation of the foetal globin gene in these tissues in spite of erythrocytosis in the host animal.

A mechanism of tumourigenesis: retrodifferentiation and reontogeny in cancer and its clinical significance.

A general theory for the origin and maintenance of the neoplastic state in tissues is presented. Cancer is described as a disease of abnormal cell and tissue differentiation, and its underlying cause is identified in the process of blocked reontogeny. Data are presented which support this hypothesis and show the striking similarities at the molecular level between neoplastic, embryonic and regenerating tissues. The hypothesis suggests that numerous potentially useful (in the clinical sense) proteins, all of them members of early development, may be associated with neoplasms. It is suggested that research aimed at extending the identification and measurement of these proteins will make a significant contribution to the clinical management of cancer and may lead to the development of easy and cheap screening techniques for the early diagnosis of neoplastic disease in man.