Enteral bioavailability of human granulocyte colony stimulating factor conjugated with poly(ethylene glycol).

PURPOSE: The focus of this paper is to demonstrate that pegylation of a therapeutic protein, recombinant human granulocyte colony stimulating factor (PEG-G-CSF), results in an increase in stability and in retention of in vivo bioactivity when administered by the intraduodenal route and may, therefore, be a suitable form of the protein for inclusion in an oral delivery formulation. METHODS: The ability of PEG-G-CSF to elicit a therapeutic response from the enteral route was investigated by two methods of intraduodenal dosing in an in vivo model to determine the optimal dosing method: by slow, constant infusion, or by a single bolus administration. RESULTS: Circulating levels of the proteins confirmed that PEG-G-CSF was delivered into the systemic circulation from the enteral route and that biological activity was retained. Bioavailability from the enteral route by the constant infusion method was calculated from the intravenous administration of the proteins to be between 1.8 and 3.5% while un-modified G-CSF failed to elicit a quantifiable response by this method. Bolus administration of PEG-G-CSF also resulted in biological activity although responses were short lived and significantly lower than with the pegylated formulation. CONCLUSIONS: The possible mechanisms of enteral delivery of PEG-G-CSF are discussed. Our results indicate that oral delivery of pegylated G-CSF may be possible and in fact, preferable to using the un-modified form of the therapeutic.

Vitamin B12 mediated oral delivery systems for granulocyte-colony stimulating factor and erythropoietin.

As a prelude to the development of orally active erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF), conjugates have been formed between these molecules and vitamin B12. During the formation of these conjugates intramolecular cross-linking of the proteins was avoided by the use of hydrazidyl derivatives of vitamin B12. A potentially biodegradable linkage was formed between vitamin B12 and G-CSF by reaction of the buried thiol in G-CSF with a long chain dithiopyridyl derivative of vitamin B12. In vitro and in vivo testing of the conjugates showed that their bioactivity was substantially maintained and that they were actively transported in an intrinsic factor dependent fashion across CaCo-2 cells and from the intestine to the circulation in a biologically active form.

Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol.

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulin-stimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.

Role of protein kinase C in the regulation of glucose transport in the rat adipose cell. Translocation of glucose transporters without stimulation of glucose transport activity.

The possible role of protein kinase C in the regulation of glucose transport in the rat adipose cell has been examined. Both insulin and phorbol 12-myristate 13-acetate (PMA) stimulate 3-O-methylglucose transport in the intact cell ein association with the subcellular redistribution of glucose transporters from the low density microsomes to the plasma membranes, as assessed by cytochalasin B binding. In addition, the actions of insulin and PMA on glucose transport activity and glucose transporter redistribution are additive. Furthermore, PMA accelerates insulin's stimulation of glucose transport activity, reducing the t1/2 from 3.2 +/- 0.4 to 2.1 +/- 0.2 min (mean +/- S.E.). However, the effect of PMA on glucose transport activity is approximately 10% of that for insulin whereas its effect on glucose transporter redistribution is approximately 50% of the insulin response. Immunoblots of the GLUT1 and GLUT4 glucose transporter isoforms in subcellular membrane fractions also demonstrate that the translocations of GLUT1 in response to PMA and insulin are of similar magnitude whereas the translocation of GLUT4 in response to insulin is markedly greater than that in response to PMA. Thus, glucose transport activity in the intact cell with PMA and insulin correlates more closely with the appearance of GLUT4 in the plasma membrane than cytochalasin B-assayable glucose transporters. Although these data do not clarify the potential role of protein kinase C in the mechanism of insulin action, they do suggest that the mechanisms through which insulin and PMA stimulate glucose transport are distinct but interactive.

Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester.

A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4- yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases approximately 5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a approximately 4-fold increase in GLUT4 while GLUT1 increases approximately 5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport approximately 3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).

Activation of adenylate cyclase and inhibition of glucose transport in rat adipocytes by forskolin analogues: structural determinants for distinct sites of action.

Forskolin and four analogues of forskolin, 7-beta-[gamma-(N'-methylpiperazino)-butyryloxy]-7-desacet ylforskolin, 7-desacetylforskolin, 7-tosyl-7-desacetylforskolin, and 1,9-dideoxyforskolin, were tested for their ability to activate adenylate cyclase, inhibit glucose transport, and inhibit cytochalasin B binding in rat adipocyte membranes. Forskolin was the most potent analogue in activating adenylate cyclase with an EC50 of 2 microM, whereas 7-beta-[gamma-(N'-methylpiperazino)butyryloxy]-7-desacety lforskolin and 7-desacetylforskolin were less potent, with EC50 values of 3 microM and 20 microM, respectively. The 7-tosyl-7-desacetylforskolin and 1,9-dideoxyforskolin did not stimulate adenylate cyclase even at the highest concentrations tested (100 microM). In contrast, forskolin and all of the analogues were able to fully inhibit glucose transport in adipocyte plasma membranes. The order of potency for the inhibition was forskolin greater than 7-beta-[gamma-(N'-methylpiperazino)butyryloxy]-7-desacety lforskolin greater than 7-desacetylforskolin greater than 7-tosyl-7-desacetylforskolin greater than 1,9-dideoxyforskolin, and the EC50 values were 0.24 microM, 1.8 microM, 7.1 microM, 8.8 microM, and 12.8 microM, respectively. Cytochalasin B binding to rat adipocyte membranes was inhibited by forskolin and the four analogues with the same order of potency as observed for the inhibition of glucose transport. Thus, the site of action of forskolin which is responsible for the inhibition of glucose transport and cytochasin B binding exhibits structural requirements for forskolin and its analogues that are different from those of the site responsible for the activation of adenylate cyclase.

The dynamics of LH-induced desensitization of adenylate cyclase and LH receptor internalization in rat Leydig cells at physiological levels of LH.

The LH-induced desensitization of adenylate cyclase and LH receptor internalization in rat Leydig cells in vitro has been investigated using sub-physiological to pharmacological concentrations of LH. Leydig cells pretreated with LH for 1 h in the presence of a phosphodiesterase inhibitor showed a dose-dependent decrease in the subsequent response to a high dose of LH; this was significant with concentrations of greater than 350 fmol/l. The maximum amount of desensitization was 60%. The time-course of LH-induced desensitization of the adenylate cyclase system was investigated; with 3 nmol LH/l cyclic AMP was increased in a linear manner up to 45 min, after which time there was either no further production or (with the higher concentrations) a decreased rate of production. In order to determine whether the 'non-desensitized' adenylate cyclase activity was dependent upon LH, the LH-treated cells were acid-washed to remove residual LH; cyclic AMP production still continued, albeit at a lower rate, thus indicating that this adenylate cyclase activity did not require the further presence of LH. The effect of various concentrations of LH on the level of surface-associated LH receptors was determined in the presence of monensin to prevent receptor recycling. A dose- and time-dependent decrease was found; this was significant after 2 h with 3.5 pmol LH/l and reached a maximum of 63% of the control with 3.5 nmol/l. A dose- and time-dependent reversal of desensitization occurred if the cells pretreated with LH were washed and reincubated; with 35 pmol LH/l and after 2 h the cells were fully responsive to a challenge with fresh LH. With higher concentrations of LH in the pretreatment, partial or no recovery was found. These studies demonstrate that physiological and sub-physiological concentrations of LH induce a rapid desensitization of Leydig cell adenylate cyclase. Internalization of occupied and unoccupied LH receptors also occurred. It is proposed that these two events are linked and may, paradoxically, and because of the low numbers of LH receptors, be necessary to maintain the normal response of Leydig cells to LH in vivo.

Effects of lutropin (LH) receptor internalization and recycling on the apparent numbers of LH receptors in rat testis Leydig cells at different temperatures.

The effect of different temperatures on the Scatchard analysis of binding studies with 125I-hCG and intact rat testis Leydig cells has been investigated. The results show that at temperatures greater than 4 degrees C an overestimation of the number of receptors/cell is likely to occur. A one-site analysis of the data gives values of 13,573, 61,924 and 3802 LH receptors/cell at 34 degrees C, 21 degrees C and 4 degrees C respectively. At 34 degrees C and 21 degrees C (but not at 4 degrees C) a two-site analysis of the data is possible, giving similar high affinity binding components at both temperatures (KD 1 X 10(-10) M) but with dissimilar low affinity binding components (34 degrees C, 2.17 X 10(-9) M and 21 degrees C, 6.3 X 10(-8) M). The calculated total number of LH receptors using a two-site analysis at 21 degrees C is equal to 114,766 receptors/cell, and at 34 degrees C it is 37,987 receptors/cell. It is proposed that the differences in the level of binding at different temperatures and the associated changes in the value of KD, reflect the temperature sensitivity of the endocytic pathway of the 125I-hCG/LH receptor complex within the rat testis Leydig cell. From previous studies it is known that at 34 degrees C and 21 degrees C, a large part of the cell-associated hormone is not receptor bound but rather it represents 'processed' hormone before it is released from the cell. At 4 degrees C the cell-associated 125I-hCG remains bound only to cell surface LH receptors and thus gives a more accurate measure of total receptor numbers/cell.

Similarities and differences in phorbol ester- and luteinizing-hormone-induced desensitization of rat tumour Leydig-cell adenylate cyclase.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was shown to mimic luteinizing hormone (LH; lutropin) in causing desensitization of LH-mediated cyclic AMP production in tumour Leydig cells. However, there were differences between LH- and TPA-induced desensitization: (1) TPA induced a more rapid effect than LH; (2) adenosine did not inhibit TPA-induced desensitization, whereas it completely inhibited the LH-induced desensitization; (3) adenylate cyclase activity in plasma membranes from TPA-desensitized cells was not decreased, whereas similar preparations from LH-desensitized cells lost their response to LH and to LH plus guanosine 5'-[beta gamma-imido]triphosphate; TPA-, but not LH-, treated cells had a decreased capacity to respond to cholera toxin and forskolin. These results indicate that LH and phorbol esters induce desensitization of adenylate cyclase in rat tumour Leydig cells by different mechanisms.

Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells.

A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature-sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone.

Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.

Inhibition of steroid production in Leydig cells by non-steroidal anti-inflammatory and related compounds: evidence for the involvement of lipoxygenase products in steroidogenesis.

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.