Intracellular levels of two cyclosporin derivatives valspodar (PSC 833) and cyclosporin a closely associated with multidrug resistance-modulating activity in sublines of human colorectal adenocarcinoma HCT-15.

P-Glycoprotein, which mediates multidrug resistance (MDR) in cancer chemotherapy, is a principal target of cyclosporin A and [3'-keto-Bmt(1)]-[Val(2)]-cyclosporin (valspodar; PSC 833). To clarify mechanisms contributing to the different MDR-modulating activities of valspodar and cyclosporin A, we investigated the relation of the intracellular levels of the two cyclosporin derivatives to their modulating effect on MDR in different P-glycoprotein-expressing human colorectal carcinoma HCT-15 cells (parental HCT-15 and adriamycin-resistant sublines). In this study, valspodar was found to be much more potent than cyclosporin A in both sensitizing resistant cells to MDR-related anticancer drugs (e.g., adriamycin, vincristine and paclitaxel (taxol)) and increasing 2-[6-amino-3-imino-3H-xanthen-9-yl]benzoic acid methyl ester (rhodamine 123) retention and [G-(3)H]vincristine sulfate ([(3)H]vincristine) accumulation in these cells. Furthermore, a good correlation was detected between P-glycoprotein levels and the MDR-reversing effect of valspodar. In contrast, the effects of cyclosporin A could not be linked to P-glycoprotein levels in the MDR cells. In addition, the intracellular accumulation of valspodar was found to be 3 - 6 fold higher than that of cyclosporin A in four sublines and verapamil, an inhibitor of P-glycoprotein-mediated transport, enhanced the accumulation of cyclosporin A, but not valspodar. These results suggested that valspodar accumulation is not actively regulated by the P-glycoprotein-mediated efflux system.

Establishment and characterization of adriamycin-resistant human colorectal adenocarcinoma HCT-15 cell lines with multidrug resistance.

The multidrug resistance (MDR) phenotype, either intrinsic and/or acquired, is discussed in relation to several MDR-associated markers such as P-glycoprotein (P-gp) encoded by mdr1, multidrug-resistance-associated protein (MRP) encoded by MRP and lung-resistance-associated protein (LRP) encoded by LRP. Well-characterized in vitro models are required to elucidate the mechanisms of MDR. The aim of the present study is the establishment of a drug-resistant subline from human colorectal adenocarcinoma HCT-15 that intrinsically expresses moderate levels of P-gp, MRP and LRP. Three adriamycin-resistant sublines (HCT-15/ADM1, HCT-15/ADM2 and HCT-15/ADM2-2) were established by stepwise exposure in growth medium that was supplemented with 25-200 ng/ml adriamycin-resulting in a 2.2- to 7.8-fold increase in IC(50) values by using the XTT assay. They were cross-resistant to MDR-related drugs, epirubicin, mitoxantrone, vincristine, etoposide and taxol, but not the MDR-unrelated drug, mytomycin C. The resistance to adriamycin was confirmed in vivo by a lack of sensitivity in athymic nude mice. Gene expression data for mdr1/P-gp, MRP/MRP and LRP/LRP on both mRNA and protein levels demonstrated that the molecules contributing to MDR in resistant sublines are mainly P-gp and partially MRP. The newly established adriamycin-resistant sublines of HCT-15 will provide clinically relevant tools to investigate how to overcome drug resistance and elucidate possible mechanisms of acquired MDR in human colon cancer.

Phase I study of intravenous PSC-833 and doxorubicin: reversal of multidrug resistance.

PSC-833 reverses multidrug resistance by P-glycoprotein at concentrations < or = 1000 ng / ml. A phase I study of PSC-833 and doxorubicin was conducted to determine the maximum tolerated dose and to investigate pharmacokinetics. PSC-833 was intravenously infused as a 2-h loading dose (LD) and a subsequent 24-h continuous dose (CD). Doxorubicin was infused over 5 min, 1 h after the LD. The starting dose was 1 mg / kg for both LD and CD with 30 mg / m(2) doxorubicin; these dosages were increased to 2 and 10 mg / kg and 50 mg / m(2), respectively. Thirty-one patients were treated. Nausea / vomiting was controllable with granisetron and dexamethasone. Neutropenia and ataxia were dose limiting. Steady-state concentrations of PSC-833 > 1000 ng / ml were achieved at a 2 mg / kg LD and a 10 mg / kg CD. Ex-vivo bioassay revealed that activity in serum for reversing multidrug resistance was achieved in all patients; IC(50) of P-glycoprotein expressing 8226 / Dox(6) in patients' serum was decreased from 5.9 to 1.3 microg / ml (P < 0.0001) by PSC-833 administration. Doxorubicin clearance was 24.3 +/- 13.7 (mean +/- SD) liter / h/m(2), which was lower than the 49.0 +/- 16.9 liter / h/m(2) without PSC-833 (P < 0.0001). The relationship between doxorubicin exposure and neutropenia did not differ between patients treated and not treated with PSC-833. The recommended phase II dose of PSC-833 was 2 and 10 mg / kg for LD and CD, respectively, which achieved a sufficient concentration in serum to reverse drug resistance, as confirmed by bioassay. The dose of doxorubicin should be reduced to 40 mg / m(2), not because of the pharmacodynamic interaction between PSC-833 and doxorubicin affecting hematopoiesis, but because of pharmacokinetic interaction.

A bioassay for the activity of PSC 833 in human serum for modulation of P-glycoprotein-mediated multidrug resistance.

We established a rapid and sensitive ex vivo bioassay to detect the multidrug resistance (MDR)-inhibitory activity of SDZ PSC 833 ([3'-keto-Bmt1]-[Val2]-cyclosporin (PSC 833)) in two RPMI 8226 human myeloma sublines (parent 8226 and doxorubicin-resistant subline Dox6) in 75% human serum. In vitro sensitivity of the tumor to doxorubicin was determined by 3-h drug exposure growth inhibition assay (MTT assay). PSC 833 in serum restored the IC50 of doxorubicin in the P-glycoprotein (P-gp)-positive resistant subline to the same level as in the sensitive cells at 1 microg/ml, which has been shown to be an achievable concentration in clinical trials. In addition, the cytotoxic effect of doxorubicin was enhanced by PSC 833 in the sera of the patient in whom the blood level was 705.7 ng/ml. However, 10 microg/ml PSC 833 in serum does not cause a complete recovery in the IC90 of doxorubicin in the resistant sublines. This MDR-inhibitory activity was supported by the finding that PSC 833 in serum does not increase accumulation of rhodamine 123 in doxorubicin-resistant cells in an in vitro functional assay. The present study provides evidence that PSC 833 in human serum is effective to modulate P-gp-mediated MDR but insufficient for the reversal of MDR from the clinicopharmacological point of view.

Interaction of cyclosporin derivatives with the ATPase activity of human P-glycoprotein.

1. P-glycoprotein, a 170-180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents. In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and [3'-keto-Bmt1]-[Val2]-cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P-glycoprotein. 2. P-glycoprotein was immunoprecipitated with a monoclonal antibody (MRK-16) and the P-glycoprotein-MRK-16-Protein A-Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay. 3. While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate-stimulated and the basal P-glycoprotein ATPase. No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P-glycoprotein ATPase activity. PSC 833 was more potent than cyclosporin A for the substrate-stimulated activity. 4. Kinetic analysis indicated a competitive inhibition of verapamil-stimulated ATPase by PSC 833. 5. The binding of 8-azido-[alpha-32P]-ATP to P-glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P-glycoprotein ATPase cannot be attributed to an effect on ATP binding to P-glycoprotein. 6. The interaction of the cyclosporin derivatives with ATPase of P-glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P-glycoprotein function.

Regression of established tumors expressing P-glycoprotein by combinations of adriamycin, cyclosporin derivatives, and MRK-16 antibodies.

BACKGROUND: Overexpression of P-glycoprotein, a transmembrane protein capable of transporting a broad spectrum of anticancer drugs out of cells, likely contributes to tumor drug resistance. Strategies for overcoming this resistance include the use of specific compounds, such as cyclosporin derivatives, that modulate P-glycoprotein function and antibodies that bind to the protein, thereby altering its activity. PURPOSE: We examined the antitumor activity of combination treatment with the anti-P-glycoprotein monoclonal antibody MRK-16, a cyclosporin derivative (either cyclosporin A [CsA] or PSC 833), and the anticancer drug Adriamycin (ADM) against human colorectal carcinoma cells in vitro and established xenografts of these cells in vivo. METHODS: The human colorectal carcinoma cell line HCT-15 and its ADM-resistant subline HCT-15/ADM2-2 were used in this study. Cellular staining with a tetrazolium dye was used to assess the antitumor (i.e., antiproliferative) effects of treatment in vitro. Caliper measurement of tumor volumes was used to assess the antitumor effects of treatment in vivo. Cell surface binding of MRK-16 was measured by means of an immunofluorescence assay. Differences in the patterns of tumor cell growth in vitro and tumor growth rates in vivo were evaluated by means of repeated measure analysis of variance. Synergy in the combined effects of treatment was evaluated by means of the fractional product method. RESULTS: HCT-15 cells were found to express P-glycoprotein intrinsically; HCT-15/ADM2-2 cells expressed approximately five times more P-glycoprotein than the parental cells. HCT-15/ADM2-2 cells were also found to be about eight times more resistant to ADM in vitro than the parental cells. Incubation of both cell types in vitro with either MRK-16 and ADM or one of the cyclosporin derivatives and ADM inhibited cell growth minimally; however, ternary treatment with MRK-16, one of the cyclosporin derivatives, and ADM dramatically reduced the growth of both cell types. An analysis of treatment effects indicated that synergistic effects were obtained with ternary treatment. When athymic mice bearing established tumors (either HCT-15 or HCT-15/ADM2-2) were treated similarly with various combinations of the tested agents, the most pronounced antitumor effects were observed with ternary treatment. In some mice bearing HCT-15/ADM2-2 xenografts, ternary treatment led to complete tumor regression. Finally, CsA and PSC 833 were both shown to enhance MRK-16 binding to HCT-15 cells and HCT-15/ADM2-2 cells in vitro. CONCLUSION: Combination treatment with a cyclosporin derivative and an anti-P-glycoprotein antibody can be effective in circumventing P-glycoprotein-mediated drug resistance.

Functional modulation of ATPase of P-glycoprotein by C219, a monoclonal antibody against P-glycoprotein.

P-glycoprotein functions as an ATP-driven efflux pump for antitumor agents. C219 is a monoclonal antibody which recognizes regions near both ATP binding domains in each half of P-glycoprotein. In this study, we have demonstrated that C219 inhibits the ATPase activity of P-glycoprotein based on the following findings: 1) the inhibition of total ATPase activity by C219 was selective to P-glycoprotein-positive membranes; 2) the C219-sensitive fraction of ATPase correlated the expression of P-glycoprotein; and 3) modulators of P-glycoprotein ATPase, verapamil and cyclosporin A, affected the C219-sensitive fraction of ATPase. The photolabeling of P-glycoprotein with 8-azido-[alpha-32P]ATP was inhibited by C219, suggesting that the inhibition of ATP binding by C219 reduced the activity. Since C219 interacts with P-glycoprotein ATPase, C219 might become a useful tool for studying the role of P-glycoprotein ATPase.

Inhibitory effect of phorbol 12,13-dibutyrate on norepinephrine-induced contraction in rabbit iris dilator muscle.

The hypothesis that the increase in Ca2+ sensitivity on norepinephrine-induced contraction of smooth muscles and also the decrease of the norepinephrine-induced sustained level of intracellular Ca2+ concentration are produced by the activation of protein kinase C was tested. Phorbol 12,13-dibutyrate (PDB; 10(-6) M) relaxed the norepinephrine-induced sustained contraction in a concentration-dependent manner. On pretreatment with PDB a transient contraction was produced by the application of norepinephrine, but the sustained contraction was significantly reduced. The sustained elevations of intracellular Ca2+ concentration ([Ca2+]i) and the contraction induced by norepinephrine in fura-2-loaded preparations were decreased by the application of PDB. These inhibitory effects were antagonized by potent protein kinase inhibitors, 2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(-indol-3-yl)-maleimide (GF 109203X) (10 (-6) M) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) (10 (-6) M), but were not affected by a protein kinase A/G inhibitor, N-(2-cinnamylaminoethyl)-5-isoquinolinesulfonamide (H-88) (10(-6) M). The slope of the regression line for norepinephrine for [Ca2+]i and tension was significantly steeper than those obtained with high K+. Also, on pretreatment with PDB the Ca2+ sensitivity of the K(+)-induced contraction was decreased, but the Ca2+ sensitivity of norepinephrine-induced contraction tended to be increased. These observations indicate that PDB induces a decrease of [Ca2+]i on Ca2+ mobility and an increase of Ca2+ sensitivity on contraction of smooth muscle through the activation of protein kinase C.

Involvement of botulinum C3-sensitive GTP-binding proteins in alpha 1-adrenoceptor subtypes mediating Ca(2+)-sensitization.

The mechanisms of alpha 1-adrenoceptor agonist-mediated sensitization of the contractile apparatus of smooth muscle to Ca2+ were studied in beta-escin-permeabilized thoracic arterial smooth muscle of rabbit. Addition of norepinephrine (10 microM) plus guanosine 5'-triphosphate (GTP, 50 microM) significantly enhanced Ca2+ sensitivity as compared with the addition of 0.3 microM Ca2+ alone (pCa6.5). In beta-escin-skinned smooth muscle of chloroethylclonidine-treated tissues, the enhancement of Ca(2+)-contraction produced by norepinephrine or clonidine was completely inhibited by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta-S, 1 mM). In addition, Clostridium botulinum C3, which inactivates low molecular weight GTP-binding protein families, abolished norepinephrine- or clonidine-induced Ca(2+)-sensitization, but did not affect clonidine-induced translocation of protein kinase C to the membrane. The norepinephrine-enhanced Ca2+ sensitivity was partially reversed by a pretreatment with a selective myosin light chain kinase inhibitor (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n- propoxy-2,3,9,10-tetrahydro-8,11-epoxy,1H,8H,11H-2,7b,11a- triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926, 500 nM), but those of clonidine and in the chloroethylclonidine-treated tissues norepinephrine were not. These results suggest that Ca(2+)-sensitization produced by the activation of the alpha 1-adrenoceptor subtypes is linked via a low molecular weight GTP-binding protein (Rho), and the regulations of phosphorylation in contractile elements.

Alpha 1A-adrenoceptor subtype effectively increases Ca(2+)-sensitivity for contraction in rabbit thoracic aorta.

1. Norepinephrine and phenylephrine increase cytosolic Ca2+ concentration ([Ca2+]i) and muscle tension, which shows positive correlation between [Ca2+]i and tension development. 2. The slopes of regression lines between [Ca2+]i and tension development for norepinephrine and phenylephrine in tissues treated with an irreversible alpha 1B-adrenoceptor selective blocking agent, 10(-4) M chloroethylclonidine, were significantly steeper than those with untreated tissues. 3. Myosin light chain kinase inhibitors, KT5926 (3 x 10(-6) M) and K252a (10(-6) M) more selectively reduced the contraction produced by norepinephrine (3 x 10(-7) M) than that produced by clonidine (3 x 10(-6) M). 4. In the chloroethylclonidine-treated tissues, KT5926 and K252a did not tend to affect the contraction induced by norepinephrine and clonidine. 5. These results suggest that the contractile response through the alpha 1A-adrenoceptor subtype causes a greater muscle tension than that through the alpha 1B-subtype at the same level of [Ca2+]i, and that the alpha 1A-adrenoceptor subtype mainly activates myosin light chain kinase independent pathways of contractile mechanisms in vascular smooth muscle of rabbit.

Analysis of the interactions of SDZ PSC 833 ([3'-keto-Bmt1]-Val2]-Cyclosporine), a multidrug resistance modulator, with P-glycoprotein.

Multidrug resistance (MDR) is considered to be an important impediment to the effective treatment of cancer. P-glycoprotein, the drug efflux pump that mediates this resistance, can be inhibited by a wide variety of pharmacological agents, resulting in the circumvention of the MDR phenotype. SDZ PSC 833 ([3'-keto-Bmt1]-Val2]-cyclosporine), a nonimmunosuppressive cyclosporine D derivative, was identified to be a potent MDR modulator (Gaveriaux et al. J. Cell Pharmacol. 2:225-234; 1991). In this study, the interactions of P-glycoprotein with two cyclosporine derivatives, SDZ PSC 833 and cyclosporine A (CsA, Sandimmune), were analyzed. SDZ PSC 833 enhanced the sensitivity of the MDR cells to anticancer drugs by increasing the accumulation and inhibiting the efflux of cytotoxic agents from resistant cells more efficiently than CsA. The two cyclosporine analogs competed with the labeling of P-glycoprotein by a photoactive cyclosporine derivative. In addition, membrane vesicles derived from resistant cells bound SDZ PSC 833. However, CsA was transported by P-glycoprotein, whereas SDZ PSC 833 was not actively transported. This resulted in a prolonged inhibitory effect by SDZ PSC 833. The studies suggest that the binding of SDZ PSC 833 to P-glycoprotein in the absence of its transport from MDR cells mediated its high potency as an MDR reversing agent. In addition, the comparison of the two cyclosporine analogs indicated that limited chemical modifications of MDR reversing agents can affect their potential to inhibit P-glycoprotein function.

Alpha 1-adrenoceptor subtypes mediating the regulation and modulation of Ca2+ sensitization in rabbit thoracic aorta.

Norepinephrine (10 microM), methoxamine (100 microM) and clonidine (100 microM) with guanosine 5'-triphosphate (GTP, 50 microM) or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma-S, 10 microM) all significantly enhanced the contraction induced by 0.3 microM Ca2+ (pCa6.5) in beta-escin-skinned smooth muscle of rabbit thoracic aorta. The enhancement of Ca2+ contraction produced by norepinephrine was greater than that produced by methoxamine or clonidine. In beta-escin-skinned strips of chloroethylclonidine-pretreated smooth muscle, the enhancement of Ca2+ contraction produced by norepinephrine was significantly decreased, whereas the amplitude was the same as that produced by methoxamine or clonidine; this enhancement was inhibited by the selective alpha 1A-adrenoceptor antagonist WB 4101 (100 nM). The enhancement of Ca2+ contraction produced by methoxamine and clonidine was not affected by chloroethylclonidine pretreatment. The effects of methoxamine, clonidine and norepinephrine in the chloroethylclonidine-pretreated tissue were all inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP beta-S, 1 mM) and 1-(5-isoquinolinylsulfonyl)-methylpiperazine (H-7, 20 microM). Furthermore, the phosphorylation of myosin light chain produced by norepinephrine was greater than that produced by clonidine. These results suggest that both alpha 1-adrenoceptor subtypes (alpha 1A and alpha 1B) increase the Ca2+ sensitivity of contractile elements, and that the Ca2+ sensitization produced by alpha 1A-subtype receptors is mediated through G-protein and protein kinase C, and plays an important role in contraction of smooth muscle of rabbit thoracic aorta.

Contractile responses and calcium movements induced by alpha 1-adrenoceptor stimulant, norepinephrine, in rabbit iris dilator muscle.

1. The mechanisms involved in contraction of rabbit iris dilator muscle induced by norepinephrine (NE) were studied. 2. The concentration-response curve of NE was not influenced by Ca2+ blockers in the normal physiological saline solution (PSS) and removal of Ca2+ from PSS. 3. In 0.01 mM EGTA containing Ca(2+)-free PSS, the NE-induced contraction was phasic, which was suppressed by TMB-8, cyclopiazonic acid, ionomycin and A23187 but still partly remained. 4. In 2 mM EGTA containing Ca(2+)-free PSS, NE increased the intracellular Ca2+ ([Ca2+]i) and muscle tension. Ryanodine abolished the increase in [Ca2+]i induced by NE but slightly inhibited the tension. 5. These results suggest that the NE-induced contraction of rabbit iris dilator in normal PSS is mainly due to the increase in the release of intracellularly sequestered Ca2+ and partly due to the Ca(2+)-independent processes.

Chloroethylclonidine discriminates between alpha 1A- and alpha 1B-adrenoceptors in the presence of guanosine 5'-triphosphate in rabbit thoracic aorta.

Studies on the displacement of [3H]prazosin binding by the alpha 1-agonist phenylephrine revealed the presence of at least high- and low-affinity binding sites in membrane preparations prepared from rabbit thoracic aorta. Although the low-affinity site was reduced by the pretreatment of tissues with chloroethylclonidine, this site was unaffected by the same pretreatment of membrane preparations that did not contain the GTP analog. However, in membrane preparations with the metabolically stable GTP analog GTP gamma-S (10(-5) M) and single cell preparations, the low-affinity site was completely eliminated by the chloroethylclonidine pretreatment. Displacement studies with the alpha 1-antagonist WB4101 also revealed high- and low-affinity binding sites labeled by [3H]prazosin. Displacement curves of WB4101 obtained from membrane preparations in the presence of GTP gamma-S (10(-5) M) did not differ from those in the absence of GTP gamma-S. These results suggest that the low affinity phenylephrine binding site labeled by [3H]prazosin was selectively bound by the chloroethylclonidine used to pretreat the tissues, membrane preparation containing GTP gamma-S and single cells, and that chloroethylclonidine is able to recognize these two distinct subtypes of alpha 1-adrenoceptors only when GTP gamma-S is present.

Inhibitory effect of pilocarpine on norepinephrine release from electrically stimulated iris dilator muscles of rabbits as a most possible mechanism of pilocarpine-induced miosis.

Instillation of pilocarpine-induced a miosis in a rabbit. In an isolated iris sphincter muscle of rabbit, pilocarpine induced a small contraction and behaved as an antagonist of carbachol. The contractile response of an isolated rabbit dilator muscle to a field stimulation was inhibited by pilocarpine. This inhibitory action of pilocarpine was antagonized by atropine in a concentration-dependent manner. An amount of norepinephrine released from the electrically stimulated dilator muscle was inhibited by pilocarpine. The inhibitory effect of pilocarpine on norepinephrine release was decreased by atropine. A following explanation is proposed as a most possible mechanism for the pilocarpine-induced miosis in the rabbit. Pilocarpine activates the prejunctional cholinoceptors to inhibit norepinephrine release from the adrenergic nerve in the dilator muscle. Thus pilocarpine decreases tonus of the dilator muscle and induces the miosis.

Effects of guanosine 5'-triphosphate on the specificity of irreversible alpha 1-antagonisms by phenoxybenzamine in rabbit thoracic aorta.

Phenylephrine displacement curves for the specific binding of [3H]prazosin in the membrane fraction prepared from rabbit thoracic aorta showed high- and low-affinity sites with slope factors significantly less than unity. The irreversible alpha 1B-antagonist phenoxybenzamine shifted the binding sites to single high affinity sites with a slope factor close to unity in the presence of the metabolically stable GTP analog GTP gamma-S. These results indicate that phenoxybenzamine may have affected selectively the low affinity site to phenylephrine in the presence of GTP gamma-S.

Alpha 1B-adrenoceptor mechanisms in rabbit iris dilator.

Rabbit isolated iris dilator strips were contracted by norepinephrine, an alpha 1A- and alpha 1B-nonselective agonist, but not by methoxamine, an alpha 1A-selective agonist. The concentration-response curve for norepinephrine was considerably inhibited by chloroethylclonidine. The pA2 values for WB4101 and 5-methylurapidil were 8.16 +/- 0.09 and 7.84 +/- 0.08 (means +/- S.E. of 8-12 experiments), respectively, and significantly smaller than the values reported in the rat renal artery and thoracic aorta, and rabbit bronchus, where the alpha 1A-subtype is predominant. These results suggest that the rabbit iris dilator contains primarily the alpha 1B-subtype. Clonidine and tizanidine did not contract the rabbit iris dilator but shifted the curve for norepinephrine in a parallel manner, suggesting that they interact with the alpha 1B-subtype and act as competitive antagonists in this muscle. Methoxamine (up to 10(-3) M) had no effect on the contractile response to norepinephrine, suggesting that methoxamine does not interact with the alpha 1B-subtype.

Relationship between potency of L-isoprenaline and beta-adrenoceptor density estimated in single cells from tracheal smooth muscles of guinea pigs of different ages.

An age-related change in potency of L-isoprenaline in the presence of ascorbic acid, desmethylimipramine, corticosterone, pargyline, and phentolamine was obtained in tracheal strips from guinea pigs of differing ages between 6 and 40 weeks. The potency in the strips from 100-week-old guinea pigs did not significantly differ from that in strips from 40-week-old animals. Single cells were prepared from the tracheal muscles of 6-, 10-, 40-, and 100-week-old guinea pigs. The specific binding of [3H]dihydroalprenolol to the single cells was saturable. The dissociation constants of [3H]dihydroalprenolol were in good agreement with those of the membrane fractions from the guinea-pig tracheal muscles, and did not change with age. An excellent relationship between the potency of L-isoprenaline and the maximum binding of [3H]dihydroalprenolol estimated in the preparations from 6- to 40-week-old guinea pigs was found, suggesting that the increase in the potency of L-isoprenaline is due to the increase in the maximum binding or receptor density. The value in the preparations from 100-week-old guinea pigs deviated significantly from the regression line. This suggests the possibility that the decrease in potency in the strips from 100-week-old animals is due to a change in post beta-receptor processes in responsiveness.

Enhanced bioavailability of subcutaneously injected insulin by pretreatment with ointment containing protease inhibitors.

The present study was undertaken to develop an ointment preparation containing a protease inhibitor for stabilizing subcutaneously injected insulin. The ointment containing the protease inhibitor, gabexate mesilate or nafamostat mesilate, was applied to the skin around the insulin injection site. Three results were obtained. First, gabexate and nafamostat inhibited insulin degradation in subcutaneous tissue homogenates in vitro. Second, after application of gabexate or nafamostat ointment, an appreciable amount of gabexate or nafamostat appeared in the subcutaneous tissue of rats or hairless mice and their concentrations were comparable to those seen in the in vitro experiment. Third, insulin degradation at the subcutaneous injection site in the rat was depressed after pretreatment with gabexate or nafamostat ointment. Pretreatment with gabexate or nafamostat ointment increased the plasma immunoreactive insulin (IRI) levels and the hypoglycermic effect of insulin in healthy volunteers. These results indicate that gabexate or nafamostat ointments stabilize subcutaneously injected insulin.

Synthesis and antitumor activity of tropolone derivatives. 3.

As part of a study on the antitumor activities of tropolone derivatives prepared from hinokitiol, which naturally occurs in the plants of Chamaecyparis species, effects of aromatic substituents of alpha,alpha-bis(7-hydroxy-5-isopropyltropon-2-yl)toluenes on the activity were examined. Several of the compounds showed high potency in the P388 leukemia assay. 4-Hydroxy analogue 4d showed the most potent activity (T/C = 195%) at a 5 mg/kg dose. The introduction of large-size substituents, of which the steric influence prevents coplanarity of the substituted aromatic function, resulted in a remarkable decrease in the potency. X-ray structural analysis of highly potent 4-methoxy analogue 4b was undertaken.