ZD1611, an orally active endothelin-A receptor antagonist, prevents chronic hypoxia-induced pulmonary hypertension in the rat.

Endothelin-1 (ET-1) is a potent vasoconstrictor and comitogen implicated in the pathogenesis of pulmonary hypertension (PH). We evaluated the effects of an ET(A)receptor-selective antagonist, ZD1611, on hypoxia-induced PH in rats. <> and <> paradigms were established in which rats were administered placebo or ZD1611 (1-3 mg/kg, q.i.dpo) concomitant with hypoxic exposure (10% O(2)1 ATM) for 14 days or beginning after 7-day hypoxic exposure for 21 days. Compared with normoxic controls, hypoxic exposure plus placebo increased (P<0.05) hematocrit, mass ratio of right ventricle over left ventricle plus septum (RV/LV+S), and right intraventricular peak systolic pressure (RVSP). These latter two effects were decreased (P<0.05) by ZD1611 in both experimental paradigms [RV/LV+S(%)::RVSP(%); prophylactic, 14::32; therapeutic, 28::37]. Hypoxic exposure did not change mean systemic arterial pressure (MSAP). ZD1611 did not affect MSAP, plasma ET-1 concentrations, or blood gases measured when rats respired room air. In mechanistic studies, ZD1611 decreased (P<0.01) smooth muscle hypertrophy of small pulmonary arteries and abolished hypoxia-induced decreases in sensitivity and maximum contraction to ET-1 in isolated extralobar branch pulmonary artery. In conclusion, the ET(A)receptor-selective antagonist, ZD1611, attenuates hypoxia-induced PH in the rat.

A nonimmunosuppressive triene-modified rapamycin analog is a potent inhibitor of peptidyl prolyl cis-trans isomerase.

A triene-modified analog of the potent immunosuppressive agent rapamycin was found to be a potent inhibitor of the peptidyl prolyl cis-trans isomerase activity of human FKBP (Ki = 12.5 nM). This analog was not immunosuppressive in a thymocyte proliferation assay itself, but was able to antagonize the effect of rapamycin. This new analog should be useful as a mechanistic probe for macrocyclic immunosuppressants.

Effects of rolipram and CI-930 on IL-2 mRNA transcription in human Jurkat cells.

Interleukin-2 (IL-2) is a major mediator of immunologic responses involved in many chronic inflammatory diseases. We have investigated the effects of rolipram, a PDE-IV inhibitor, and CI-930, a PDE-III inhibitor, on IL-2 gene expression in the Jurkat human T cell line. The immunosuppressant cyclosporin A (CsA) was included as a positive control. Jurkat cells were stimulated with 1 microgram/ml phytohemagglutinin (PHA) and 50 ng/ml phorbol 12-myristate, 13-acetate (PMA) for 6 h, and mRNA was analyzed using reverse transcription and polymerase chain reaction (RT/PCR). IL-2 transcription was greatly inhibited by 1 microM CsA, whereas neither 10 microM rolipram nor 10 microM CI-930 had any effect on steady-state levels of IL-2 mRNA. Therefore, PDE inhibitors do not affect synthesis of IL-2 mRNA in this model of activated T cells. This is of interest given that these agents inhibit the proliferation of primary T cells. For murine splenocytes stimulated by 2.5 micrograms/ml concanavalin A (Con A), rolipram had an IC50 of 0.09 microM and CI-930 an IC50 of 4.4 microM. These concentrations are below those at which IL-2 mRNA synthesis was shown to be unaffected. Therefore, the mechanism by which inhibitors of PDE-III and PDE-IV affect T cell proliferation is not likely to involve suppression of IL-2 mRNA transcription.

Bacterial lipopolysaccharide potentiates type II collagen-induced arthritis in mice.

Collagen-induced arthritis (CIA) is an immunologically relevant animal model of human rheumatoid arthritis. Studies comparing the disease incidence in genetically susceptible male and female DBA/1LacJ mice demonstrated that under low density/low stress housing conditions, female mice had earlier onset (day 35) and higher disease incidence (25%) than the male mice (17% at day 49) when immunized with bovine type II collagen. A single subcutaneous or intraperitoneal injection of bacterial lipopolysaccharide (LPS) 17-24 days after collagen immunization greatly potentiated this standard CIA model in a dose related manner. 20-40 mug of LPS accelerated the onset of disease from day 35 to day 21 and exacerbated the clinical severity score from 0.27 to 2.00 at day 42. A similar administration of 6 mug of recombinant interleukin-beta produced a comparable potentiated CIA model. The acute phase protein, serum amyloid P (SAP), was elevated in the serum at day 26 to 440 mug ml(-1) for the LPS potentiated CIA mice compared to 65 mug ml(-1) in the non-potentiated immunized CIA mice. There was a significant correlation (r = 0.78) between SAP levels and disease expression in the LPS treated CIA mice. The rapidity and uniformity of disease expression in this LPS potentiated CIA model will allow more and different drugs to be evaluated with a smaller number of animals.

Inhibition of interleukin-1 (IL-1) induced neutral proteases from rabbit articular chondrocytes by WY-46,135 and WY-48,989.

The effects of potential anti-osteoarthritic compounds both on the direct inhibition of collagenase and neutral protease activities and on IL-1 induced release of neutral proteases from rabbit articular chondrocytes were investigated. WY-46,135 ((+)-N-[[[(5-chloro-2-benzothiazolyl)thio]phenyl]acetyl]-L- cysteine) directly inhibited collagenase activity (IC50 = 15.4 microM). This inhibition was reversible upon dialysis. WY-46,135 also directly inhibited neutral protease activity (IC50 = 16.8 microM) but did not significantly block bacterial collagenase activity at a concentration of 80 microM. In contrast, WY-48,989 (4-[[2-(7-chloro-2-phenyl-2H-pyrazolo[4,3-c]quinolin-4- yl)ethyl]amino]benzonitrile) did not directly inhibit either collagenase (10 microM) or neutral protease (100 microM) activity. Both WY-48,989 and WY-46,135 inhibited IL-1 stimulated release of neutral proteases (IC50 = 3 microM). The activities of these compounds represents two potential approaches for the treatment of osteoarthritis. WY-46,135 combines direct metalloprotease inhibitory activity with the inhibition of IL-1 stimulated neutral protease release from articular chondrocytes while WY-48,989 selectively inhibits the IL-1 induced release of metalloproteases.

Degradation of rat chondrosarcoma proteoglycans by a neutral metalloprotease from rabbit chondrocytes.

Rat chondrosarcoma proteoglycan aggregate with radiolabeled core protein was digested with a chondrocyte metalloprotease (CMP) or clostripain (CP) at neutral pH. The rates of product formation and the sizes and antigenicities of the products were studied using column chromatography and monoclonal antibodies. Sixteen percent of [35S]methionine label and 17-18% of [3H]serine label in core protein were freed from glycosaminoglycan bound peptides by 50 U/ml (760 micrograms/ml) of CP or 10 micrograms/ml (estimated) of CMP in 180 min. The CP reaction was almost complete at 5 minutes while the CMP reaction proceeded slowly from 5 to 180 min. The chondroitin-sulfate rich fragments were smaller after CP than CMP treatment. The 180 min CMP digest contained protein that migrated in 2 peaks on Sepharose CL6B. These two peaks corresponded to the peaks where hyaluronic acid binding region produced by CP and link protein migrate. Metalloenzyme inhibitors inhibited CMP with IC50s of 5 x 10(-5)M, 1 x 10(-3)M, and 80 micrograms/ml for phenanthroline, EDTA, and alpha 2-macroglobulin, respectively.

Interleukin-1 induces chondrocyte protease production: the development of collagenase inhibitors.

Supernatants from the P388D1 macrophage cell line as well as human interleukin-1 (IL-1) stimulated primary rabbit articular chondrocytes to produce collagen- (C-ase) and proteoglycan- (PG-ase) degrading proteases. The P388D1 derived factor had a molecular weight of 16,000-20,000 and a pI of 4.5-5.0. Both protease activities were metal dependent and inhibited by EDTA, phenanthroline, and alpha 2-macroglobulin but not by PMSF, TLCK, pepstatin, or alpha 1-antitrypsin. Size exclusion chromatography indicated the molecular weights for latent PG-ase and C-ase were 44,000-56,000 and 34,000-44,000, respectively. Chemical synthesis efforts produced two classes of C-ase inhibitors--thiols and hydroxamic acids. The former had IC50 values of 10(-5)-10(-6) M while the latter approached 10(-7) M.

Proteoglycan degradation by a chondrocyte metalloprotease. Effects of synthetic protease inhibitors.

Synthetic inhibitors of a chondrocyte metalloprotease (CMP) were assessed for potency. Proteoglycan core protein was used as substrate. The IC50 values were between 2 X 10(-6) and 7 X 10(-6) M for two types of inhibitors, thiol tripeptides and N-carboxyalkyl peptides. Hydroxamic acid peptides were more potent, with IC50 values of 3.2 X 10(-8) to 6.0 X 10(-8) M. These results confirm inhibitory concentrations reported using a proteoglycan-polyacrylamide bead assay. The slopes of the dose-response curves for the thiol compounds were steeper than the slopes for the other two types of compounds. All of the culture media tested inhibited CMP to some extent. Some media also interfered with inhibitor activity. In Ham's F10 nutrient medium, minimum CMP inhibition occurred, and all four hydroxamic acid peptides retained their activity for 1-2 days at 37 degrees. One thiol peptide compound assayed lost activity in 1 hr in thiocyanate-treated serum. All four hydroxamic acid peptides assayed retained activity in thiocyanate-treated serum after 3 days at 37 degrees. The hydroxamic acid peptides may provide a way to block endogenous CMP activity in vivo and to assess the role of CMP in normal and experimentally altered cartilage. They are more potent than other known CMP inhibitors. They retain activity in culture media and serum conditions used for in vivo and in vitro tests of CMP activity and toxicity.

Effect of synthetic metalloprotease inhibitors on cartilage autolysis in vitro.

The role of chondrocyte metalloprotease (CMP) in mediating cartilage autolysis was studied. Proteoglycan (PG) release and synthesis by rabbit articular cartilage explants were measured. After a 1-day preculture in control medium, 3.3 X 10(-6) M retinoic acid (RET) treatment for 1 day stimulated PG release several fold. RET also caused a large decrease in PG synthesis that returned to the control level after a 3-day recovery period. The effect on PG synthesis was observed at serum levels of 5 and 0.05%. The effect of RET on PG release required protein synthesis, inasmuch as it was lost in cultures maintained in media without amino acids or in a low volume of media. Interleukin-1 (IL-1) and lipopolysaccharide (LPS) treatment for 2 days also stimulated PG release. More PG was released after RET than after IL-1 or LPS, and only RET produced an effect that was evident by day 1. The amount of CMP that produced the same size effect on PG release as these stimulators was below the detection level of PG protease assays. Three potent CMP inhibitors reduced RET-, IL-1- and LPS-stimulated PG release to control levels. These inhibitors did not block another action of RET on chondrocytes, namely the inhibition of PG synthesis by RET immediately after treatment. The inhibitors did not act by reducing cell viability, because recovery of the rate of PG synthesis 3 days post-treatment occurred in inhibitor-treated cultures. These studies suggest that CMP is involved in cartilage autolysis that is stimulated by RET and IL-1.

Interleukin-1 stimulates the secretion of proteoglycan- and collagen-degrading proteases by rabbit articular chondrocytes.

Supernatants from the P388D1 murine macrophage cell line as well as commercially prepared human interleukin-1 (IL-1) stimulated primary rabbit articular chondrocytes to produce collagen- and proteoglycan-degrading proteases. The P388D1-derived factor had a molecular weight of 16,000-20,000 and a pI of 4.5-5.0, and was sensitive to phenylglyoxal treatment. Human IL-1 and the P388D1 supernatants enhanced glycosaminoglycan (GAG) release from bovine nasal cartilage explants. The proteoglycan- and collagen-degrading proteases required Ca2+ for activity. Latent proteoglycanase and collagenase had molecular weights of 44,000-56,500 and 34,000-44,000, respectively. The activated proteases had molecular weights of 30,000-40,000 and 22,000-36,000, respectively. Heparin-Sepharose affinity chromatography yielded two latent proteoglycanase-degrading protease activities and a collagen-degrading peak. The two proteoglycanase peaks also degraded fibronectin, laminin, gelatin, and azocoll but not type I collagen. The collagenase peak also degraded proteoglycan, gelatin, fibronectin, laminin, and azocoll. The activity of the proteoglycan- and collagen-degrading peaks was inhibited by phenanthroline and alpha 2-macroglobulin but not by phenylmethylsulfonylfluoride (PMSF), tosyllysylchloromethylketone (TLCK), pepstatin, or alpha 1-antitrypsin. The control of factors which augment protease production may offer a novel therapeutic approach to arthritis.