2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid with inbuilt ß-N-hydroxy-γ-keto-acid pharmacophore as HCV NS5B polymerase inhibitors.

The inbuilt 2-N-hydroxy-1-oxo-3-carboxylic acid of isoquinolone was designed as pyrophosphate mimic for hepatitis C NS5B polymerase. Various 2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid derivatives 11a-p were synthesized and evaluated as HCV NS5B polymerase inhibitors. Compound 11c exhibited moderate inhibitory potency based on the inorganic pyrophosphate generation (IC50 = 9.5 µM) and based on NTP incorporation by NS5B enzyme (IC50 = 5.9 µM). Compound 11c demonstrated antiviral activity (EC50 = 15.7 µM) and good selectivity in HCV genotype 1b replicon Ava.5 cells. Compound 11c reduced the interaction of NTP to NS5B polymerase. Docking model showed that 11c situated in similar orientation to the bound uridine triphosphate in the active site of NS5B polymerase. As a result, 2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid was disclosed as a novel inbuilt ß-N-Hydroxy-γ-keto-acid pharmacophore for HCV NS5B polymerase inhibitors.

NS5B RNA dependent RNA polymerase inhibitors: the promising approach to treat hepatitis C virus infections.

Hepatitis C virus (HCV), a causative agent for non-A and non-B hepatitis, has infected approximately 3% of world's population. The current treatment option of ribavirin in combination with pegylated interferon possesses lower sustained virological response rates, and has serious disadvantages. Unfortunately, no prophylactic vaccine has been approved yet. Therefore, there is an unmet clinical need for more effective and safe anti-HCV drugs. HCV NS5B RNA dependent RNA polymerase is currently pursued as the most popular target to develop safe anti-HCV agents, as it is not expressed in uninfected cells. More than 25 pharmaceutical companies and some research groups have developed ≈50 structurally diverse scaffolds to inhibit NS5B. Here we provide comprehensive account of the drug development process of these scaffolds. NS5B polymerase inhibitors have been broadly classified in nucleoside and non nucleoside inhibitors and are sub classified according to their mechanism of action and structural diversities. With some additional considerations about the inhibitor bound NS5B enzyme X-ray crystal structure information and pharmacological aspects of the inhibitors, this review summarizes the lead identification, structure activity relationship (SAR) studies leading to the most potent NS5B inhibitors with subgenomic replicon activity.

Anti-tumour activity and toxicity of the new prodrug 9-aminocamptothecin glucuronide (9ACG) in mice.

Cancer chemotherapy is limited by the modest therapeutic index of most antineoplastic drugs. Some glucuronide prodrugs may display selective anti-tumour activity against tumours that accumulate beta-glucuronidase. We examined the toxicity and anti-tumour activity of 9-aminocamptothecin glucuronide, a new glucuronide prodrug of 9-aminocamptothecin, to evaluate its potential clinical utility. 9-aminocamptothecin glucuronide was 25-60 times less toxic than 9-aminocamptothecin to five human cancer cell lines. Beta-glucuronidase activated 9-aminocamptothecin glucuronide to produce similar cell killing as 9-aminocamptothecin or topotecan. The in vivo toxicity of 9-aminocamptothecin glucuronide in BALB/c mice was dose-, route-, sex- and age-dependent. 9-aminocamptothecin glucuronide was significantly less toxic to female than to male mice but the difference decreased with age. 9-aminocamptothecin glucuronide and 9-aminocamptothecin produced similar inhibition (approximately 80%) of LS174T human colorectal carcinoma tumours. 9-aminocamptothecin glucuronide cured a high percentage of CL1-5 human lung cancer xenografts with efficacy that was similar to or greater than 9-aminocamptothecin, irinotecan and topotecan. The potent anti-tumour activity of 9-aminocamptothecin glucuronide suggests that this prodrug should be further evaluated for cancer treatment.

Conjugated polyhydroxybenzene derivatives block tumor necrosis factor-alpha-mediated nuclear factor-kappaB activation and cyclooxygenase-2 gene transcription by targeting IkappaB kinase activity.

Because the transcription factor, nuclear factor (NF)-kappaB, plays a key role in cellular inflammatory and immune responses, components of the NF-kappaB-activating signaling pathways are frequently used as targets for anti-inflammatory agents. This study shows that 2-(3',4'-dihydroxyphenyl)-5-hydroxybenzo[b]furan (GF-015) and 2,3-di(3',4'-dihydroxy-transstyryl) pyridine (GF-90), two conjugated polyhydroxybenzene derivatives, inhibited a common step in NF-kappaB activation in human NCI-H292 epithelial cells by preventing tumor necrosis factor (TNF)-alpha- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced IkappaB kinase (IKK) complex activation. Both agents inhibited the TNF-alpha- or TPA-induced expression of cyclooxygenase (COX)-2 mRNA and protein, COX-2 promoter activity, and prostaglandin E2 (PGE2) production. Overexpression of wild-type NF-kappaB-inducing kinase, IKKalpha, and IKKbeta led, respectively, to 3.5-, 2.6-, and 2.6-fold increases in COX-2 promoter activity, and these effects were inhibited by both compounds. GF-015 and GF-90 also prevented the TNF-alpha- and TPA-induced activation of IKK and NF-kappaB-specific DNA-protein binding activity. These results suggest that the inhibitory effect of GF-015 and GF-90 on TNF-alpha-induced COX-2 protein expression was caused by suppression of IKK activity and NF-kappaB activation in the COX-2 promoter, resulting in attenuation of COX-2 gene expression and PGE2 production.

Antihypertensive action and blockade of alpha1-adrenoceptors by DL-017, a quinazoline derivative.

3-[[4-(2-Methoxyphenyl)piperazin-1-yl]methyl]-5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (DL-017), a quinazoline derivative, exhibits alpha 1 -adrenoceptor antagonistic and type I antiarrhythmic effects on mammalian cardiac tissues. In the current study, the effects of DL-017 on the hemodynamic profile in anesthetized, spontaneously hypertensive rats were evaluated. Intravenous administration of DL-017 induced dose-dependent reductions of heart rate and blood pressure, which persisted over 2 h. DL-017 exerted a maximal antihypertensive effect at 0.1 mg/kg, which was similar to that of 0.1 mg/kg of prazosin. DL-017 was able to block the pressor response to phenylephrine but not to angiotensin II. Regional cerebral blood flow of the right parietal cortex decreased by 14 +/- 4% 10 min after bolus injection and then rapidly returned to control levels while the arterial pressure was still low. These results indicate that blockade of the alpha 1 -adrenoceptor by DL-017 contributes to reduction of arterial pressure. The antihypertensive effect without reflex tachycardia and loss of autoregulation of cerebral blood flow makes DL-017 suitable for chronic long-term treatment of hypertension.

Novel lead generation through hypothetical pharmacophore three-dimensional database searching: discovery of isoflavonoids as nonsteroidal inhibitors of rat 5 alpha-reductase.

A hypothetical pharmacophore of 5 alpha-reductase inhibitors was generated and served as a template in virtual screening. When the pharmacophore was used, eight isoflavone derivatives were characterized as novel potential nonsteroidal inhibitors of rat 5 alpha-reductase. This investigation has demonstrated a practical approach toward the development of lead compounds through a hypothetic pharmacophore via three-dimensional database searching.

Blockade of alpha1-adrenoceptors and cardiac depressant effect by a newly synthetic antihypertensive drug, DL-017 of quinazoline derivative.

The electromechanical effects of 3-[[4-(2-methoxy phenyl)piperazin-1-yl]methyl]-5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (DL-017), a newly synthesized quinazoline-derived antihypertensive agent, on mammalian cardiac tissues were evaluated. In driven canine Purkinje fibers, DL-017 decreased twitch tension, the maximal rate of upstroke of the action potential (Vmax), and intracellular Na+ activity (a(i)Na) in a concentration-dependent manner. The action potential duration was decreased in canine Purkinje fibers but increased in guinea pig papillary muscles. In guinea pig ventricular papillary muscles, phenylephrine in the presence of 1 microM propranolol increased the twitch tension in a concentration-dependent manner. At 10 microM, phenylephrine significantly decreased a(i)Na and shortened the action potential duration. DL-017 at 0.01 microM inhibited these phenylephrine-induced effects and shifted the concentration-dependent curve to the right. In sinoatrial nodes, DL-017 inhibited pacemaker activity, involving decreases in the slope of diastolic depolarization and Vmax and an increase in a delay of repolarization. These results suggest that, in addition to blockade of alpha1-adrenoceptors and Na+ channels, DL-017 reduces cardiac excitability and contractility in association with inhibition of slow inward Ca2+ and outward K+ channels. Since two order higher concentrations are required, the contribution of DL-017 to cardiac depressant from blockade of ionic channels seems to be less important when this compound is clinically used as an antihypertensive drug.

Pharmacological characterization of EK112, a new combined angiotensin II and thromboxane A(2) receptor antagonist.

The pharmacological characterization of EK112, a new combined angiotensin II and thromboxane A(2) receptor blocking agent, was examined in this study. EK112 was found to be a angiotensin II receptor antagonist, as revealed by its competitive antagonism of angiotensin II-induced smooth muscle contraction (pA(2) value of 7. 63 +/- 0.14) in rabbit aorta. It also had an angiotensin II blocking action in guinea pig ileum (pA(2) value of 7.87 +/- 0.67). Additionally, EK112 also possessed thromboxane A(2) receptor blocking activity, since it competitively antagonized aortic contractile responses elicited by U46619 and PGF(2alpha)(pK(B) values of 6.67 +/- 0.09 and 6.24 +/- 0.09, respectively) in rat. In contrast, EK112 did not affect the contractile responses to many other receptor agonists. EK112 did not mimic that of the angiotensin-converting enzyme (ACE) inhibitor, captopril, to enhance the muscle contraction elicited by bradykinin in guinea pig ileum, suggesting that EK112 did not inhibit ACE. Neither cyclic AMP nor cyclic GMP content in rat aortic rings was changed by EK112. These data demonstrate that EK112 is a selective antagonist of angiotensin II > thromboxane A(2) thromboxane A(2) receptor.

Efficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy.

The F(ab')(2) fragment of the anti-TAG-72 antibody, B72.3, was covalently linked to Escherichia coli-derived beta-glucuronidase that was modified with methoxypoly(ethylene glycol). The conjugate (B72.3-betaG-PEG) localized to a peak concentration in LS174T xenografts within 48 h after injection, but enzyme activity persisted in plasma such that prodrug administration had to be delayed for at least 4 days to avoid systemic prodrug activation and associated toxicity. Conjugate levels in tumors decreased to 36% of peak levels at this time. Intravenous administration of AGP3, an IgM mAb against methoxypoly(ethylene glycol), accelerated clearance of conjugate from serum and increased the tumor/blood ratio of B72. 3-betaG-PEG from 3.9 to 29.6 without significantly decreasing the accumulation of conjugate in tumors. Treatment of nude mice bearing established human colon adenocarcinoma xenografts with B72. 3-betaG-PEG followed 48 h later with AGP3 and a glucuronide prodrug of p-hydroxyaniline mustard significantly (p< or =0.0005) delayed tumor growth with minimal toxicity compared to therapy with a control conjugate or conventional chemotherapy.

Design and synthesis of water-soluble glucuronide derivatives of camptothecin for cancer prodrug monotherapy and antibody-directed enzyme prodrug therapy (ADEPT).

Glucuronide prodrugs of 9-aminocamptothecin were synthesized. Prodrug 4, in which 9-aminocamptothecin was connected to glucuronic acid by an aromatic spacer via a carbamate linkage, was stable in both aqueous solution and human plasma. Prodrug 4 and its potassium salt 12 were 20-80-fold less toxic than 9-aminocamptothecin to human tumor cell lines. The simultaneous addition of beta-glucuronidase and 4 or 12 to tumor cells resulted in a cytotoxic effect equal to that of 9-aminocamptothecin alone. Prodrugs 4 and 12 were over 80 and 4000 times more soluble than 9-aminocamptothecin in aqueous solutions at pH 4.0, respectively. Compounds 4 and 12 may be useful for prodrug monotherapy of tumors that accumulate extracellular lysosomal beta-glucuronidase as well as for antibody-directed enzyme prodrug therapy (ADEPT) of cancer.

Characterization of an antineoplastic glucuronide prodrug.

The specificity of tumor therapy may be improved by preferentially activating antineoplastic prodrugs at tumor cells pretargeted with antibody-enzyme conjugates. In this study, the conditions required for the efficient activation of p-hydroxyaniline mustard glucuronide (BHAMG) to p-hydroxyaniline mustard (pHAM) were investigated. pHAM induced cross-links in linearized double-stranded DNA at about 180-fold lower concentrations than BHAMG, indicating that the nucleophilicity of pHAM was decreased by the presence of a glucuronide group. The partition coefficient of BHAMG was about 1890 times lower than pHAM in an octanol-water two-phase system, suggesting that the reduced toxicity of BHAMG was due to both hindered diffusion across the lipid bilayer of cells and decreased reaction with nuclear DNA. BHAMG was significantly less toxic to BHK cells that expressed cytosolic Escherichia coli-derived beta-glucuronidase (betaG) compared with cells that were engineered to secrete betaG, demonstrating that extracellular localization of betaG was required for optimal activation of BHAMG. The extended retention of mAb RH1 on the surface of AS-30D cells was also consistent with extracellular activation of BHAMG. Taken together, our results indicate that the low toxicity of BHAMG was due to hindered cellular uptake and low alkylating activity. BHAMG must be enzymatically activated outside of tumor cells for maximum cytotoxicity, and non-internalizing antibodies are preferred for human tumor therapy by targeted antibody-enzyme activation of BHAMG.

Accelerated clearance of polyethylene glycol-modified proteins by anti-polyethylene glycol IgM.

Tumor therapy by the preferential activation of a prodrug at tumor cells targeted with an antibody-enzyme conjugate may allow improved treatment efficacy with reduced side effects. We examined antibody-mediated clearance of poly(ethylene glycol)-modified beta-glucuronidase (betaG-sPEG) as a method to reduce serum concentrations of enzyme and minimize systemic prodrug activation. Enzyme-linked immunosorbent assay and immunoblot analysis of two monoclonal antibodies generated by immunization of BALB/c mice with an antibody-betaG-sPEG conjugate showed that mAb 1E8 (IgG1) bound betaG and betaG-sPEG whereas mAb AGP3 (IgM) bound poly(ethylene glycol). Neither antibody affected the betaG activity. mAb 1E8 and AGP3 were modified with 36 and 208 galactose residues (1E8-36G and AGP3-208G) with retention of 72 and 48% antigen-binding activity, respectively, to target immune complexes to the asialoglycoprotein receptor on liver cells. mAb 1E8 and AGP3 cleared betaG-PEG from the circulation of mice as effectively as 1E8-36G and AGP3-208G, respectively. mAb AGP3, however, cleared betaG-sPEG more completely and rapidly than 1E8, reducing the serum concentration of betaG-sPEG by 38-fold in 8 h. AGP3 also reduced the concentration of an antibody-betaG-sPEG conjugate in blood by 280-fold in 2 h and 940-fold in 24 h. AGP3-mediated clearance did not produce obvious damage to liver, spleen, or kidney tissues. In addition, AGP3 clearance of betaG-sPEG before administration of BHAMG, a glucuronide prodrug of p-hydroxyaniline mustard, prevented toxicity associated with systemic activation of the prodrug based on mouse weight and blood cell numbers. AGP3 should be generally useful for accelerating the clearance of PEG-modified proteins as well as for improving the tumor/blood ratios of antibody-betaG-PEG conjugates for glucuronide prodrug therapy of cancer.

Portal hypotensive effects of combined terlipressin and DL-028, a synthetic alpha 1 adrenoceptor antagonist administration on anesthetized portal hypertensive rats.

AIMS/BACKGROUND: The purpose of this study was to investigate the therapeutic effects of terlipressin, alone or in combination with DL-028, a synthetic alpha 1-adrenoceptor antagonist on anesthetized portal hypertensive rats. METHODS: Portal hypertension was induced by either partial portal vein ligation (PVL) or bile duct ligation (BDL) in Sprague-Dawley rats. Each portal hypertensive rat received only one of the two regimens: vehicle plus terlipressin or DL-028 plus terlipressin. Terlipressin dosage was 0.017 mg/kg/min infused for 3 min, while vehicle or DL-028 (0.50 microgram/kg/min) was continuously infused for 40 min, starting 10 min before terlipressin infusion. RESULTS: In PVL rats, infusions of vehicle plus terlipressin induced significant, maximum reduction of portal venous pressure (PVP, -11.0 +/- 1.8%) and prominent elevation of mean arterial pressure (MAP, 50.3 +/- 9.0%) from baseline. Infusions of DL-028 plus terlipressin induced maximum PVP reduction (-17.5 +/- 2.8%) and MAP elevation (39.8 +/- 7.4%). In BDL rats, infusion of vehicle plus terlipressin also induced significant, maximum reduction of PVP (-6.8 +/- 2.1%) and prominent elevation of MAP (61.4 +/- 7.8%) from baseline. Infusions of DL-028 plus terlipressin induced maximum PVP reduction (-17.9 +/- 2.2%) and MAP elevation (47.9 +/- 7.4%). Compared to vehicle plus terlipressin, DL-028 significantly enhanced portal hypotensive effects of and attenuated systemic pressor effects of terlipressin in both PVL and BDL rats. CONCLUSIONS: Our results suggest that terlipressin, alone or in combination with DL-028, induced portal hypotensive effects in portal hypertensive rats. The combination of terlipressin with DL-028 was beneficial in enhancing the portal hypotensive effects and ameliorating the systemic pressor effects of terlipressin.

Bystander killing of tumour cells by antibody-targeted enzymatic activation of a glucuronide prodrug.

RHI-betaG-PEG, formed by linking poly(ethylene glycol)-modified beta-glucuronidase to Mab RH1, was employed to examine bystander killing of antigen-negative N1S1 rat hepatoma cells by activation of a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at antigen-positive AS-30D rat hepatoma cells. Sequential treatment of cells with 10 microg ml(-1) RH1-betaG-PEG and 20 microM BHAMG was not toxic to N1S1 cells but killed 99% of AS-30D cells. Over 98% of N1S1 cells, however, were killed in mixed populations containing as few as 2% AS-30D cells after identical treatment, demonstrating an in vitro bystander effect. Subcutaneous injection of AS-30D and N1S1 cells in BALB/c nu/nu mice produced solid tumours containing both cells. Uptake of radiolabelled RH1-betaG-PEG in solid AS-30D and mixed AS-30D/N1S1 tumours was 11.6 and 9.3 times greater than a control antibody conjugate 120 h after i.v. injection. Intravenous treatment with RH1-betaG-PEG and BHAMG cured seven of seven nude mice bearing solid s.c. AS-30D tumours and significantly delayed, compared with control conjugate and prodrug treatment, the growth of mixed N1S1/AS-30D tumours with one cure, showing that targeted activation of BHAMG kills bystander tumour cells in vivo.

Studies on quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides. 8.1, 2 synthesis and pharmacological evaluation of tricyclic fused quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides as potential alpha1-adrenoceptor antagonists.

A series of 2-substituted methyl 2,3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-ones (4), 3-substituted methyl 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-ones (5), 3-substituted methyl 2,3-dihydro-5H-thiazolo[2,3-b]quinazolin-5-ones (15a,b), 3-substituted methyl 2,3-dihydroimidazo[2,1-b]quinazolin-5(1H)-ones (16a,b), 3-substituted methyl 2,3-dihydro-1H-imidazo[1,2-b][1,2, 4]benzothiadiazine 5,5-dioxides (33a,b), 2-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (42-45a,b), 3-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (50-53a,b), 3-substituted methyl 5H-thiazolo[2,3-b]quinazolin-5-ones (55-56a,b), and 3-substituted methyl 5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (57) were synthesized as compound 1conformational rigid congeners for pharmacological evaluation as potential alpha1-adrenoceptor antagonists. Compounds 4, 5, 33a,b, 44a,b, 45a,b, 52a,b, 53a,b, and 57 were found to possess high affinity for the alpha1-adrenoceptor. Compounds 5 and 57 were the most highly selective and potent alpha1 antagonists with Ki = 0.21 +/- 0.02 and 0.90 +/- 0.08 nM, respectively. The S-enantiomers of these two compounds (Ki = 0.13 +/- 0.01 nM for (S)-(-)-5; Ki = 1.0 +/- 0.2 nM for (S)-(+)-57) were 144-200-fold more potent than the R-enantiomers (Ki = 26 +/- 8 nM for (R)-(+)-5; Ki = 144 +/- 23 nM for (R)-(-)-57). Compound 4 showed 8-fold higher affinity to alpha1A-AR better than alpha1B-AR. These compounds possessed weak to no activity against the 5-HT1A receptor.

Portal hypotensive effects of DL-028 and prazosin on portal hypertensive rats.

The portal hypotensive effects of prazosin and DL-028 (chem- ical name: 3-[[4-(2-methoxyphenyl)piperazin-1-yl]methyl]- 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-one(27b)), a synthetic alpha1-adrenoceptor antagonist, were assessed in portal hypertensive rats. Portal hypertension was induced by partial portal vein ligation in Sprague-Dawley rats. Two weeks after ligation, when the hyperdynamic state was stabilized the rats were anesthetized after an overnight fast and cannulated for measuring mean arterial pressure (MAP), portal venous pressure (PVP), cardiac index (CI) and heart rate (HR). Both DL-028 and prazosin (1, 3.3 and 10 microgram/kg) induced dose-dependent decreases of PVP and MAP after intravenous infusion, with effects lasting for longer than 30 min. The maximum percentage reduction of PVP after DL-028 was 10, 10 and 15%, respectively, for the dosages given (1, 3.3 and 10 microgram/kg), and 5, 12 and 25%, respectively, after prazosin. CI was not changed by either drug. HR was not changed by either drug except DL-028 at 10.0 microgram/kg with a bradycardiac effect. Our results showed that both DL-028 and prazosin reduced PVP in portal hypertensive rats.

Hemodynamic effects of 8-day DL-028 and octreotide administration in rats with portal hypertension.

BACKGROUND: DL-028 (chemical name: 3-[[4-(2-methoxyphenyl)piperazin-1-yl]methyl]-2,3-dihydroimidaz o[1,2-c]quinazolin-5(6H)-one (27b)) is a synthetic alpha1-adrenoceptor antagonist. The present study was undertaken to investigate the hemodynamic effects of chronic DL-028 administration, alone or in combination with octreotide, in rats with portal hypertension. METHODS: Portal hypertension was induced by partial portal-vein ligation. Portal-hypertensive rats were allocated to one of the four groups: vehicle group (saline, 0.5 ml/12 h), octreotide group (30 microg/kg/12 h), DL-028 group (0.4 mg/kg/12 h), and octreotide (30 mg/kg/l2 h) plus DL-028 (0.4 mg/kg/12 h) group, with eight rats in each group. DL-028 or saline was administered by gavage and octreotide by subcutaneous injection. Drugs were given immediately after ligation and for 8 consecutive days thereafter. Systemic and splanchnic hemodynamic variables were measured thereafter. RESULTS: Portal-vein-ligated rats showed a typical hyperdynamic state as compared with sham-operated rats. The portal venous pressure, portal tributary blood flow, and cardiac index were significantly reduced by treatment with octreotide, DL-028, or octreotide plus DL-028 in portal-hypertensive rats. Hyperdynamic variables of systemic, renal, hepatocollateral, and portal territory vascular resistances and renal and hepatic arterial blood flow were ameliorated by treatment with octreotide or octreotide plus DL-028 in portal-hypertensive rats. Octreotide plus DL-028 treatment exerted better hemodynamic effects on the cardiac index but worse effects on systemic and hepatocollateral vascular resistance than octreotide treatment alone. CONCLUSION: Although either DL-028 or octreotide ameliorated portal hypertension and splanchnic hyperemia in portal-hypertensive rats, octreotide treatment exerted more beneficial hemodynamic effects than DL-028 treatment. The combination of octreotide and DL-028 conferred no better hemodynamic benefits than octreotide alone, except on the cardiac index.

Cure of malignant ascites and generation of protective immunity by monoclonal antibody-targeted activation of a glucuronide prodrug in rats.

We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.

Electrical and mechanical effects of a novel antihypertensive quinazoline derivative, 3-[[4-(2-methoxyphenyl) piperazin-1-ly]methyl]-5-(methylthio)-2,3-dihydroimidazo [1,2-c]quinazoline, on guinea pig ventricular muscles.

Electrical and mechanical effects of 3-[[4-(2-methoxyphenyl)piperazin-1-ly]methyl]-5-(methylthio)-2,3-+ ++dihydroimidazo[1,2-c]quinazoline (DL-017), a new synthesized antihypertensive agent, were studied in guinea-pig ventricular papillary muscles. In muscle fibers driven at 1 Hz, DL-017 decreased the twitch force in a concentration-dependent manner and significantly increased the action-potential duration and decreased intracellular Na+ activity (ai(Na)) and maximal rate of upstroke of action-potential (Vmax) when concentrations were greater than 1 microM. Phenylephrine in the presence of 1 microM propranolol produced a concentration-dependent positive inotropy. DL-017 (0.01 microM) antagonized the positive inotropic effect of phenylephrine and shifted the concentration-response curve to the right. In K+-depolarized muscle fibers, 0.1 microM DL-017 significantly decreased the contractile force without changing the slow action potential. In low-[K+]o and high-[Ca2+]o solutions, a train of stimuli triggered a spontaneous rhythm that could be abolished by 3 microM DL-017. Our results suggest that DL-017 not only exhibits an alpha1-antagonistic effect but also induces negative inotropy by a decrease in myofibrillar calcium sensitivity and inhibits Na+ channels at higher concentrations, contributing to the drug's negative inotropic and type-I antiarrhythmic effects.

Poly(ethylene glycol) modification of beta-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs.

Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of antibody-betaG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect betaG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified betaG was coupled via a thioether bond to mAb RH1, an IgG2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-betaG-3PEG), 5.2 (RH1-betaG-5PEG) or 9.8 (RH1-betaG-10PEG) PEG molecules per betaG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-betaG. In contrast to the rapid serum clearance of RH1-betaG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-betaG-3PEG and RH1-betaG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-betaG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-betaG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-betaG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective.