The oncofetal protein, 5T4, is a suitable target for antibody-guided anti-cancer chemotherapy with calicheamicin.

The oncofetal protein, 5T4, is a tumor-associated protein displayed on the cell membrane of various carcinomas. This molecule is a promising target for anti-tumor vaccine development and for targeted therapy with staphylococcus exotoxin. The potential use of 5T4 as a target for antibody-guided chemotherapy has not been demonstrated. We report oncolytic efficacy and selectivity in vitro and in vivo with immuno-conjugates of calicheamicin (CM) and the anti-5T4 antibody, H8. CM is a potent cytotoxic drug that causes double strand breaks in DNA. Conjugates of CM and H8 were constructed with acid-labile as well as acid-stabile linkers. In vitro, when applied to monolayers of 5T4(+) cells, CM-conjugates targeting 5T4 were consistently more toxic than either free drug or a non-binding control CM-conjugate. This difference was less pronounced on 5T4-deficient cells. In vivo, four 5T4-positive subcutaneous tumor models were treated with conjugates. Efficacy was demonstrated by reduction of tumor growth relative to controls treated with drug vehicle. To evidence selectivity, the efficacy of the anti-5T4 conjugates was compared to the efficacy of H8, a mixture of H8 and calicheamicin, calicheamicin alone or calicheamicin conjugated to the anti-CD33 antibody, hP67.6. In addition, the efficacy and selectivity of an acid-labile conjugate of H8 was evaluated in an orthotopic model for 5T4(+) lung cancer. Increased survival following treatment was used as a parameter of efficacy. Calicheamicin conjugates of H8 were effective and selective in all the examined tumor models. Differences in efficacy between the acid-labile and acid-stabile conjugates depended on the investigated tumor model.

Therapeutic potential of CD22-specific antibody-targeted chemotherapy using inotuzumab ozogamicin (CMC-544) for the treatment of acute lymphoblastic leukemia.

CMC-544 (inotuzumab ozogamicin) is a CD22-specific cytotoxic immunoconjugate of calicheamicin intended for the treatment of B-lymphoid malignancies. This preclinical study investigated antitumor activity of CMC-544 against CD22+ acute lymphoblastic leukemia (ALL). CMC-544 inhibited in vitro growth of ALL cell lines more potently than that of Ramos B-lymphoma cells. When administered to nude mice with established sc xenografts of REH ALL, CMC-544 caused dose-dependent inhibition of xenograft growth producing complete tumor regression and cures in tumor-bearing mice at the highest dose of 160 microg/kg of conjugated calicheamicin. In contrast, a nonbinding control conjugate was 16-fold less effective than CMC-544 in inhibiting growth of REH ALL xenografts. When REH cells were injected intravenously in scid mice and allowed to disseminate systemically, mice developed hind-limb paralysis that was effectively prevented by treatment with CMC-544. Flow cytometric analysis of cells recovered from the bone marrow from mice with disseminated disease verified the presence of engrafted ALL cells. Significantly reduced numbers of ALL cells were recovered from the bone marrow of CMC-544-treated mice than from vehicle-treated mice with disseminated disease. The anti-leukemia activity of CMC-544 demonstrated here further supports clinical evaluation of CMC-544 for the treatment of CD22+ leukemia.

N-phenyl-2-pyridinecarbothioamides as gastric mucosal protectants.

A series of substituted 2-pyridinecarbothioamides was synthesized and evaluated for gastric mucosal protectant activity in the rat. Out of this investigation N-(3,5-difluorophenyl)-2- pyridinecarbothioamide (23, AY-31,574) was identified. This compound was much more potent than sucralfate and ranitidine against ethanol-induced lesions. Compound 23 was equipotent with ranitidine against gastric injury caused by stress. Unlike ranitidine, 23 was found to be devoid of antisecretory activity in the pylorus-ligated rat model, making it a selective mucosal protectant. Such a potent selective mucosal protectant may provide a novel clinical approach in treating ulcers.

The effect of rapamycin on kidney function in the Sprague-Dawley rat.

The effects of rapamycin (RAPA) on kidney function and histology were investigated in the Sprague-Dawley rat and compared with cyclosporine. Drugs were administered orally in a Cremophor-ethanol formulation for 14 days in two separate studies. RAPA, at 1 mg/kg, had no effect either functionally or histologically on the kidney. At 10 mg/kg, RAPA depressed the gain in body weight by 20% in the rat but had only minor functional disturbances on urine output, plasma creatinine, and creatinine clearance in the kidney. It did not induce any histomorphologic abnormalities. CsA, at 25 mg/kg, produced functional alterations in the kidney including elevated plasma creatinine and depressed clearance of creatinine as well as depressed body weight gain (17%). Histologically, CsA induced proximal tubule damage. These results demonstrate that RAPA (10 mg/kg) does not produce nephrotoxicity in the Sprague-Dawley rat at doses three times higher than its effective immunosuppressive doses established in the rat.

Therapeutic blood levels of sirolimus (rapamycin) in the allografted rat.

A study was conducted to determine the relationship among oral dose, trough whole blood levels, graft survival, and side effects in sirolimus-treated allografted rats. The heterotopic heart allograft model using Brown Norway donors and Lewis rat recipients was used. Rats were dosed daily with sirolimus or vehicle until graft failure or up to a maximum of 28 days. Upon graft failure, rats were bled for measurement of trough blood levels of drug and tissues sent for histopathologic analysis. Sirolimus blood concentration correlated positively with dose and graft survival. Significant graft survival occurred at whole blood trough levels of 0.5 ng/ml achieved at the 0.3 mg/kg/day dose. Analysis of the concentration-effect data using a sigmoidal Emax model calculated a whole blood EC50 of 2.0 ng/ml for graft survival. With mean trough concentrations of 7 ng/ml and higher, grafts survived after cessation of drug treatment. At the 0.8 mg/kg/day dose, there was a significant decrease in body weight gain in the rats. Histopathologic examination of sirolimus-treated animals detected thymic and lymphoid atrophy, both considered pharmacologic extensions of sirolimus's immunosuppressive activity and focal myocardial degeneration, an exacerbation of a spontaneous occurring lesion. These results demonstrate that sirolimus prolongs graft survival in rat in a concentration dependent manner with therapeutic whole blood levels of about 10 ng/ml.

Alpha-2 receptors in the gastrointestinal system: a new therapeutic approach.

Alpha-2 receptor activation mediates the inhibition of a number of gastrointestinal functions including gastric and intestinal secretions. Alpha-2 receptors are located in the brain and presynaptically on cholinergic nerve terminals; activation of either inhibits vagus nerve activity. Intestinal secretions are inhibited by postsynaptic alpha-2 receptors located on intestinal epithelial cells. Agents which selectively activate alpha-2 receptors in the gut may therefore be beneficial in treating gastric ulcers and diarrheal states. Two such agents which activate alpha-2 receptors in the gut are WHR-1370A [1-n-butoxy-3-(2,6-dimethylphenylcarbamoyl) guanidine hydrochloride] and lidamidine. WHR-1370A is a potent gastric antisecretory and antiulcer agent which inhibits the release of acetylcholine from the vagus nerve. WHR-1370A's activity is blocked by yohimbine. Lidamidine is a clinically effective antidiarrheal agent. Lidamidine's response is partially inhibited by yohimbine in animal diarrheal models. Alpha-2 agonists represent a new class of drugs which have a promising future in the treatment of gastrointestinal disorders.

Hyperglycemic effect of lidamidine in the rat.

Lidamidine, a new antidiarrheal agent, produced a dose-dependent increase in plasma glucose levels in fed rats. The hyperglycemic response was evident 10 min after oral administration of lidamidine and lasted for 4 hr. Lidamidine's effect was absent in alloxanized rats. Insulin administration prevented the hyperglycemia. Pretreatment with the alpha 2-adrenoceptor antagonists yohimbine or RX781094A blocked the hyperglycemic response, while prazosin, propranolol, and hexamethonium pretreatment had no effect. These results indicate that the hyperglycemic effect of lidamidine is primarily due to the activation of peripherally located alpha 2-adrenoceptors that inhibit the release of insulin from pancreatic beta-cells.

Effect of epidermal growth factor on non-steroidal anti-inflammatory drug-induced intestinal damage.

Epidermal growth factor (EGF, 10 micrograms/kg po, ip, or sc, BID, and 20 micrograms/kg iv) had no protective activity in the indomethacin-induced intestinal lesion model (6 h model). In the ethanol-induced gastric lesion model, EGF (10 micrograms/kg sc) reduced lesions by 52% and reduced gastric acid secretion by 68% (5 micrograms/kg iv). In the 24 h indomethacin-induced intestinal lesion model, pretreatment with EGF (10 micrograms/kg sc, BID; 1 day before and during indomethacin treatment) had no beneficial effects. Therefore, EGF had no protective effects against non-steroidal antiinflammatory drug (NSAID)-induced intestinal lesions at doses that protect against the necrotizing action of ethanol and that inhibit gastric acid secretion in the rat.

Gastric antisecretory and antiulcer/cytoprotective effects of 2-cyano-3-(ethylthio-3-methylthio)-2-propenoic acid methyl ester.

AY-28,200 (2-cyano-3-(ethylthio-3-methylthio)-2-propenoic acid methyl ester), a new gastric antisecretory/antiulcer agent, inhibited basal and pentagastrin-stimulated gastric acid secretion in the conscious rat (ED50 = 7.6 and 1.9 mg/kg i.g., respectively). For inhibition of basal secretion, peak activity was attained in 5 to 6 h after dosing and was maintained for more than 10 h, with no gastric antisecretory activity occurring at 24 h. The K+ stimulated H+-K+ ATPase activity from rabbit gastric microsomes was inhibited by AY-28,200 (IC50 = 22 mumol/l). AY-28,200 inhibited ethanol-induced gastric lesions, at 3 mg/kg p.o. AY-28,200's cytoprotective effects against ethanol lasted for more than 4 h. AY-28,200 blocked acetylsalicylic acid and stress-induced gastric ulcers but was inactive against indomethacin-induced gastric ulcers. These results suggest that AY-28,200 is a parietal cell proton pump inhibitor with cytoprotective properties, and may produce its cytoprotective effect by stimulating the formation of endogenous prostaglandins.

Gastric antisecretory and antiulcer effects of WHR1582A, a compound exerting alpha-2 adrenoceptor agonist activity.

The gastric antisecretory and antiulcer effects of a novel compound, [1-(2,-dimethylphenyl)-3-isobutoxyamidinourea]hydrochloride (WHR1582A), are described. WHR1582A was active in preclinical ulcer models induced by 18-hr pylorus ligation, aspirin, indomethacin, reserpine, stress or cysteamine. WHR1582A inhibited acid secretion in the pylorus-ligated rat and in the anesthetized, lumen-perfused rat. The antisecretory effects of WHR1582A were antagonized by yohimbine, RX781094A and phentolamine. Propranolol, prazosin, corynanthine, methysergide, sulpiride and pimozide were unable to block its activity. WHR1582A blocked acid secretion stimulated by 2-deoxy-D-glucose but was inactive against the direct parietal cell stimulants carbachol and dimaprit. WHR1582A also inhibited electrically stimulated contractions that were mediated via the vagus in the isolated rat stomach preparation. The antisecretory activity of WHR1582A was not due to a reduction in gastric mucosal blood flow. These results demonstrate that WHR1582A is an effective antiulcer-antisecretory agent that exerts its gastric effects through the activation of alpha-2 adrenoceptors located presynapitcally on the vagus.

Cytoprotective and antiulcer activities of the antacid magaldrate in the rat.

The cytoprotective and antiulcer activities of the antacid magaldrate (ES Riopan) as well as its effects on gastric mucosal blood flow and mucus secretions, were determined in the rat. Magaldrate afforded protection against gastric necrotic lesions induced by absolute ethanol (ED50, as magaldrate, 419 mg/kg); gastric ulcers induced by acidified acetylsalicylic acid (ED50 540 mg/kg), stress (cold restraint, ED50 388 mg/kg), indometacin (ED50 281 mg/kg), and pylorus ligation; and intestinal ulcers induced by cysteamine (ED50 243 mg/kg) and indometacin (ED50 184 mg/kg). At a dose of 8 ml/kg (1728 mg/kg magaldrate), the cytoprotective effect of magaldrate against ethanol was evident 1 min after oral administration and lasted more than 8 h. The cytoprotection induced by magaldrate was decreased by pretreatments with the depletor of endogenous thiols, n-ethylmaleimide, or with the cyclooxygenase inhibitor, indometacin. Magaldrate did not affect gastric mucosal blood flow, but it increased gastric mucus secretion. This later effect may be a factor responsible for the cytoprotective activity of the agent. The efficacy of magaldrate may be due not only to its antacid, bile sequestering, and antipeptic activities, but also to its cytoprotective activity. The present results suggest that magaldrate could be effective in preventing gastric damage caused by alcohol and antiinflammatory drugs.

Activated aluminum complex derived from solubilized antacids exhibits enhanced cytoprotective activity in the rat.

Removing the buffering capacity of aluminum-containing antacids by acidification greatly increased their cytoprotective activity over the parent antacid. Commercially available antacids were acidified with 6 N HCl. Peak mucosal protective activity occurred at pH 2.5, and declined at lower pH. At pH 2.5, the antacid suspensions became solubilized and no acid-neutralizing capacity remained. This solution was named activated aluminum complex. Based on aluminum ion content, each aluminum-containing antacid suspension tested demonstrated a comparable increase in potency on acidification against ethanol-induced lesions. HCl (pH 2.5) was inactive against ethanol-induced lesions. At cytoprotective doses, activated aluminum complex did not cause gastric lesions when orally administered by itself, demonstrating that it is not acting as a local mucosal irritant. The data suggest that solubilization of aluminum-containing antacids in acidic medium enhances their mucosal protective activity, probably by releasing an activated species of aluminum ion reported to be a hexaaquoaluminum cation.

Renal effects of rapamycin in the spontaneously hypertensive rat.

The effects of rapamycin (RAPA), administered at therapeutic doses, were investigated in the spontaneously hypertensive rat (SHR). Additionally, the reversibility of RAPA's renal effects was investigated at a supratherapeutic dose. At doses that were active in preventing heart and kidney allograft rejection in the rat (0.01-0.08 mg/kg i.v.), RAPA had no effect on kidney function or rat body weight gain. At higher doses (0.8 mg/kg), RAPA produced significant changes in kidney function parameters and caused a loss in body weight. Histopathologic changes, including necrotizing vasculopathy and tubular atrophy, were noted at therapeutic doses. The effects of RAPA on kidney function were completely reversible after a 2-week washout period, though the histopathologic changes were still evident. These studies demonstrate that RAPA does not impair kidney function at therapeutic doses when administered for 2 weeks but does appear to accelerate the naturally occurring renal lesions of the SHR.

Kidney dysfunction in the arthritic rat.

Kidney function and histopathology were investigated in the adjuvant-induced arthritic rat. Rats injected with Mycobacterium butyricum exhibited symptoms of arthritis (i.e. paw edema and loss of body weight) by day 9 which worsened and included systemic manifestations of the disease on days 16 and 30. Definite signs of kidney dysfunction were observed by day 16 which included elevated urine output and plasma creatinine values and decreased creatinine clearance. By day 30, these parameters were similar to the values obtained from normal rats; however, kidney weights from arthritic rats than those from normal rats. Histopathologic abnormalities observed in the kidneys of arthritic rats on day 30 included tubular lesions consisting of focal basophilia, edema, granular deposits and basement membrane thickening. Changes in the glomerulus included granular deposits with focal glomerulopathy. These findings are the first to clearly demonstrate kidney dysfunction and histopathologic alterations associated with the early expression of the adjuvant-induced disease process in the rat. Our observations in the rat suggest that renal dysfunction in man can be mediated by the inflammatory disease process and is not solely a drug treatment-induced side effect.

Mucosal protective activity of activated aluminum complex.

The antacid ES Riopan was acidified ex vivo to pH 2.5 to completely eliminate its buffering capacity and was then tested as a mucosal protective agent. The pH 2.5 acidified antacid solution was named activated aluminum complex. Activated aluminum complex was 8.2 times more potent than its parent antacid in protecting against acidified aspirin-induced gastric lesions in the rat. Activated aluminum complex had a duration of action greater than 10 h in the ethanol-induced gastric lesion model, while ES Riopan was active for 6 h. Activated aluminum complex was able to inhibit both acid- and nonacid-mediated ulcers in the stomach and intestine. Its mucosal protective activity was not blocked by pretreatment with indomethacin. These results demonstrate that the nonbuffering antacid activated aluminum complex exerted a more potent and longer-lasting mucosal protective activity than its parent antacid. The activity was probably due to the presence of a hexaaquoaluminum cation and supports the argument that antacids possess mucosal protective effects independent of their acid-neutralizing capacity.

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.

The synthesis and biological activity of a novel series of diazepine MMP inhibitors.

A novel series of diazepine-based hydroxamic acid inhibitors of MMP-1, MMP-9, and MMP-13 were prepared and evaluated both in vitro and in vivo.

The asymmetric synthesis and in vitro characterization of succinyl mercaptoalcohol and mercaptoketone inhibitors of matrix metalloproteinases.

A series of succinyl based mercaptoketones and diastereomeric mercaptoalcohols were prepared and evaluated in vitro as inhibitors of the matrix metalloproteinases collagenase-1 (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9).

The discovery of anthranilic acid-based MMP inhibitors. Part 1: SAR of the 3-position.

A novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, and MMP-13 was prepared and evaluated both in vitro and in vivo. The most potent compound, 6e, has in vivo activity in a rat sponge-wrapped cartilage model.