A novel trans-platinum coordination complex possessing in vitro and in vivo antitumor activity

As part of a drug discovery program to discover more effective platinum-based anticancer drugs, a series of platinum complexes of trans coordination geometry centered on trans-ammine(cyclohexylaminedichlorodihydroxo)platinum(IV) (JM335) has been evaluated in vitro against a panel of cisplatin-sensitive and cisplatin-resistant human tumor cell lines (predominantly ovarian). In vitro, against 5 human ovarian carcinoma cell lines, JM335 was comparably cytotoxic to cisplatin itself and over 50-fold more potent than transplatin (mean 50% inhibitory concentrations: JM335, 3.1 microM; cisplatin, 4.1 microM; transplatin, 162 microM). With the use of seven pairs of human tumor cell lines (parent and subline with acquired resistance to cisplatin and encompassing all of the known major mechanisms of resistance to cisplatin) JM335 exhibited a different cross-resistance pattern to that of its cis isomer (JM149). JM335 showed non-cross-resistance in six of the seven resistant lines, cross-resistance in the A2780cisR line possibly being associated with high levels of glutathione. Preliminary intracellular DNA binding studies showed that in contrast to transplatin, JM335 was efficient at forming DNA-DNA interstrand cross-links. In vivo, JM335 produced growth delays in excess of 15 days against 4 of 6 human ovarian carcinoma xenografts and was unique among the complexes studied in retaining some efficacy against a cisplatin-resistant subline of the murine ADJ/PC6 plasmacytoma. JM335 is the first trans-platinum complex to demonstrate marked antitumor efficacy against both murine and human s.c. tumor models and represents a significant structural lead to complexes capable of circumventing cross-resistance to cisplatin.     

Meprin A, the major matrix degrading enzyme in renal tubules, produces a novel nidogen fragment in vitro and in vivo

We examined the effect of meprin A, the major matrix degrading metalloproteinase in rat kidney, on the laminin-nidogen complex. N-terminal sequence information from the most abundant 55 kDa fragment revealed that it was a breakdown product of nidogen rather than laminin. In comparison with over 50 nidogen cleavage sites produced by other proteases, the meprin A-induced nidogen cleavage site at amino acid position 899-900, a glutamine-glycine site in the G3 domain, is unique. In addition, these data demonstrate that meprin A degrades the G3 domain of nidogen even in the presence of laminin binding, which usually accords protection from proteolytic degradation. Meprin A also degraded purified nidogen into similar breakdown products. Given that the tubular basement membrane is located on the basilar side of the cell, the location of meprin A on the apical brush border makes it difficult to envision a role for meprin A in injury-induced basement membrane component breakdown. Thus, we examined the possibility that following renal tubular epithelial cell injury, meprin A undergoes a translocation to reach the underlying basement membrane. After renal ischemia-reperfusion there was a marked alteration in meprin A staining with meprin A now distributed throughout the renal tubular cell cytoplasm and directly adherent to the tubular basement membrane. This was in contrast to the usual linear staining of the brush border of tubules in the corticomedullary junction. These data provide unequivocal evidence that following injury, meprin A undergoes redistribution and/or adherence to the tubular basement membrane. Since in our in vitro studies, we identified a distinct meprin-induced 55 kDa nidogen breakdown product, the urine was also examined for the presence of nidogen degradation products after rat renal ischemia-reperfusion injury. Western blots showed a marked increase in the urinary 55 kDa nidogen fragment as early as the first day following ischemia-reperfusion injury and continuing for six days. Taken together, these in vivo data strongly support the notion that the nidogen breakdown products are the result of partial degradation of tubular basement membrane by meprin A following renal tubular ischemia-reperfusion injury.     

Somatostatin receptor subtype specificity and in vivo binding of a novel tumor tracer, 99mTc-P829

Recent data suggest that somatostatin receptors (SSTRs) are expressed on various tumor cells. High-level expression of SSTR on the tumor cell surface provides the basis for the successful clinical use of radiolabeled ligands for the in vivo localization of tumor sites. We have characterized the in vitro binding properties of the novel SSTR ligand 99mTc-P829 using primary human tumors (carcinoids, breast cancers, intestinal adenocarcinomas, pheochromocytomas, small cell and non-small cell lung cancer, and melanomas; n = 28), various tumor cell lines, and COS7 cells transfected with the human SSTR (hSSTR) subtypes 1, 2, 3, 4, and 5. 99mTc-P829 bound to primary tumor cells and tumor cell lines with high affinity and high capacity. The dissociation constants (Kd) ranged between 1 and 20 nM. 99mTc-P829 also bound with high affinity to the transfected hSSTR2 (Kd, 2.5 nM), hSSTR5 (Kd, 2 nM), and hSSTR3 (Kd, 1.5 nM). Binding of 99mTc-P829 to hSSTR3 was found to be displaceable by unlabeled P829/([ReO]-P829), SST-14, and vasoactive intestinal peptide (VIP; IC50, 2 nM) and, less effectively, by Tyr3-octreotide (IC50, 20 nM). In contrast, the binding of 99mTc-P829 to hSSTR2 and hSSTR5 could be displaced by P829/([ReO]-P829) and Tyr3-octreotide but not by VIP. 99mTc-P829 scintigraphy revealed in vivo binding to primary or metastatic tumor sites in seven of eight patients with breast cancer and six of six patients with melanoma. In summary, our data show that 99mTc-P829 binds with high affinity to many different types of primary and cloned tumor cells. Furthermore, our data identify hSSTR2, the VIP acceptor hSSTR3, and hSSTR5 as the respective target receptors. Because these receptors are frequently expressed at high levels on primary tumor cells, 99mTc-P829 appears to be a promising novel peptide tracer for tumor imaging.     

In vitro and in vivo antibacterial activities of Q-35, a novel fluoroquinolone

Q-35, a new fluoroquinolone, was evaluated for its in vitro and in vivo antibacterial activities. In vitro antibacterial activity against gram-positive bacteria was almost equal to that of sparfloxacin or tosufloxacin, but its activity against gram-negative bacteria was 2 times or more lower than that of other quinolones tested. In experimental septicemia, the in vivo activity of Q-35 reflected its in vitro antibacterial activity. In respiratory tract infections with Streptococcus pneumoniae TMS-3 in mice, Q-35 showed a therapeutic effect similar to sparfloxacin and tosufloxacin. Q-35 showed almost the same activity as that of ofloxacin in mice with pyelonephritic infection due to Escherichia coli TMS-3. The peak levels of Q-35 in murine serum, lungs and kidneys after a single oral administration were intermediate compared to those of tested quinolones.     

In vitro and in vivo antibacterial activities of CS-834, a novel oral carbapenem

CS-834 is a novel oral carbapenem antibiotic. This compound is an ester-type prodrug of the active metabolite R-95867. The antibacterial activity of R-95867 was tested against 1,323 clinical isolates of 35 species and was compared with those of oral cephems, i.e., cefteram, cefpodoxime, cefdinir, and cefditoren, and that of a parenteral carbapenem, imipenem. R-95867 exhibited a broad spectrum of activity covering both gram-positive and -negative aerobes and anaerobes. Its activity was superior to those of the other compounds tested against most of the bacterial species tested. R-95867 showed potent antibacterial activity against clinically significant pathogens: methicillin-susceptible Staphylococcus aureus including ofloxacin-resistant strains, Streptococcus pneumoniae including penicillin-resistant strains, Clostridium perfringens, Neisseria spp., Moraxella catarrhalis, most members of the family Enterobacteriaceae, and Haemophilus influenzae (MIC at which 90% of strains are inhibited, < or =0.006 to 0.78 microg/ml). R-95867 was quite stable to hydrolysis by most of the beta-lactamases tested except the metallo-beta-lactamases from Stenotrophomonas maltophilia and Bacteroides fragilis. R-95867 showed potent bactericidal activity against S. aureus and Escherichia coli. Penicillin-binding proteins 1 and 4 of S. aureus and 1Bs, 2, 3, and 4 of E. coli had high affinities for R-95867. The in vivo efficacy of CS-834 was evaluated in murine systemic infections caused by 16 strains of gram-positive and -negative pathogens. The efficacy of CS-834 was in many cases superior to those of cefteram pivoxil, cefpodoxime proxetil, cefdinir, and cefditoren pivoxil, especially against infections caused by S. aureus, penicillin-resistant S. pneumoniae, E. coli, Citrobacter freundii, and Proteus vulgaris. Among the drugs tested, CS-834 showed the highest efficacy against experimental pneumonia in mice caused by penicillin-resistant S. pneumoniae.     

Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP-specific phosphodiesterase, in vitro and in vivo

The effect of A02131-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl thieno (3,2-c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131-1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131-1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131-1 suppressed both the generation of inositol 1,4,5-triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131-1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131-1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131-1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131-1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131-1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131-1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131-1 is a cGMP-specific PDE (type V) inhibitor in human platelets.     

Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme

In this study, the efficacy of an anti-ras ribozyme in reversing the neoplastic phenotype was investigated. Murine NIH3T3 cells were transfected with cellular DNA from the FEMX-I human melanoma cell line expressing the activated H-ras gene. The transformed cells displayed the neoplastic phenotype in vitro and were tumorigenic in nude mice in vivo. When the transformants were transfected by a ribozyme designed to cleave only activated H-ras RNA, the transformed phenotype was abrogated. In contrast, expression of a mutant ribozyme, essentially acting only as antisense, into the transformed cells resulted in less dramatic changes in cell growth and tumorigenicity. These results reinforce the potential role of anti-oncogene ribozymes as suppressors of neoplastic growth, with possible implications for gene therapy.     

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.     

Enhanced intracoronary thrombolysis with urokinase using a novel, local drug delivery system. In vitro, in vivo, and clinical studies

BACKGROUND: Current pharmacological regimens for treating intracoronary thrombus in the cardiac catheterization laboratory generally involve the administration of thrombolytic agents that result in a systemic fibrinolytic state and/or require prolonged arterial drug infusion. The purpose of the present study was to assess a new technique for treating intracoronary thrombus consisting of the local infusion of limited quantities of urokinase with a novel drug delivery device. METHODS AND RESULTS: THe Dispatch coronary infusion catheter is a new local drug delivery system that allows for the prolonged infusion of therapeutic agents at an angioplasty site while distal coronary flow is maintained. Three experimental protocols were performed to determine the in vitro, in vivo, and clinical efficacy of this device. First, in vitro thrombolysis of fresh, porcine thrombus trapped in a 4-mm plastic tube with a 50% constriction and perfused with 20% porcine plasma was measured. Twenty-three thrombi were weighed before and after no treatment (n = 5), "systemic" urokinase administration (n = 4), local infusion of 150,000 U urokinase with a standard end-hole catheter (n = 4), local infusion of saline with the Dispatch catheter (n = 5), and local infusion of 150,000 U urokinase with the Dispatch catheter (n = 5). Second, 25 porcine coronary arteries in 23 pigs were dilated in vivo with conventional balloon angioplasty and then treated with 123I-labeled urokinase that was administered either by the Dispatch catheter (150,000 U; n = 16), intravenous systemic bolus (1,000,000 U; n = 3), guiding catheter infusion (500,000 U; n = 3), or local end-hole catheter infusion (150,000 U; n = 3). All vessels were subsequently harvested to quantify intramural deposition and subsequent washout of urokinase at the angioplasty site. Finally, 19 patients with angiographic evidence of intracoronary thrombus were treated with local urokinase infusion with the Dispatch catheter either before or after balloon angioplasty or directional atherectomy. In vitro studies demonstrated that infusion of urokinase with the Dispatch catheter decreased thrombus weight by 66% compared with no treatment (-25%), "systemic" urokinase administration (25%), end-hole catheter urokinase infusion (32%), or infusion of saline by the Dispatch catheter (32%) (P < or = .005). In vivo studies demonstrated immediate deposition of 0.12% of the urokinase delivered by the Dispatch catheter to the angioplasty site, compared with 0.0007% with systemic bolus, 0.003% with guiding catheter infusion, and 0.007% with local infusion with an end-hole catheter (P < .001). Urokinase deposited by the Dispatch catheter persisted intramurally for at least 5 hours. Patient studies demonstrated reduction of thrombus-containing stenoses and complete disappearance of intracoronary thrombus in all cases in which 150,000 U urokinase was locally infused over 30 minutes. There was no evidence of abrupt closure, distal embolization, or no reflow in any patient. CONCLUSIONS: Local urokinase delivery with the Dispatch catheter can result in rapid and complete intracoronary thrombolysis using substantially less drug than standard thrombolytic techniques. Intramural deposition of drug with this technique creates a local reservoir of urokinase that may provide prolonged thrombolytic activity at the infusion site.     

A comparative in vitro and in vivo pharmacological characterization of the novel dopamine D3 receptor antagonists (+)-S 14297, nafadotride, GR 103,691 and U 99194

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell-substrate adhesion leading to cell rounding (hence Rnd for "round"). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.     

A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".     

Activity and cleavage site specificity of an anti-HIV-1 hairpin ribozyme in human T cells

The DD genotype is a polymorphism of the angiotensin-converting enzyme (ACE) gene, and is associated with a significantly increased risk of myocardial infarction. As endothelial dysfunction is an important event in both early atherogenesis and late atherosclerosis, we hypothesised that the adverse effect associated with the ACE/DD genotype might be mediated via endothelial damage. Using high resolution ultrasound, we studied the brachial arteries of 184 subjects aged 15-73 (mean 38 +/- 14) years, who were all normotensive, non-diabetic lifelong non-smokers. Arterial diameter was measured at rest, during reactive hyperaemia (with flow increase causing endothelium-dependent dilation) and after sublingual glyceryl trinitrate (GTN, an endothelium-independent vasodilator). The ACE genotype was determined in each case by DNA amplification; 49/184(27%) had DD, 89 (48%) had ID and 46 (25%) had II genotype. Flow-mediated dilation (FMD) was 8.5% +/- 3.9% in the DD, 7.8% +/- 4.1% in the ID and 7.8% +/- 4.1% in the II subjects (P = NS). GTN-induced dilation was also similar in the 3 groups. On multivariate analysis, endothelium-dependent dilation was inversely related to age (r = -0.33, P < 0.001), vessel size (r = -0.41, P < 0.001) but not ACE genotype (r = 0.002, P = 0.97). The ACE genotype is unrelated to endothelium-dependent dilation in the systemic arteries of clinically well adults. This suggests that the risk associated with this polymorphism may be mediated by other mechanisms.     

In vitro and in vivo antidermatophyte activities of NND-502, a novel optically active imidazole antimycotic agent

In vitro and in vivo antidermatophyte activities of NND-502, a new imidazole antimycotic agent, were compared with those of two existing antifungal agents, lanoconazole and terbinafine. NND-502 exhibited strong in vitro antifungal activity against Trichophyton spp.; its MIC was 1 to 4 times lower than that of lanoconazole or terbinafine. In an in vivo study with a guinea pig model of tinea pedis, 7-day topical treatment with a 0.5% solution of NND-502 (dissolved in polyethylene glycol 400) was more effective than that with a 0.5% solution of either lanoconazole or terbinafine for eradicating fungi from the infected feet. When the duration of treatment was shortened to 3 days, a topical 1% solution of NND-502 achieved a complete mycological cure, while topical 1% solutions of lanoconazole and terbinafine did not.     

In vitro and in vivo antibacterial activities of OPC-20011, a novel parenteral broad-spectrum 2-oxaisocephem antibiotic

OPC-20011, a new parenteral 2-oxaisocephem antibiotic, has an oxygen atom at the 2- position of the cephalosporin frame. OPC-20011 had the best antibacterial activities against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae: MICs at which 90% of the isolates were inhibited were 6.25, 6.25, and 0.05 microg/ml, respectively. Its activity is due to a high affinity of the penicillin-binding protein 2' in MRSA, an affinity which was approximately 1,050 times as high as that for flomoxef. Against gram-negative bacteria, OPC-20011 also showed antibacterial activities similar to those of ceftazidime. The in vivo activities of OPC-20011 were comparable to or greater than those of reference compounds in murine models of systemic infection caused by gram-positive and -negative pathogens. OPC-20011 was up to 10 times as effective as vancomycin against MRSA infections in mice. This better in vivo efficacy is probably due to the bactericidal activity of OPC-20011, while vancomycin showed bacteriostatic activity against MRSA. OPC-20011 produced a significant decrease of viable counts in lung tissue at a dose of 2.5 mg/kg of body weight, an efficacy similar to that of ampicillin at a dose of 10 to 20 mg/kg on an experimental murine model of respiratory tract infection caused by non-ampicillin-susceptible S. pneumoniae T-0005. The better therapeutic efficacy of OPC-20011 was considered to be due to its potent antibacterial activity and low affinity for serum proteins of experimental animals (29% in mice and 6.4% in rats).     

A comparison of in vivo and in vitro strain with posterior fixed partial dentures

Formulated diets associated with a high risk (HR) or low risk (LR) for colon cancer were used to assess the effect of diet on putative metabolic biomarkers in human flora-associated rats: The HR diet was high in fat and sucrose and low in calcium and fiber; the LR diet was low in fat and high in starch, calcium, and fiber. The nutrient-to-energy ratio and energy intake were the same for both diets. Body and liver weights were significantly higher in animals fed the HR diet, possibly due to greater energy availability from fat. Cecal weights were significantly higher in animals fed the LR diet, presumably due to a bulking effect of the fiber and increased bacterial biomass. The HR diet significantly altered cecal bacterial enzyme activity: beta-glucuronidase activity increased 2.5-fold, and beta-glucosidase activity was halved. Ammonia production and the bacterial metabolism of 2-amino-3-methyl-7H-imidazo[4,5-f] quinoline (IQ) to 7-hydroxy-IQ (7OHIQ) were significantly higher in animals fed the HR diet. The HR diet, which contained factors common to diets consumed throughout the Western world, increased beta-glucuronidase activity, elevated cecal ammonia concentrations, and enhanced the genotoxic risk from 7OHIQ formation, three putative metabolic biomarkers of colorectal cancer. The significance of the reduction in beta-glucosidase is unclear.     

Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).     

Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo

Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance. The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR-associated genes such as the MDR-gene mdr1 encoding the P-glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR-related protein or the LRP gene encoding the lung resistance protein. Numerous experimental and clinical approaches aiming at reversing resistance require well-characterised in vitro and in vivo models. The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF-7 and its multidrug resistant subline MCF-7/AdrR. MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5- to 35-fold increase in IC50 values using the MTT-test. Cell lines were cross-resistant toward another MDR-related drug, vincristine, but remained sensitive to non-MDR-related compounds such as cisplatin and methotrexate. The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system. Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT-PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype. Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT-1/ADR, MT-3/ADR and MaTu/ADR.     

In vitro and in vivo properties of murine monoclonal antibody for a novel immature thymocyte-differentiated antigen, JL1

Bactericidal activities of 8 antibiotics (ticarcillin, piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, imipenem, amikacin, ciprofloxacin) and of 7 combinations have been studied by time-killing curve technique for 16 Pseudomonas aeruginosa selected for their beta-lactams resistance phenotypes (3 wild strains, 5 penicillinases, 2 extended-spectrum beta-lactamases, 3 high level cephalosporinases and 3 non enzymatic resistance). Bactericidal activities of beta-lactams used alone were as follows: imipenem > cefepime > ceftazidime, piperacillin, piperacillin-tazobactam > ticarcillin. All the antibiotics combinations were synergistic or additive. However, only the combination of imipenem with amikacin was bactericidal for all strains. For the strains with penicillinases, piperacillin-tazobactam was not more effective than piperacillin. For the extended-spectrum beta-lactamases, we have observed a synergy with piperacillin-tazobactam and ciprofloxacin or amikacin.     

Infrared A radiation-controlled permeation of tetracaine in vitro and in vivo

The incidence and case fatality rates of meningococcal disease were assessed in the county of Northern Jutland, Denmark, during the 16-year period from 1980 to 1995. A total of 320 patients were identified from the Meningococcal Research Database, which comprises information from the following sources: (i) the Department of Public Health, to whom notification of meningococcal disease is obligatory; (ii) the Regional Hospital Discharge Registry; and (iii) the register of the regional department of clinical microbiology. In order to assess prognostic indicators assessable at admission, information was collected for each patient from hospital records regarding contacts, symptoms and signs on arrival, laboratory data, and course of disease. The mean incidence was 4.3 cases per 100000 persons per year (range, 2.7-7.7). The incidence increased slightly during the period studied. Overall, the case fatality rate was 9.7%, with a significant rise occurring during the period (P=0.016) and a peak occurring in 1992. Advanced age (> or = 50 years), seizures, impaired consciousness, and skin bleeding on arrival at hospital were predictors of death.     

Anticancer efficacy in vivo and in vitro, synergy with 5-fluorouracil, and safety of recombinant methioninase

The elevated exogenous-methionine dependency of tumors for growth has been observed in all major cancer cell types. We have previously cloned a methioninase (rMETase) from Pseudomonas putida to deplete methionine. Growth inhibition followed by apoptotic cell death was induced by treatment of tumor cells with rMETase in vitro. A single i.p. injection of 300 units of rMETase can lower the serum methionine level in the mice from 70 microM to less than 1 microM within 2 h and maintain this depleted level for 8 h. Repeated dosing of rMETase of tumor-bearing mice could be administered without acute immune-hypersensitivity. rMETase treatment demonstrated growth inhibitory activity against human tumors in nude mice, including those which were multiple drug-resistant. No body weight loss or hematotoxicity, except a slight anemia, was found throughout the therapy. The combined treatment of the Lewis lung carcinoma with a fixed rMETase dose and increasing doses of 5-fluorouracil (5-FU) resulted in a dose-dependent enhanced antitumor efficacy for survival as well as tumor growth inhibition. Thus, methionine depletion by rMETase potentiates the antitumor efficacy of 5-FU. The data presented in this report thus indicate that rMETase is active alone, is synergistic in combination with 5-FU, and has negligible toxicity suggesting a novel clinical approach for effective cancer therapy.