Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)-3

Tissue inhibitor of metalloproteinases-3(TEMP-3), a novel memberof TEMP family genes, has been recently cloned and shown tobe expressed in preneoplastic but not in neoplastic mouse JB6epidermal cells (Sun et al. 1994 Cancer Res., 54, 11139). Thisdown regulation of the gene appears to be attributable at leastin part to alteration of gene methylation (Sun et al. 1995 J.Biol Chem., 270, 19312). Little is known, however, about therole of TEMP-3 in human cancers. We screened several human tumorcell lines for TEMP-3 expression and found that a colon carcinomaline, DLD-1, did not express TEMP-3. If down regulation of TIMP-3is causally related to carcino-genesis, re-expression by transfectionmay reverse the tumor cell phenotype. We therefore overexpressedhuman TEMP-3 in DLD-1 cells. TEMP-3 transfectants showed a serum-dependentgrowth inhibition in monolayer culture and a decreased growthpotential in nude mice in a manner dependent on the level ofTEMP-3 expression. A transfectant expressing a high level ofactive hTEMP-3 completely lost the ability to form tumors followings.c. injection into nude mice. We also tested TEMP-3 expressingcells and neocontrol TEMP-3 negative cells for their abilityto grow in liquid suspension culture, since both cells grewin semi-solid soft agar. As compared to neocontrol cells, TIMP-3over-expressors formed large aggregates, followed by cell death.This effect was not mimicked by BB94, a broad MMP inhibitor.We conclude from this study that (i) TEMP-3 overexpression inhuman colon carcinoma cells induces growth arrest in low serumconditions and inhibits in vivo tumor growth and (ii) the TEMP-3-inducedlarge aggregate formation and subsequent cell death under suspensiongrowth cannot be explained by its MMP inhibitory activity.     

Cocaine-induced anxiety: alleviation by diazepam,but not buspirone,dimenhydrinate or diphenhydramine

Clinical reports and animal experiments indicate that both cocaine administration and cocaine withdrawal increase anxiety. We investigated the ability of a number of putative anxiolytic agents to alleviate these anxiety states using the elevated plus-maze. Rats in the cocaine condition received either saline or cocaine (20 mg/kg) 40 min prior to testing; those in the withdrawal condition were tested 48 h following a chronic treatment regime (saline or cocaine 20 mg/kg per day for 14 days). Prior to testing, animals received a benzodiazepine (1.0 or 2.0 mg/kg diazepam), a serotonergic agonist (0.5 or 1.0 mg/kg buspirone), an antihistamine (50 mg/kg dimenhydrinate or 27 mg/kg diphenhydramine) or a saline injection. All drugs were administered intraperitoneally. Cocaine administration and cocaine withdrawal reduced the percentage time spent on and the number of entries into the open arms. Diazepam dose-dependently alleviated cocaine withdrawal-induced anxiety and non-significantly attenuated cocaine-induced anxiety. Buspirone, dimenhydrinate and diphenhydramine did not consistently alleviate the anxiety caused by either cocaine pre-treatment regime; in the saline conditions, however, each of these treatments was anxiogenic. In summary, benzodiazepines alleviated cocaine-induced anxiety, while future research on the ability of serotonergic and antihistaminergic drugs to alleviate these anxiety states is warranted.     

Synthesis,beta-adrenoceptor pharmacology and toxicology of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride,a short acting beta(1)-specific antagonist

The synthesis of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride (D140S.HCl 6), a novel short acting beta(1)-specific adrenoceptor antagonist, has been described. The antagonist potency for D140S.HCl 6 has been compared with esmolol, another short acting agent, and other well known beta-adrenoceptor antagonists in isolated rat tissue preparations. The pharmacokinetics of D140S.HCl 6 in 7 day continuous intravenous infusions and 4 weeks intravenous bolus injection studies in conscious rats and dogs have been examined in toxicology studies. The effect on the isoprenaline-induced heart rate increase and the pharmacodynamic half-life of D140S.HCl 6 has been compared with esmolol in a conscious rat model. In addition, the results of a range of toxicological studies are presented. The results indicate that D140S.HCl 6 is a highly specific beta(1)-adrenoceptor antagonist (pA(2) = 8.15+/-0.22, beta(1)/beta(2) selectivity > 4400). The in vitro studies suggest D140S.HCl is ca. ten times more potent and 60 times more beta(1)-specific than racemic esmolol. Pharmacokinetic non-linearity was seen when given as a 7 day intravenous infusion at toxicological doses above 10 mg kg(-1) h(-1) in the rat and 2.5 mg kg(-1) h(-1) in the dog. Both D140S.HCl 6 and esmolol have very short durations of action after intravenous infusion in the rat (pharmacodynamic half-life is < 15 min for D140S.HCl and 10 min for esmolol). The toxicological tests indicate that D140S.HCl 6 shows no unexpected toxicity and none of the tissue irritancy problems reported for esmolol formulations.     

The Elusive Extracellular AV Nodal Potential: Studies from the Canine Heart, Ex Vivo

Various forms of extracellular recordings from the AV node (AVN) have been reported. However, lack of consistent validation have precluded the use of such recordings in experimental and clinical studies. In 14 Langendorff perfused dog hearts, the triangle of Koch (TOK) was exposed and an octapolar electrode catheter (2 mm rings, 2 mm spacing) was inserted under the endocardium so that the bipolar pairs recorded electrograms from the apex to the base of the TOK. All recording were filtered between 0.05 and 250 Hz, except for a His bundle (Hb) recording (30–250 Hz) made from another bipolar electrode catheter placed in the aortic root. Transmembrane action potentials (AP) were recorded close to the sites of extracellular electrograms. Pin electrodes at the periphery of the bath were arranged to register two ECG leads from the volume conductor. During recovery of electrical activity 11 of 14 preparations developed a junctional rhythm that initially manifested only an AV nodal extracellular and corresponding intracellular AV nodal potentials followed gradually by conduction to the Hb and ventricles but no retrograde atrial activation; 3 preparations initially produced Hb rhythms based on extracellular and transmembrane AP recordings from the AVN and Hb. The amplitude and duration of the AVN extracellular potentials (average: 97 ± 26 V and 92 ± 25 msec, respectively) during AVN rhythms, significantly differed from those during atrial pacing (262 ± 185 V and 78 ± 26 msec, p < 0.05). Histologic sections of the sites underlying the electrodes recording AVN potentials showed AVN tissue throughout. We conclude that extracellular AV nodal potentials are independent waveforms with specific qualitative and quantitative characteristics that distinguish them from adjacent atrial, transitional, Hb or ventricular potentials.     

Angiotensin 1-9 and 1-7 release in human heart: role of cathepsin A

Human heart tissue enzymes cleave angiotensin (Ang) I to release Ang 1-9, Ang II, or Ang 1-7. In atrial homogenate preparations, cathepsin A (deamidase) is responsible for 65% of the liberated Ang 1-9. Ang 1-7 was released (88% to 100%) by a metallopeptidase, as established with peptidase inhibitors. Ang II was liberated to about equal degrees by ACE and chymase-type enzymes. Cathepsin A's presence in heart tissue was also proven because it deamidated enkephalinamide substrate by immunoprecipitation of cathepsin A with antiserum to human recombinant enzyme and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. The products of Ang I cleavage, Ang 1-9 and Ang 1-7, potentiated the effect of an ACE-resistant bradykinin analog and enhanced kinin effect on the B(2) receptor in Chinese hamster ovary cells transfected to express human ACE and B(2) (CHO/AB), and in human pulmonary arterial endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and nitric oxide (NO) release by kinin. Direct assay of NO liberation by bradykinin from endothelial cells was potentiated at 10 nmol/L concentration, 2.4-fold (Ang 1-9) and 2.1-fold (Ang 1-7); in higher concentrations, Ang 1-9 was significantly more active than Ang 1-7. Both peptides had traces of activity in the absence of bradykinin. Ang 1-9 and Ang 1-7 potentiated bradykinin action on the B(2) receptor by raising arachidonic acid and NO release at much lower concentrations than their 50% inhibition concentrations (IC(50)s) with ACE. They probably induce conformational changes in the ACE/B(2) receptor complex via interaction with ACE.     

Patterns of elevation of plasma 2'-deoxyuridine, a surrogate marker of thymidylate synthase (TS) inhibition, after administration of two different schedules of 5-fluorouracil and the specific TS inhibitors raltitrexed (Tomudex) and ZD9331.

5-Fluorouracil (5-FU) exerts cytotoxic effects through inhibition of thymidylate synthase (TS) and incorporation of metabolites into RNA. TS inhibition may be greater for infusional 5-FU, with bolus regimens more likely to cause RNA effects. Elevation of plasma 2'-deoxyuridine (dUrd) is a surrogate marker of TS inhibition. Nineteen patients were treated with continuous infusion (CI) 5-FU 300mg/m(2)/day or bolus 5-FU 425mg/m(2)/day plus leucovorin (LV) 20mg/m(2)/day days 1-5. Pretreatment (day 1) and day 2, 3, 4, 5, 8, 15, 22, and 29 plasma samples were assayed for dUrd by reverse-phase high-performance liquid chromatography. In patients treated with bolus 5-FU/LV, dUrd elevation at 24 and 48 h was 235 +/- 125 and 254 +/- 119%, respectively, falling to 138 +/- 58%, 156 +/- 89%, and 92 +/- 25% on days 8, 15, and 22, respectively. dUrd elevation with CI 5-FU was 229 +/- 86% at 24 h and 239 +/- 86, 240 +/- 98%, and 255 +/- 109% at days 15, 22, and 29, respectively. Duration of dUrd elevation was generally less than 8 days for bolus 5-FU/LV. A single dose of raltitrexed (3 mg/m(2)) gave a similar profile to this regimen. ZD9331 (130 mg/m(2), days 1 and 8) gave dUrd elevation for 14 of 21 days, with some recovery prior to day 8. Thus, both 5-FU regimens inhibit TS, and prolonged TS inhibition is achieved by CI 5-FU without significant toxicity. This suggests that the mechanism of antiproliferative toxicity from bolus 5-FU/LV is partly non-TS mediated. These results clarify underlying pharmacodynamic processes and could guide scheduling of 5-FU and TS inhibitors.     

Training in back care to improve outcome and patient satisfaction. Teaching old docs new tricks

BACKGROUND: We examined clinical outcomes and patient perceptions of back care given by physicians before and after an intensive course of training in back care and limited manual therapy techniques. METHODS: From a prospective observational cohort study of low back pain involving 208 physicians (115 primary care) and their patients and a subsequent clinical trial of treatment of low back pain given by 31 physicians specially trained in manual therapy and enhanced back care, outcome data from the patients of 13 physicians participating in both studies were compared. In the observational study, the 13 physicians cared for 120 patients. In the manual therapy trial (191 patients) a control group of 94 patients received enhanced back care and an intervention group of 97 patients received enhanced back care plus manual therapy. Pearson's chi-square comparisons and linear and Cox proportional hazard modeling were used to examine effects of variables and recovery time. RESULTS: Characteristics of the 13 physicians' patients in the cohort group and the manual therapy trial showed some differences in income, workers' compensation, previous employment, and baseline dysfunction. Both control and intervention patients in the manual therapy trial showed more rapid improvement in functional status over time and greater satisfaction with their care than those in the previous cohort study. However, there was no difference between the studies in patient-reported time to return to performing usual daily activities. CONCLUSIONS: A structured clinical approach to low back care may bring modestly improved clinical outcomes and patient satisfaction.     

Outpatient needle biopsy of the lung: its safety and utility

Four hundred forty-seven needle biopsies of the lung were carried out on 348 patients in a nonhospital outpatient setting. There were no fatalities or serious complications. A reliable working diagnosis was obtained in 74% of patients (86% true positive for malignancy). Diagnostic thoracotomy was avoided in 31%, and the diagnostic interval was shortened in most. Impressive economic benefits resulted. Careful postbiopsy evaluation revealed pneumothorax in 41% of patients, with 10% requiring chest tube drainage, usually on an ambulatory basis. With management of the infrequent complications by the radiologist, outpatient needle biopsy of the lung can be safe, reliable, and highly cost effective.