Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice.

BACKGROUND: Extracellular matrix synthesis and degradation contribute to the morphological changes that occur after myocardial infarction (MI). METHODS AND RESULTS: We tested the hypothesis that inhibition of matrix metalloproteinases (MMPs) attenuates left ventricular remodeling in experimental MI. Seventy-one male FVB mice that survived ligation of the left anterior coronary artery were randomized to a broad-spectrum MMP inhibitor (CP-471,474) or placebo by gavage. Echocardiographic studies were performed before randomization (within 24 hours of surgery) and 4 days later and included short-axis imaging at the midpapillary and apical levels. Infarction as defined by wall motion abnormality was achieved in 79% of the procedures (n=56), and mortality rate during the 4-day protocol was 23% (9 of 36 on treatment vs 7 of 35 on placebo; P=NS). Baseline end-diastolic and end-systolic dimensions and areas were similar (P=NS) between treated and placebo groups. At follow-up, infarcted mice allocated to MMP inhibitor had significantly smaller increases in end-systolic and end-diastolic dimensions and areas at both midpapillary and apical levels compared with infarcted mice allocated to placebo (all P<0.05). In addition, infarcted animals that received MMP inhibitor had no change in fractional shortening (-3+/-13%), whereas animals that received placebo had a decrease in fractional shortening (-12+/-12%) (P<0.05). In an analysis stratified by baseline end-diastolic area, the effects of MMP inhibition on the changes in end-systolic area and end-diastolic area were most prominent in animals that had more initial left ventricular dilatation (both P<0.05). CONCLUSIONS: -Administration of an MMP inhibitor attenuates early left ventricular dilation after experimental MI in mice. Further studies in genetically altered mice and other models will improve understanding of the role of MMPs in left ventricular remodeling.

Inhibition of MMP-1 and MMP-13 with phosphinic acids that exploit binding in the S2 pocket.

Through the use of empirical and computational methods, phosphinate-based inhibitors of MMP-1 and MMP-13 that bind into the S2 pocket of these enzymes were designed. The synthesis and testing of 2 suggested that binding was occurring as hypothesized. Structure determination of a co-crystal of 2 bound to the catalytic domain of MMP-1 confirmed the binding mode. Substituents binding into S2, S1', S2' and S3', were optimized yielding compounds with low double-digit nM IC50's against these enzymes.

The preclinical pharmacological profile of the potent and selective leukotriene B4 antagonist CP-195543.

CP-195543 [(+)-2-(3-benzyl-4-hydroxy-chroman-7-yl)-4-trifluoromethyl-benzoic acid] is a structurally novel, selective and potent leukotriene B4 (LTB4) receptor antagonist. In vitro CP-195543 inhibited [3H]LTB4 binding to high-affinity LTB4 receptors on human neutrophils (HN) and murine spleen membranes with IC50 values of 6.8 nM (Ki = 4.9 nM) and 37.0 nM (Ki = 26.9 nM), respectively. CP-195543 inhibited human and mouse neutrophil chemotaxis mediated by LTB4 with IC50 values of 2.4 nM and 7.5 nM, respectively. Evidence of noncompetitive antagonist effects on the HN high-affinity LTB4 receptor was obtained by Scatchard analysis of [3H]LTB4 binding to and chemotaxis of HN to LTB4. Scatchard analyses of [3H]LTB4 binding to low-affinity receptors on HN indicated that CP-195543 acted as a competitive antagonist at this receptor, and inhibition of LTB4-mediated CD11b up-regulation on HN was inhibited competitively by CP-195543 (pA2 = 7.66). In whole blood, CP-195543 also blocked CD11b up-regulation on HN (pA2 = 7.12) and murine neutrophils (pA2 = 7.06) with a similar potency. LTB4-mediated CD11b up-regulation on human monocytes and eosinophils in whole blood were inhibited by CP-195543 with IC50 values of 270 nM and 420 nM, respectively. CP-195543 at 10 microM failed to inhibit HN chemotaxis and CD11b up-regulation mediated through alternative (i.e., complement fragment 5a, interleukin-8, platelet-activating factor) G-protein-coupled chemotactic factor receptors. In vivo, after oral administration, CP-195543 blocked LTB4-mediated neutrophil infiltration in guinea pig and murine skin with ED50 values of 0.1 mg/kg and 2.8 mg/kg p.o., respectively. When administered in osmotic pumps, CP-195543 reduced the clinical symptoms and attendant weight loss in an IL-1-exacerbated murine model of collagen-induced arthritis with half-maximal effects associated with plasma drug levels of 0.4 to 0.5 microg/ml. Collectively these data provide evidence of the in vitro potency and in vivo efficacy of a novel LTB4 antagonist and support its clinical evaluation in a variety of inflammatory diseases in man.

Model development and analysis of tenidap-induced proteinuria in the rat.

Tenidap is a novel antirheumatic agent that causes a mild, reversible proteinuria in human clinical trials. In order to achieve a mechanistic understanding and safety perspective of the proteinuric effects of tenidap observed in clinical trials, female Sprague-Dawley rats were treated with up to 100 mg/kg/day of tenidap in the diet for 4 to 6 weeks followed by a 1- to 6-week reversal period. Pharmacokinetics and measurements of renal function and histology were assessed during the study. Sustained high plasma concentrations of tenidap [area under the plasma concentration curve (0-24 hr) of 941-1021 micrograms. hr/ml and peak plasma concentration of 61-67 micrograms/ml] increased urinary protein, albumin and phosphate excretion (2- to 8-fold) in rats. These renal effects were reversible within 9 days after removal of the drug. These effects preceded later occurring changes in renal morphology (papillary degeneration and necrosis). There was no evidence of glomerular damage, proximal tubule degeneration or necrosis or tubulointerstitial nephritis at the light microscopic level. Other indices of overall renal function (glomerular filtration rate, electrolyte and glucose excretion) were unaffected. Examination in situ of microperfused proximal tubules from treated rats revealed a 68% decrease in the rate of proximal tubule albumin absorption compared to controls (19 +/- 4 vs. 59 +/- 7 pg/min/mm, respectively). Fluid absorption rate and bicarbonate handling by the proximal tubule, along with blood bicarbonate concentrations, pH, PCO2 and PO2, were unaffected by treatment. It was concluded that tenidap caused a rapid, stable and reversible phosphaturia, microalbuminuria and proteinuria in the rat. The proteinuric effects were due to impaired proximal tubule albumin reabsorption that were not associated with other signs of impaired renal function or histological evidence of tubulointerstitial nephritis or proximal tubule/glomerular damage.

Preparation of biodegradable microcapsules containing zidovudine (AZT) using solvent evaporation technique.

Sustained release biodegradable microcapsules of AZT were prepared using different concentrations of copolymer of poly(lactic/glycolic) acid (PLGA 50:50 and PLGA 90:10). Solid microcapsules were collected following the complete evaporation of the solvent. The yield of microcapsules was increased two fold with a two-fold increase of the polymer concentration. The efficiency of encapsulation of AZT was also increased with the increase of the polymer concentration. These microcapsules were characterized using scanning electron microscopy. The dissolution of AZT from the microcapsules of PLGA (50: 50) was higher than the microcapsules of PLGA (90:10); the PLGA (50:50) microcapsules containing 1:10 drug/polymer ratio showed higher dissolution than the microcapsules containing 1:20 drug/polymer ratio. The PLGA (90:10) microcapsules containing 1:6 drug/polymer ratio showed higher dissolution than the microcapsules containing 1:10 drug/polymer ratio. In conclusion, the dissolution of AZT was dependent on the type of the copolymer used and the relative concentrations of the drug and the copolymer.

In vitro and in vivo effects of leukotriene B4 antagonism in a primate model of asthma.

To test the hypothesis that leukotriene (LT) B4 antagonists may be clinically useful in the treatment of asthma, CP-105,696 was evaluated in vitro, using chemotaxis and flow cytometry assays, and in vivo, using a primate asthma model. CP-105,696 inhibited LTB4-mediated monkey neutrophil chemotaxis (isolated cells, LTB4 = 5 nM) and CD11b upregulation (whole blood, LTB4 = 100 nM) with IC50 values of 20 nM and 16.5 microM, respectively. Using a modification of a previously described in vivo protocol (Turner et al. Am. J. Respir. Crit. Care Med. 1994. 149: 1153-1159), we observed that treatment with CP-105,696 inhibited the acute increase in bronchoalveolar lavage (BAL) levels of IL-6 and IL-8 by 56.9 +/- 13.2% and 46.9 +/- 14.5%, respectively, 4 h after challenge with Ascaris suum antigen (Ag). CP-105,696 tended to reduce the increase in BAL protein levels 0.5 h after Ag challenge by 47.5 +/- 18.3%, but this was not statistically significant. In addition, CP-105,696 prevented the significant 11-fold increase in airway responsiveness to methacholine after multiple Ag challenge. These results suggest that LTB4 partially mediates acute and chronic responses to antigen in an experimental primate asthma model and support the clinical evaluation of LTB4 antagonists in human asthma.

Effects of tenidap on canine experimental osteoarthritis. I. Morphologic and metalloprotease analysis.

OBJECTIVE: To examine the effects of tenidap and diclofenac on osteoarthritic lesions and metalloprotease activity in experimental osteoarthritis (OA). METHODS: The anterior cruciate ligament of the right stifle joint of 25 mongrel dogs was sectioned by a stab wound. Seven dogs received no treatment, 6 were treated with oral omeprazole (20 mg/day), another 6 were treated with diclofenac (0.25 mg/kg/twice daily) plus omeprazole (20 mg/day), and 6 received oral tenidap (3 mg/kg/twice daily) plus omeprazole (20 mg/day). The dogs received medication for 8 weeks; all dogs were killed at the end of this period. Eight normal dogs were used as controls. Lesions were evaluated macroscopically for the incidence and size of osteophytes and the area and grade of cartilage erosions on the condyles and plateaus, along with histologic evaluation of the severity of the cartilage lesions and synovial inflammation. Stromelysin and collagenase activities and the collagenase messenger RNA (mRNA) level were measured in cartilage and synovial membrane. RESULTS: Compared with the untreated or omeprazole-treated OA groups, the dogs treated with tenidap exhibited significant reduction in the incidence (P < or = 0.001) and size (P < or = 0.0001) of osteophytes. Tenidap also significantly decreased the size and grade of cartilage macroscopic lesions, as well as the histologic severity of cartilage lesions on both condyles and plateaus. The histologic severity of synovial inflammatory reaction was also significantly reduced (P < or = 0.003) in the tenidap group. Tenidap markedly decreased stromelysin and collagenase activity in both cartilage (stromelysin P < or = 0.003; collagenase P < or = 0.01) and synovial membrane (stromelysin P < or = 0.003; collagenase P < or = 0.005). Moreover, tenidap also decreased the collagenase mRNA level in cartilage (P < or = 0.005) and synovial membrane (P < or = 0.002). Diclofenac slightly reduced the incidence and size of osteophytes and cartilage lesions, but these changes were not statistically significant. Diclofenac had no effect on the severity of synovial inflammation, metalloprotease activity, or collagenase expression. CONCLUSION: This study showed that tenidap had a more potent anti-osteoarthritic effect than diclofenac in this model. The effect of the drug in suppressing metalloprotease synthesis, a process known to play a major role in the pathophysiology of osteoarthritic lesions, may explain its mechanism of action.

Treatment of the cutaneous pain of acute herpes zoster with 9% lidocaine (base) in petrolatum/paraffin ointment.

BACKGROUND: Treatment of the pain of acute herpes zoster by local anesthetic injections has drawbacks. Topical percutaneous local anesthesia (TPLA) may offer another strategy of providing regional analgesia in affected patients. OBJECTIVE: We evaluate the analgesic efficacy and safety of 9% (wt/vol) lidocaine (base) in petrolatum/paraffin ointment in patients with acute herpes zoster. METHODS: Ointment was applied to the affected skin of 22 patients. Pain, tenderness, sensitivity to pinprick and cold, and blood lidocaine concentration were measured repeatedly during a 20-hour interval and intermittently thereafter. RESULTS: Mean pain, tenderness, and cutaneous sensation scores were reduced at measurements taken from 4 to 20 hours after ointment application (p < 0.05), but not every patient obtained relief. No patient had local skin irritation or systemic toxic effects related to the local anesthetic. CONCLUSIONS: TPLA is a promising therapy for control of cutaneous pain of acute herpes zoster. Controlled studies should be performed to prove efficacy, determine optimal TPLA formulation, and define dosage limits.

Pharmacokinetics and pharmacodynamics in copper deficiency. I. Antiinflammatory activity of aspirin.

The effect of nutritional copper (Cu) deficiency on the antiinflammatory activity and pharmacokinetics of aspirin (ASA) was investigated in rats. Male, weanling Sprague-Dawley rats were fed either a Cu-deficient (CuD) or Cu-sufficient (CuS) diet for 49-50 d. The antiinflammatory activity of ASA was studied using the carrageenan-induced paw edema (CPE) test. ANOVA analyses of edema volumes at 2, 3, 4, 5, and 21 h postcarrageenan indicated significant differences between groups. The percent inhibition of edema due to ASA treatment in CuS was lower than that in CuD rats at 5 h, AUC5h, and AUC21h. ASA was found to be significantly more effective in inhibiting the CPE in CuD rats when compared to the CuS rats. Thus, we hypothesized that the increase in ASA's antiinflammatory activity in CuD rats was a result of a decrement in its elimination during nutritional Cu deficiency. The elimination of ASA in CuD and CuS rats was studied using an iv dose of 200 mg/kg. Concentrations of ASA and salicylic acid (SA) were determined in blood; whereas the concentrations of SA, salicylic phenol-glucuronide (SPG), and salicyluric acid (SUA) were determined in urine by HPLC. The results of the pharmacokinetic analyses from blood and urinary data indicated no significant differences in the disposition of ASA between CuD and CuS rats. For instance, the total body clearance for ASA (mean +/- SD, mL/min/kg) was 37.9 +/- 9.4 and 38.5 +/- 13.9 (p > 0.05); and the volume of distribution (Vd) for ASA (mean +/- SD, mL/kg) was 385.5 +/- 110.3 and 397.1.1 +/- 137.9 (p > 0.05) for CuD and CuS groups, respectively. Thus, contrary to our hypothesis, the enhanced antiinflammatory activity of ASA in CuD rats does not appear to be mediated via a decrement in the elimination of the drug. In addition, plasma ASA-esterase activity was found to be independent of Cu nutritional status.

Stability of fluconazole in an extemporaneously prepared oral liquid.

The stability of fluconazole in an extemporaneously prepared oral liquid was studied. An aqueous liquid formulation of fluconazole was prepared by reconstituting the powder from triturated 100-mg tablets with deionized water; the nominal fluconazole concentration was 1 mg/mL. Glass vials of the liquid were stored in the dark at 4, 23, and 45 degrees C and sampled immediately and after 1, 2, 3, and 15 days. Samples were analyzed in duplicate for fluconazole concentration by high-performance liquid chromatography. The concentration of fluconazole was virtually unchanged under all the storage conditions. The results were confirmed by analysis of variance. Fluconazole 1 mg/mL in an extemporaneously prepared oral liquid was stable at 4, 23, and 45 degrees C for up to 15 days.

Effect of age on distribution of zidovudine (azidothymidine) into the cerebrospinal fluid of Macaca nemestrina.

The brain tissue is an important target for anti-HIV drug therapy. Since the permeability of the blood-brain and blood-cerebrospinal fluid (CSF) barriers may differ between neonates and adults, we have determined the effect of age on the distribution of zidovudine (ZDV or azidothymidine) into the CSF in the macaque (M. nemestrina). Five newborn macaques were administered ZDV (iv bolus, 5 mg/kg) at various ages (2 days to 4 months). Both CSF (cisternal) and venous blood samples were obtained at approximately 60 and 90 min after drug administration. In another series of experiments, adult female macaques received ZDV as either an iv bolus (5 and 10 mg/kg) or an infusion for at least 12 hr. CSF (lumbar) and venous blood samples were obtained at approximately 60 and 90 min after iv bolus and at more than 12 hr after iv infusion. ZDV concentration in the CSF and the plasma samples was determined by high-performance liquid chromatography. The CSF/plasma concentration ratio of ZDV in the newborn and adult macaques, after iv bolus administration, was independent of time. In addition, no significant (P > 0.05) difference was observed in the pooled iv bolus ZDV CSF/plasma concentration ratio between the adult group (0.236 +/- 0.058) and the newborns (0.213 +/- 0.039). Moreover, the ZDV CSF/plasma concentration ratio in the adults and the newborns, after iv bolus administration, was found not to be significantly (P > 0.05) different from the ratio obtained at steady state in the adults (0.224 +/- 0.094). These data indicate that the distribution of ZDV into the CSF in macaque neonates and adults is similar.

Pharmacokinetics of zidovudine (azidothymidine). III. Effect of pregnancy.

Administration of zidovudine (ZDV or azidothymidine) to pregnant women with HIV infection may be of benefit to both the mother and her unborn child. Since pregnancy can have a substantial effect on the pharmacokinetics of a drug, the effect of pregnancy on the pharmacokinetics of ZDV (10 mg/kg, i.v. bolus) was studied in the macaque (Macaca nemestrina). The plasma clearance, steady-state volume of distribution, and terminal half-life of ZDV were found not to be significantly affected by pregnancy. Based on these findings, we predict that the pharmacokinetics of ZDV in women will not be affected by pregnancy. If this prediction is found to be correct, the dose of ZDV need not be adjusted when the drug is administered to pregnant women.

Simple and effective procedure for complete urine collection from infant macaques (Macaca nemestrina).

A diaper method, used in pediatric medicine, has been adapted and validated for total urine collection from infant macaques (Macaca nemestrina). The device consists of cellulose sponges and polyethylene sheets. The method proposed is non-invasive, simple, and does not significantly hinder the movement of the infant. The method should be useful when one is conducting pharmacokinetic studies in which total urine collection is required.

Pharmacokinetics of zidovudine (azidothymidine). I. Transplacental transfer.

Administration of zidovudine (ZDV) to pregnant women with human immunodeficiency virus infection may be of benefit to both the mother and the unborn child. Before testing this hypothesis, however, it is necessary to determine the transplacental transfer, fetal toxicity, and fetal accumulation of ZDV (if any) in a representative animal model. Therefore, the transplacental transfer and the fetal accumulation of ZDV were determined at steady state in near-term pregnant macaques (Macaca nemestrina). ZDV was administered to five dams at a rate predicted to produce a steady-state plasma concentration of about 1 microgram/ml. When steady state was predicted to have been achieved, a cesarean section was performed on each dam. At this time, blood samples from the dam (peripheral vein) and the fetus (umbilical vein) were obtained simultaneously. The plasma concentration of ZDV and its major metabolite, zidovudine glucuronide (ZDVG), were determined by a specific high-performance liquid chromatography (HPLC) method. The ratio of steady-state plasma concentration (Crss) of ZDV in the fetus (Cssf) to that in the dam (Cssd) (Crss = Cssf/Cssd) was found to be close to unity (0.826 +/- 0.067). Similar results were obtained for the ratio of steady-state unbound ZDV plasma concentration (0.852 +/- 0.083). We conclude that ZDV readily crosses the placenta, probably by passive diffusion, and that ZDV does not accumulate in the fetus when administered to near-term pregnant macaques.

Pharmacokinetics of zidovudine (azidothymidine). II. Development of metabolic and renal clearance pathways in the neonate.

The pharmacokinetics of zidovudine (ZDV or azidothymidine) were investigated in newborn macaques (Macaca nemestrina) at various ages ranging from less than 1 week to 4 months. The mean ZDV total plasma clearance, renal clearance, and the metabolic clearance of ZDV to the glucuronide (ZDVG) were significantly (p less than 0.05) smaller during the first week of life (6.15 +/- 1.03, 4.25 +/- 0.36, and 1.19 +/- 0.67 ml/min/kg, respectively) than the corresponding estimates at the age of 4 months (19.62 +/- 3.5, 8.28 +/- 1.90, and 8.28 +/- 2.24 ml/min/kg, respectively). The mean estimates of these parameters at 4 months were close to those found in adult macaques (23.55 +/- 1.48, 10.05 +/- 0.75, and 10.5 +/- 1.9 ml/min/kg, respectively), indicating that these clearance pathways develop rapidly, within 4 months of birth. If similar results are obtained in human neonates, therapeutic drug monitoring should be instituted when administering ZDV to this population so that the dose of ZDV may be adjusted to correspond with age-related changes in total plasma clearance of ZDV.

Quantification of riboflavin, riboflavin 5'-phosphate and flavin adenine dinucleotide in plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method with fluorimetric detection for the quantification of riboflavin (RB), riboflavin 5'-phosphate (FMN), and flavin adenine dinucleotide (FAD) in plasma, whole blood, and urine is described. Under isocratic conditions with a reversed-phase column, the compounds are completely resolved and eluted within 9 min. Plasma proteins are precipitated with acetonitrile followed by shaking the aqueous phase with chloroform. Urine samples are diluted and injected directly. The reproducibility of this method for the quantification of RB in plasma has a between-day coefficient of variation of 6%. The application of this method is illustrated by analyzing plasma and urine samples from a human subject who received an intravenous dose of FMN equivalent to 25 mg of RB.

Ascorbic and dehydroascorbic acids simultaneously quantified in biological fluids by liquid chromatography with fluorescence detection, and comparison with a colorimetric assay.

We describe a "high-performance" liquid-chromatographic method for separating and quantifying ascorbic acid (AA) and dehydroascorbic acid (DHA) in plasma and urine. We used a reversed-phase C18 column with an ion-pair reagent and detected the analytes by post-column reaction with 4,5-dimethyl-o-phenylenediamine to form a fluorescent derivative (measured at excitation and emission wavelengths of 365 and 440 nm, respectively). Isoascorbic acid (IA) is the internal standard. Retention times for DHA, AA, and IA are 5.6, 15.5, and 19.9 min, respectively. Between-day CVs for AA in plasma in concentrations of 8 and 20 mg/L were 9% and 7%, respectively. The limit of detection is 10 and 4 ng for AA and DHA, respectively. Results by the present method and the methoxyaniline colorimetric method for AA are comparably accurate.

Vigilancia de niveles plasmaticos de difenilhidantoina en pacientes epilepticos. / Surveillance of diphenylhydantoin serum levels in epileptic patients

Se presentan los resultados de un estudio realizado en 83 pacientes epilepticos que acudem periodicamente al Servicio de Electroencefalografia del Hospital Infantil de Mexico "Federico Gomez". El objetivo del mismo fue determinar las concentraciones plasmaticas de difenilhidantoina (DFH), obtenidas con diferentes dosis y relacionarlas con el estado clinico del paciente y su electroencefalograma; las muestras sanguineas se analizaron por el metodo de cromatografia de gas liquido. De acuerdo a los hallazgos encontrados, se describe que 55.4% de los pacientes estudiados presentan una concentracion plasmatica de DFH inferior a la considerada como terapeutica; el 38.4% de 39 pacientes no tienen un adecuado control de sus crisis convulsivas y el 46.1% de este grupo presentaron un trazo electroencefalografico anormal y una concentracion plasmatica de DFH inferior a la descrita como terapeutica por diferentes autores