Local delivery of gene vectors from bare-metal stents by use of a biodegradable synthetic complex inhibits in-stent restenosis in rat carotid arteries.

BACKGROUND: Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. METHODS AND RESULTS: We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37 degrees C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial Ad(GFP) expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. CONCLUSIONS: Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature.

Pharmacokinetics and metabolism of a RAS farnesyl transferase inhibitor in rats and dogs: in vitro-in vivo correlation.

Compound I (1-(3-chlorophenyl)-4-[(1-(4-cyanobenzyl)-1H-imidazol-5-yl)methyl]piperazin-2-one) is a potent and selective inhibitor of farnesyl-protein transferase (FPTase). The pharmacokinetics and metabolism of compound I displayed species differences in rats and dogs. After oral administration, the drug was well absorbed in dogs but less so in rats. Following i.v. administration, compound I was cleared rapidly in rats in a polyphasic manner with a terminal t(1/2) of 41 min. The plasma clearance (CL(p)) and volume of distribution (V(dss)) were 41.2 ml/min/kg and 1.2 l/kg, respectively. About 1% of the dose was excreted in rat bile and urine as unchanged drug over a period of 24 h, suggesting that biotransformation is the major route of elimination of compound I. Using liquid chromatography (LC)-tandem mass spectometry, nineteen metabolites of compound I were identified in urine and bile from dogs and rats. Structures of two major metabolites were confirmed by LC-NMR. N-Dealkylation and phase II metabolism were the major metabolic pathways. Animal and human liver microsomal intrinsic clearance values were scaled to predict hepatic clearance and half-life in humans, and the predicted values were in good agreement to the in vivo data.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

Sex-dependent pharmacokinetics of indinavir: in vivo and in vitro evidence.

Indinavir, a potent and specific inhibitor of human immunodeficiency virus protease, is used for the treatment of AIDS. This study was designed to investigate the sex-related differences in kinetics and metabolism of indinavir in rats, dogs, and monkeys to support the toxicity studies. When given intravenously, indinavir was cleared rapidly in a polyphasic manner in all species. Indinavir exhibited significant differences in elimination kinetics among species. The rat had the highest plasma clearance (CLp; 41-89 ml/min/kg), and the dog had the lowest CLp (15-26 ml/min/kg), with the monkey exhibiting an intermediate value (36-39 ml/min/kg). Furthermore, marked sex-related differences in CLp were observed in rats and dogs, but not in monkeys. The CLp was 89 ml/min/kg for male rats and 41 ml/min/kg for female rats. In contrast to rats, female dogs cleared indinavir more rapidly than male dogs; the CLp was 26 ml/min/kg for female dogs and 15 ml/min/kg for male dogs. Consistent with the in vivo observations, hepatic microsomes from male rats had a substantially higher metabolizing activity toward indinavir than that from females, whereas liver microsomes from female dogs catalyzed the drug at a higher rate than that from male dogs. Qualitatively, in vitro metabolic profiles of indinavir were similar among species and between male and female animals. Studies with an anti-rat cytochrome P450 (CYP) 3A1 antibody pointed to the probable involvement of isoforms in the CYP3A subfamily in the oxidative metabolism of indinavir in both males and females of all species. The functional activity of CYP3A measured by the formation of testosterone 6beta-hydroxylation and immunoblot analysis of the level of CYP3A proteins strongly suggested that gender differences in the levels of CYP3A isoforms may contribute to the observed sex-related differences in indinavir metabolism in rats and dogs.

Regiospecific intestinal absorption of the HIV protease inhibitor L-735,524 in beagle dogs.

PURPOSE: To evaluate regional intestinal absorption and the feasibility of sustained release dosage form development for an HIV protease inhibitor, L-735,524, METHODS: L-735,524 free base or sulfate salt was administered orally as suspension, solution or in solid dosage forms to fasted or fed Beagle dogs. Delayed-release dosage forms with "slow" or "fast" in vitro dissolution rates were evaluated in vivo to assess plasma concentration profiles. In addition, drug was administered directly into the jejunum or colon of animals, and drug concentrations determined in portal circulation to characterize absorption from these sites. RESULTS: L-735,524 sulfate was well absorbed orally form a solution or capsule formulation if fasted animals' stomachs were preacidified with citric acid solution. A free base suspension, delivered in divided doses to fed animals, was also well absorbed. Prototype extended release dosage forms of L-735,524 produced a reduction in peak plasma levels but failed to prolong absorption and extend plasma concentrations compared to an immediate release capsule. Administration of L-735,524 sulfate solution (pH < 3) as bolus solution or by infusion into the jejunum resulted in rapid but incomplete absorption compared to oral gavage. The free base suspension (pH 6.5) delivered into jejunal or colonic regions did not produce measurable systemic plasma concentrations. CONCLUSIONS: Extended release formulations did not prolong absorption of L-735,524 in dogs. Optimal L-735,524 absorption was dependent on solubility in an acidic environment in the duodenum.

Dose-dependent toxicokinetics of L-689,502, a potent human immunodeficiency virus protease inhibitor, in rats and dogs.

L-689,502, N-[2(R)-hydroxy-1(S)-indanyl]-5(S)-(1,1-dimethylethoxy- carbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-(4-[2-(4- morpholinyl)ethoxy]phenyl)methyl hexanamide, is a potent inhibitor of human immunodeficiency virus-1 protease. The effect of dose on the elimination kinetics of L-689,502 was studied in rats and dogs. After i.v. administration, total plasma clearance of L-689,502 in rats decreased with increasing dose; the clearance decreased from 181 ml/min/kg at 1 mg/kg to 86 ml/min/kg at 20 mg/kg. Similar results were observed in dogs; clearance fell from 29 ml/min/kg at 0.5 mg/kg to 17 ml/min/kg at 10 mg/kg. Bile flow in rats was retarded in a dose-dependent manner after a single i.v. injection of L-689,502. The cholestatic effect was reversible and maximal at 5 mg/kg i.v. Consistent with the cholestatic effect, L-689,502 caused an increase in serum levels of aminotransferase. After i.v. administration of L-689,502 (10 mg/kg), alanine aminotransferase increased from 50 to 370 IU/liter and aspartate aminotransferase from 120 to 700 IU/liter. Moreover, pretreatment of rats with L-689,502 resulted in a significant decrease in the elimination kinetics of antipyrine and diflunisal, as well as of L-689,502 itself. Collectively, these results suggest that the dose-dependent kinetics of L-689,502 in rats and dogs are more likely due to hepatotoxicity caused by the drug than to capacity-limited metabolism.

Physiological disposition and metabolism of L-365,260, a potent antagonist of brain cholecystokinin receptor, in laboratory animals.

L-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl)-N'-(3-methylphenylurea)], a potent nonpeptide antagonist of the CCKB receptor, is currently under investigation to treat anxiety and panic disorders. This study describes absorption and disposition of the drug in rats, dogs, and monkeys. Following iv administration (5 mg/kg), L-365,260 was cleared very rapidly in rats, dogs, and monkeys. In all species, the concentrations of the drug in plasma declined in a polyphasic manner. There was no difference in total blood clearance among species, whereas considerable species differences were observed in volume of distribution and terminal half-lives. Binding of 14C-L-365,260 to plasma protein was extensive for all test species (greater than 96%). Interspecies differences in absorption were also observed. The bioavailability for rats, dogs, and monkeys was approximately 14%, 9%, and 2%, respectively. HPLC radiohistograms of urine and bile revealed that only trace amounts of intact drug were present; the drug was mainly eliminated by biotransformation. NMR and mass spectral analyses indicate that hydroxylation and glucuronide conjugation are the major biotransformation pathways.

Serum osteocalcin measurements in prostate carcinoma patients with skeletal deposits shown by bone scintigram: comparison with serum PSA/PAP measurements.

The correlation of technetium-99m-HMDP bone scintigraphic findings, serum osteocalcin as a measure of bone turnover and prostate-specific antigen (PSA) and/or prostate acid phosphatase (PAP) was determined in 19 men with bone metastasis due to prostatic carcinoma. Six of the 19 patients with metastases on bone scan showed elevation of osteocalcin. These patients had extensive metastatic disease. All 19 men with positive bone scans had high serum PSA and/or PAP levels. Serum osteocalcin measurement is less sensitive to detection of bone deposits than PSA/PAP measurements (p less than 0.0008).

Hematoporphyrin derivative uptake and photodynamic therapy in pancreatic carcinoma.

Little information is currently available concerning the uptake of porphyrins by pancreatic tumors, or the effect of photodynamic therapy (PDT) on pancreatic cancer. In Syrian golden hamsters (n = 33), the organ distribution of 125I-labeled dihematoporphyrin ether (DHE) was studied in a pancreatic cancer model. In the same animal model the effect of PDT was studied using a gold vapor laser for energy delivery 3 hr after the injection of DHE (n = 7). DHE was 2.4 times more concentrated in the pancreatic tumor than in the nontumorous pancreas at 3 hr. Simultaneously there was a considerable accumulation of DHE in the surrounding gastrointestinal tract, causing perforation of the duodenum and jejunum with resultant death in four (57%) animals after PDT. Photodynamic therapy caused extensive tumor necrosis without any obvious effect on the nontumor-bearing pancreas. Damage to the surrounding tissue in the hamster indicates that precautions should be taken if PDT is to be used clinically in pancreatic cancer. Intratumoral injection of DHE may give higher drug concentrations with greater specificity for tumor treatment.

Differential effects of phenobarbital on ester and ether glucuronidation of diflunisal in rats.

The relative contribution of ether and ester glucuronidation to diflunisal metabolism was assessed by studying the effects of enzyme inducers, phenobarbital (PB), 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF). Treatment with either PB, 3-MC or BNF increased markedly the unbound intrinsic clearance of diflunisal. Saline-treated control rats showed a greater unbound intrinsic clearance of diflunisal than oil-treated controls indicating that repetitive treatment with oil had an effect on enzyme activity. Treatment with 3-MC and BNF appeared to cause a decrease in the biliary clearance of ether and ester glucuronide, but PB had little effect on the biliary clearance of glucuronides. Rats pretreated with PB showed a 3-fold increase in the fractional metabolite formation clearance of ether glucuronide and a 2-fold increase in the fractional metabolite formation clearance of ester glucuronide, suggesting differential effects of PB on ester and ether glucuronidation. A similar trend, but to a smaller extent, was also observed for 3-MC- and BNF-treated rats. These results suggest the possibility of selective induction of multiple forms of UDP-glucuronyltransferase involved in metabolism of diflunisal.

Calcium, phosphorus, magnesium, and calcitonin concentrations in the serum and cerebrospinal fluid of children.

Mineral metabolism in the cerebrospinal fluid (CSF) of children is poorly understood. Recent reports have suggested a neuroregulatory role for calcitonin. We examined the hypotheses that in children (1) CSF levels of calcium and phosphorus might be low, (2) CSF levels of magnesium might be higher than serum levels of magnesium, and (3) immunoreactive calcitonin might be present in the CSF. We examined serum and CSF samples of 45 children, aged 8 days to 16 years, undergoing spinal taps for suspected meningitis or as part of leukemia therapy. Both serum and CSF levels of calcium correlated with those of magnesium. There was no correlation for CSF levels vs serum levels of calcium, magnesium, or phosphorus. The CSF levels of calcium and phosphorus were lower than the serum levels of these elements, but the CSF levels of magnesium were higher than the serum levels of magnesium. Calcitonin was detected in the CSF of 8% of samples assayed (range, 14 to 175 ng/L [14 to 175 pg/mL]). Two of these five samples had bacteriologically proven meningitis, and two samples were from patients less than 2 months of age. The CSF levels of calcitonin did not correlate with the serum levels of calcitonin. Thus, in children CSF levels of calcium and phosphorus are low, CSF levels of magnesium are higher than the serum levels, and the level of immunoreactive calcitonin is usually not present in the CSF but possibly is elevated in meningitis and early infancy.

Studies on the possible role of thyroid hormone in altered muscle protein turnover during sepsis.

Five days after thyroidectomy (Tx) or sham-Tx in young male Sprague-Dawley rats, sepsis was induced by cecal ligation and puncture (CLP). Control animals underwent laparotomy and manipulation of the cecum without ligation or puncture. Sixteen hours after CLP or laparotomy, protein synthesis and degradation were measured in incubated extensor digitorum longus (EDL) and soleus (SOL) muscles by determining rate of 14C-phenylalanine incorporation into protein and tyrosine release into incubation medium, respectively. Triiodothyronine (T3) was measured in serum and muscle tissue. Protein synthesis was reduced by 39% and 22% in EDL and SOL, respectively, 16 hours after CLP in sham-Tx rats. The response to sepsis of protein synthesis was abolished in Tx rats. Protein breakdown was increased by 113% and 68% in EDL and SOL, respectively, 16 hours after CLP in sham-Tx animals. The increase in muscle proteolysis during sepsis was blunted in hypothyroid animals and was 42% and 49% in EDL and SOL, respectively. T3 in serum was reduced by sepsis, both in Tx and sham-Tx rats. T3 in muscle, however, was maintained or increased during sepsis. Abolished or blunted response of muscle protein turnover after CLP in hypothyroid animals may reflect a role of thyroid hormones in altered muscle protein metabolism during sepsis. Reduced serum levels of T3, but maintained or increased muscle concentrations of the hormone, suggests that increased T3 uptake by muscle may be one mechanism of low T3 syndrome in sepsis, further supporting the concept of a role for thyroid hormone in metabolic alterations in muscle during sepsis.

Clinical utility of serum prostatic acid phosphatase measurements for detection (screening), diagnosis, and therapeutic monitoring of prostatic carcinoma; assessment of monoclonal and polyclonal enzymes and radioimmunoassays.

Serum prostatic acid phosphatase (PAP), measured by three different technics, was used to screen 560 men over 50 years of age for prostatic cancer. All three PAP procedures failed to detect the one subject found to have prostatic cancer on physical examination and confirmed by biopsy, and all assays had a significant number of "falsely" elevated PAP levels. Four PAP assays were assessed in 80 men undergoing prostatic biopsy/removal in whom histologic examination of prostatic tissue was made. Clinical sensitivity and specificity for detecting those patients with and without prostatic cancer ranged from 20-57% and 54-95%, respectively, with the monoclonal enzyme immunoassay being the most sensitive but the least specific, and the traditional enzyme assay being the most specific but insensitive. In men with diagnosed prostatic cancer undergoing radiation or chemotherapy, the assays were useful in distinguishing between those with active and inactive disease. The authors conclude that serum PAP assays, irrespective of the type of procedure used, has no place in screening for prostatic cancer, a minimal role in establishing a definitive diagnosis, but a useful role in monitoring therapy.

Sensitive radioimmunoassay for measurement of circulating peptide YY.

A radioimmunoassay with sufficient sensitivity to detect the recently discovered, circulating peptide YY in the majority of healthy adults has been developed. The antiserum to synthetic peptide YY raised in rabbits essentially showed no cross-reactivity with bovine or porcine synthetic pancreatic polypeptide, neurotensin, or neuropeptide Y, but cross-reacted to the extent of 0.03% with synthetic peptide HI. The minimum detectable limit of the assay for plasma peptide YY was 20 pg/ml with coefficients of variation less than 16%. The immunoreactivity of peptide YY isolated from healthy adult plasma had a gel-filtration chromatographic profile similar to that of synthetic peptide YY. Fasting levels of peptide YY in plasma from 127 of 137 (92.7%) ostensibly healthy adults were measurable and showed a log-normal distribution. Using the maximum-likelihood estimation method, we calculated the normal range to be 13-178 pg/ml with a geometric mean value of 49 pg/ml. The fasting plasma peptide YY levels showed no significant age or sex dependency.

Relation between effective radiation dose and outcome of radioiodine therapy for thyroid cancer.

We used a combination of radioiodine scanning and quantitative radiation dosimetry to evaluate responses to therapeutic irradiation with 131I in 76 patients with thyroid adenocarcinoma. Fifty patients received 131I treatment for ablation of residual thyroid tissue after surgical thyroidectomy, and 26 had 131I treatment for metastatic thyroid cancer. Successful ablation was observed in patients receiving higher radiation doses to the thyroid--about 4.4 times those in patients whose lesions were not ablated--largely because of a longer effective half-life of 131I in residual thyroid tissue in the patients with ablated lesions. Patients with metastases that persisted after 131I therapy tended to have more advanced disease and received significantly lower radiation doses per millicurie of administered 131I than did persons whose lesions responded to treatment. Initial 131I treatment resulting in radiation doses of at least 30,000 rad to thyroid remnants and 8000 rad to metastases was associated with a significant increase in the rate of response to therapy.

Effects of growth hormone replacement therapy on 1,25-dihydroxyvitamin D and calcium metabolism.

To elucidate the changes in mineral metabolism and blood concentrations of calciotropic hormones which accompany GH therapy, we studied 12 GH-deficient children for 5 days before and for 1 week after high dose (5 IU/day) GH therapy, and again at 1 month, 3 months, and 1 yr of replacement therapy (0.1 IU/kg to a maximum dose of 2 IU three times weekly). All responded with acceleration of height velocity, and bone ages advanced appropriately. Fasting serum ionized calcium levels did not change: 4.11 +/- 0.06 (SEM) mg/dl before, 4.19 +/- 0.05 for the week of high dose therapy, and 4.20 +/- 0.14 during replacement therapy. Likewise, fasting serum parthormone did not vary: 38.9 +/- 2.6 muleq/ml before to 44.1 +/- 9.2 at 1 yr. Twenty four-hour nephrogenous cyclic AMP (NcAMP) did not vary over the first week (1.2 +/- 0.7 nmol/dl glomerular filtrate before, 1.3 +/- 0.4 after 1 week), but increased to 5.3 +/- 1.9 after 1 yr (alpha less than 0.001). The response of ionized calcium and parathormone to a standardized disodium EDTA infusion of 50 mg/kg also did not change. The mean fasting serum calcitonin level was not different before therapy (29.4 +/- 2.8 pg/ml), after 1 week (21.5 +/- 1.8), or after 1 yr (42.4 +/- 11.0). However, the mean serum 1,25-dihydroxyvitamin D concentration rose from 33.1 +/- 3.3 pg/ml before therapy to 68.3 +/- 12.3 on the seventh day of high dose therapy (alpha less than 0.01), returning to pretherapy values by 1 month. We conclude that high dose GH therapy in GH-deficient children raises 1,25-dihydroxyvitamin D concentration acutely, but that long term, physiological replacement therapy does not cause such an effect. Because NcAMP excretion rose in the absence of an increase in serum parathormone concentration, we conclude that GH sensitizes the kidney to a cAMP-mediated effect of parathormone.

Low iodine diet in I-131 ablation of thyroid remnants.

A low-iodine diet was developed for used in decreasing iodine intake and excretion in patients undergoing evaluation with radioactive I-131 for ablation of thyroid remnants as treatment for thyroid cancer. It has been demonstrated to effectively lower iodine excretion to less than 25% of basal values. Preliminary calculations suggest that such iodine depletion may be potentially useful in increasing the radiation dose per mCi of administered activity in I-131 ablative therapy.