Complication rates of percutaneous brachial artery puncture: effect of live ultrasound guidance.

PURPOSE: The current literature on the use of brachial artery access is controversial. Some studies found increased puncture site complications. Others found no higher complication rates than in patients with femoral or radial access. The purpose of this study was to determine the impact of ultrasound (US)-guidance on access site complications. MATERIALS AND METHODS: This is a single-center retrospective study of all consecutive patients with brachial arterial access for interventional procedures. Complications were classified into minor complications (conservative treatment only) and major complications (requiring surgical intervention). The brachial artery was cannulated in the antecubital fossa under US-guidance. After the intervention, manual compression or closure devices, both followed by a compression bandage for 3 h, either achieved hemostasis. RESULTS: Seventy-five procedures in seventy-one patients were performed in the study period using brachial access. Access was successful in all cases (100%). Procedures in different vascular territories were performed: neurovascular (10/13.5%), upper extremity (32/43.2%), visceral (20/27.0%), and lower extremity (12/16.3%). Sheath size ranged from 3.2F to 8F (mean: 5F). Closure devices were used in 17 cases (22.7%). In total, six complications were observed (8.0%), four minor complications (5.3%, mostly puncture site hematomas), and two major complications, that needed surgical treatment (2.7%). No brachial artery thrombosis or upper extremity ischemia occurred. CONCLUSION: Exclusive use of US-guidance resulted in a low risk of brachial artery access site complications in our study compared to the literature. US-guidance has been proven to reduce the risk of access site complications in several studies in femoral access. In addition, brachial artery access yields a high technical success rate and requires no additional injection of spasmolytic medication. Sheath size was the single significant predictor for complications.

In Vitro and In Vivo Activity of a Novel Catheter Lock Solution against Bacterial and Fungal Biofilms.

Central-line-associated bloodstream infections are increasingly recognized to be associated with intraluminal microbial biofilms, and effective measures for the prevention and treatment of bloodstream infections remain lacking. This report evaluates a new commercially developed antimicrobial catheter lock solution (ACL), containing trimethoprim (5 mg/ml), ethanol (25%), and calcium EDTA (Ca-EDTA) (3%), for activity against bacterial and fungal biofilms, using in vitro and in vivo (rabbit) catheter biofilm models. Biofilms were formed by bacterial (seven different species, including vancomycin-resistant Enterococcus [VRE]) or fungal (Candida albicans) species on catheter materials. Biofilm formation was evaluated by quantitative culture (CFU) and scanning electron microscopy (SEM). Treatment with ACL inhibited the growth of adhesion-phase biofilms in vitro after 60 min (VRE) or 15 min (all others), while mature biofilms were completely inhibited after exposure for 2 or 4 h, compared to control. Similar results were observed for drug-resistant bacteria. Compared to the heparinized saline controls, ACL lock therapy significantly reduced the catheter bacterial (3.49 ± 0.75 versus 0.03 ± 0.06 log CFU/catheter; P = 0.016) and fungal (2.48 ± 1.60 versus 0.55 ± 1.19 log CFU/catheter segment; P = 0.013) burdens in the catheterized rabbit model. SEM also demonstrated eradication of bacterial and fungal biofilms in vivo on catheters exposed to ACL, while vigorous biofilms were observed on untreated control catheters. Our results demonstrated that ACL was efficacious against both adhesion-phase and mature biofilms formed by bacteria and fungi in vitro and in vivo.

[Clinical experience in the diagnosis and treatment of kidney trauma]. / Klinische Erfahrungen mit Diagnostik und Therapie von Nierentraumen.

BACKGROUND: Kidney trauma belongs to the rarely arising urological illnesses. In recent years guidelines for the treatment of urological injuries have been compiled by the Société Internationale d'Urologie (SIU) and the European Association of Urology (EAU). In the current literature increasingly more meaning is granted to a conservative in relation to a surgical therapy procedure, also with grade 4 and 5 kidney injuries. PATIENTS: From 2002 to 2009, 83 patients with kidney injuries were treated in our hospital. There were 56 patients with grade 1 and grade 2 injuries, 12 patients with grade 3 and grade 4 injuries, and in addition, a total of 15 patients with a grade 5 injury. RESULTS: In 75% of the cases a conservative procedure could be arranged depending on the further clinical course. In 25% of the cases, however, relative and/or absolute indications for operative intervention stood in the foreground, so that this group had an exploratory laparotomy. In 7 cases total nephrectomy had to be performed; however, in 11 cases organ-preserving surgery was possible.

Increase of intracellular Ca2+ by P2X and P2Y receptor-subtypes in cultured cortical astroglia of the rat.

Astrocytes express purinergic receptors that are involved in glial-neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed "astroglia" throughout. Astroglia expressed predominantly P2X(4,6,7) as well as P2Y(1,2) receptor-subtypes. Less intensive immunostaining was also found for P2X(5) and P2Y(4,6,13,14) receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca(2+) ([Ca(2+)](i)). Of the agonists tested, only the P2X(1,3) receptor-selective alpha,beta-methylene ATP was ineffective. Experiments with Ca(2+)-free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca(2+)-ATPase, indicated that the [Ca(2+)](i) response to most nucleotides, except for ATP and 2',3'-O-(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca(2+) from intracellular stores. A Gprotein-mediated release of Ca(2+) is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y(1,2,6,14) and probably also P2Y(4)) receptors. Several unidentified P2X receptors, including P2X(7), may also be present, although they appear to only moderately participate in the regulation of [Ca(2+)](i). The rise of [Ca(2+)](i) is due in this case to the transmembrane flux of Ca(2+) via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca(2+)](i) of cultured astroglia and thereby may play an important role in cell-to-cell signaling.

[Bony sacroiliac corridor. A virtual volume model for the accurate insertion of transarticular screws]. / Der sakroiliakale Knochenkorridor. Ein virtuelles Volumenmodell zur sicheren Implantation transartikulärer Schrauben.

BACKGROUND: Minimally invasive sacroiliac (SI) screw fixation carries a high risk for implant malposition. Only idealised shape conceptions of the safe bony corridor exist. METHODS: Two SI corridor models were generated based on a 3D CT reconstruction of a human pelvis. Therefore two penetration depths of the screws into the sacrum were defined. RESULTS: By inserting screws into the centre of the first sacral body an osseous volume of 121 cm3 and an iliac entrance area of 53 cm2 were utilizable. Screw positioning beyond the opposite sacral isthmus leads to a reduction of the bony volume to 72 cm3 (60%) and a decrease of the iliac screw entrance to 20 cm2 (38%). CONCLUSION: The computed realistic 3D models provide exact references to confining bone structures for safe screw positions. The implementation of a software algorithm for fully automated calculation of such volumes based on fluoroscopic or CT images could enhance the performance of computer-assisted navigation systems.

Study of gelatin supplemented diet on amino acid homeostasis in the horse.

REASONS FOR PERFORMING STUDY: Gelatin supplementation is a common measure in an attempt to assist cartilage repair, but little scientific evidence exists to support its efficacy. OBJECTIVES: To investigate the effects of gelatin administration on post prandial homeostasis. METHODS: Twelve Standardbred horses (mean 404 kg bwt) were fed a hay-concentrate diet supplemented by soy bean meal and oil (control [C], n = 6) or with the addition of 60 g gelatin/day (G, n = 6). The horses were trained by an alternate order of interval and prolonged exercise every second day. The velocities of the treadmill corresponding to 2 and 10 mmol lactate/l blood were derived from lactate curves during a standardised exercise test at the start and middle of the 64 day training period. Blood samples for amino acid analysis were obtained weekly at rest (2 h post prandial). In the second part of the training period, a post prandial sampling was conducted on a day without exercise (prior feeding up to 8 h post prandial). Plasma free amino acids (AA) were determined by HPLC. RESULTS: The change from pre- to the training diet induced an increase in many AA during the total training period. At rest free glycine and proline in blood increased with gelatin supplementation during 7 days after the start of supplementation. The AA in plasma showed a post prandial curve with peak concentrations 2-3 h after feeding. Significant post prandial effects of gelatin intake were detectable for glycine, proline and arginine. CONCLUSIONS: The AA from gelatin are absorbed quickly and become available for AA metabolism. POTENTIAL RELEVANCE: It is evident that in the horse, gelatin influences the homeostasis of those amino acids required for cartilage synthesis. Further research is needed to elucidate the utilisation of those amino acids for the prevention or repair of cartilage damage.

Ventilator-induced lung injury upregulates and activates gelatinases and EMMPRIN: attenuation by the synthetic matrix metalloproteinase inhibitor, Prinomastat (AG3340).

Mechanical ventilation has become an indispensable therapeutic modality for patients with respiratory failure. However, a serious potential complication of MV is the newly recognized ventilator-induced acute lung injury. There is strong evidence suggesting that matrix metalloproteinases (MMPs) play an important role in the development of acute lung injury. Another factor to be considered is extracellular matrix metalloproteinase inducer (EMMPRIN). EMMPRIN is responsible for inducing fibroblasts to produce/secrete MMPs. In this report we sought to determine: (1) the role played by MMPs and EMMPRIN in the development of ventilator-induced lung injury (VILI) in an in vivo rat model of high volume ventilation; and (2) whether the synthetic MMP inhibitor Prinomastat (AG3340) could prevent this type of lung injury. We have demonstrated that high volume ventilation caused acute lung injury. This was accompanied by an upregulation of gelatinase A, gelatinase B, MT1-MMP, and EMMPRIN mRNA demonstrated by in situ hybridization. Pretreatment with the MMP inhibitor Prinomastat attenuated the lung injury caused by high volume ventilation. Our results suggest that MMPs play an important role in the development of VILI in rat lungs and that the MMP-inhibitor Prinomastat is effective in attenuating this type of lung injury.

Evaluation of intraocular pharmacokinetics and toxicity of prinomastat (AG3340) in the rabbit.

To determine the ocular pharmacokinetics, physiological and histological effects of prinomastat (a matrix metalloprotease inhibitor), a total of seventy-seven eyes of New Zealand White rabbits received intravitreous and subtenon injections of prinomastat or of acidified water vehicle as control, Doses of 0.5 mg in 0.05 mL of prinomastat or acidified water were used for intravitreal injection. For the subtenon injections, doses of 5 mg prinomastat in 0.5 mL of acidified water were administered in the superotemporal quadrant. Intraocular pharmacokinetics were determined by analyzing vitreous samples at different postinjection time points using Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS). The toxicity was evaluated by biomicroscopy, electroretinography (ERG), pneumatonometry, and histology. No toxicity was found with either administration method. At day 14 after intravitreal injection, levels of prinomastat in the vitreous and choroid were 1.4 ng/mg and 7.8 ng/mg, respectively. The retinal levels of prinomastat were 22 ng/mg at 24 hr and dropped below 1 ng/mg at 48 hr. Prinomastat remained well above minimum effective concentration in the choroid for at least four weeks after a single intravitreal injection, suggesting that local intravitreal injection may have potential in treating choroidal neovascularization.

The effect of prinomastat (AG3340), a synthetic inhibitor of matrix metalloproteinases, on posttraumatic proliferative vitreoretinopathy.

In a search for a pharmacologic adjuvant in the management of posttraumatic proliferative vitreoretinopathy (PVR), we investigated the effect of intravitreal injection of prinomastat (AG3340) on an experimental model. Posterior penetrating eye trauma was created in one eye each of 24 New Zealand white rabbits. One week after the surgery, all rabbits were randomized (1:1) to receive 0.5 mg prinomastat or the vehicle of the drug intravitreally every week for 6 weeks. The degree of PVR for each hemiretina was scored, and the two scores were summed to obtain a total eye score. The mean total eye score was 3.58 in the treatment group and 5.75 in the control group (p = 0.0307). The numbers of eyes with tractional retinal detachment in the prinomastat-treated (n = 12) and control (n = 12) groups were 3 and 9, respectively (p = 0.0391). These results suggest that intravitreally administered prinomastat has an inhibitory effect on posttraumatic PVR.

The effect of prinomastat (AG3340), a potent inhibitor of matrix metalloproteinases, on a subacute model of proliferative vitreoretinopathy.

PURPOSE: To determine the efficacy of prinomastat (AG3340), a synthetic inhibitor of matrix metalloproteinase, in the treatment of experimental proliferative vitreoretinopathy (PVR) induced by intravitreal dispase injection. METHODS: One eye each of 53 New Zealand white rabbits was injected in the vitreous cavity with 0.07 unit of dispase to induce PVR. One week after PVR induction, 53 rabbits were randomized (27:26) to receive 0.5 mg prinomastat or the vehicle of the drug (acidified water) intravitreally every two weeks. The scores of PVR severity (scale of 1-5) were graded to compare the prinomastat-treated animals with the control group. RESULTS: The average PVR scores in the treatment and control groups were 2.62 and 3.57 respectively (p = 0.038; Wilcoxon rank sum). Clinically significant PVR with retinal detachment (PVR > or = grade 3) developed in 76% of rabbits in the control group versus 51% of rabbits treated with prinomastat. CONCLUSIONS: Intravitreally administered prinomastat decreased development of PVR in an experimental model which made use of dispase to induce PVR.

Marked antiangiogenic and antitumor efficacy of AG3340 in chemoresistant human non-small cell lung cancer tumors: single agent and combination chemotherapy studies.

Effective therapy is needed to improve the survival of patients with advanced lung cancers. We studied the effects of a selective metalloprotease inhibitor, AG3340, on chemoresistant human non-small cell lung cancer tumors (line MV522) in vivo. Mice bearing s.c. tumors were given twice-daily oral doses of AG3340. As a single agent, AG3340 inhibited angiogenesis (up to 77%) and tumor growth (up to 65%) in a dose-dependent manner at well-tolerated daily doses up to 400 mg/kg/day and induced significant tumor necrosis. In contrast, tumors were relatively insensitive to carboplatin with approximately 25% growth inhibition observed at a maximum tolerated dose of approximately 30 mg/kg/week (given i.p., twice weekly). Carboplatin inhibited tumor growth markedly only at toxic doses, demonstrating a superior therapeutic index of AG3340 to carboplatin in this tumor model. A suboptimal dose of AG3340, when used in combination with an ineffective maximum tolerated dose of carboplatin, resulted in greater tumor growth inhibitions than those produced by either agent alone. Similarly, growth inhibition was enhanced when AG3340 was used in combination with paclitaxel. Cotreatment with carboplatin did not alter AG3340 plasma concentrations achieved acutely after oral dosing. These data demonstrate an antiangiogenic and antitumor effect of AG3340 when used as a single agent and enhanced growth inhibitions when AG3340 is used in combination with cytotoxic agents. These data suggest that treatment with this novel matrix metalloprotease inhibitor may be beneficial in advanced lung cancers and other chemoresistant malignancies.

Broad antitumor and antiangiogenic activities of AG3340, a potent and selective MMP inhibitor undergoing advanced oncology clinical trials.

We studied AG3340, a potent metalloproteinase (MMP) inhibitor with pM affinities for inhibiting gelatinases (MMP-2 and -9), MT-MMP-1 (MMP-14), and collagenase-3 (MMP-13) in many tumor models. AG3340 produced dose-dependent pharmacokinetics and was well tolerated after intraperitoneal (i.p.) and oral dosing in mice. Across human tumor models, AG3340 produced profound tumor growth delays when dosing began early or late after tumor implantation, although all established tumor types did not respond to AG3340. A dose-response relationship was explored in three models: COLO-320DM colon, MV522 lung, and MDA-MB-435 breast. Dose-dependent inhibitions of tumor growth (over 12.5-200 mg/kg given twice daily, b.i.d.) were observed in the colon and lung models; and in a third (breast), maximal inhibitions were produced by the lowest dose of AG3340 (50 mg/kg, b.i.d.) that was tested. In another model, AG3340 (100 mg/kg, once daily, i.p.) markedly inhibited U87 glioma growth and increased animal survival. AG3340 also inhibited tumor growth and increased the survival of nude mice bearing androgen-independent PC-3 prostatic tumors. In a sixth model, KKLS gastric, AG3340 did not inhibit tumor growth but potentiated the efficacy of Taxol. Importantly, AG3340 markedly decreased tumor angiogenesis (as assessed by CD-31 staining) and cell proliferation (as assessed by bromodeoxyuridine incorporation), and increased tumor necrosis and apoptosis (as assessed by hematoxylin and eosin and TUNEL staining). These effects were model dependent, but angiogenesis was commonly inhibited. AG3340 had a superior therapeutic index to the cytotoxic agents, carboplatin and Taxol, in the MV522 lung cancer model. In combination, AG3340 enhanced the efficacy of these cytotoxic agents without altering drug tolerance. Additionally, AG3340 decreased the number of murine melanoma (B16-F10) lesions arising in the lung in an intravenous metastasis model when given in combination with carboplatin or Taxol. These studies directly support the use of AG3340 in front-line combination chemotherapy in ongoing clinical trials in patients with advanced malignancies of the lung and prostate.

Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2: a key enzyme in angiogenesis.

BACKGROUND: Angiogenesis is involved in tumor growth, macular degeneration, retinopathy and other diseases. Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to specific receptors (VEGFRs) on the surface of vascular endothelial cells. VEGFRs are receptor tyrosine kinases that, like the platelet-derived growth factor receptors (PDGFRs), contain a large insert within the kinase domain. RESULTS: We report here the generation, kinetic characterization, and 2.4 A crystal structure of the catalytic kinase domain of VEGF receptor 2 (VEGFR2). This protein construct, which lacks 50 central residues of the 68-residue kinase insert domain (KID), has comparable kinase activity to constructs containing the entire KID. The crystal structure, determined in an unliganded phosphorylated state, reveals an overall fold and catalytic residue positions similar to those observed in other tyrosine-kinase structures. The kinase activation loop, autophosphorylated on Y1059 prior to crystallization, is mostly disordered; however, a portion of it occupies a position inhibitory to substrate binding. The ends of the KID form a beta-like structure, not observed in other known tyrosine kinase structures, that packs near to the kinase C terminus. CONCLUSIONS: The majority of the VEGFR2 KID residues are not necessary for kinase activity. The unique structure observed for the ends of the KID may also occur in other PDGFR family members and may serve to properly orient the KID for signal transduction. This VEGFR2 kinase structure provides a target for design of selective anti-angiogenic therapeutic agents.

Marked inhibition of tumor growth in a malignant glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor AG3340.

Synthetic matrix metalloproteinase (MMP) inhibitors have activity against a variety of tumors in preclinical models but have not been studied in gliomas. We determined the effect of AG3340, a novel synthetic MMP inhibitor with Ki values against gelatinases in the low picomolar range, on the growth of a human malignant glioma cell line (U87) in SCID-NOD mice. Mice were injected s.c. with U87 cells. Tumors were allowed to grow to a size of approximately 0.5 x 0.5 cm (after about 3 weeks), and the mice were randomized to receive either: (a) 100 mg/kg AG3340 in vehicle; or (b) vehicle control (0.5% carboxymethyl cellulose, 0.1% pluronic F68), both given daily i.p. Tumor area was measured twice weekly, and animals were sacrificed when moribund, or earlier if premorbid histology was examined. In vivo inhibition of tumor growth was profound, with AG3340 decreasing tumor size by 78% compared with controls after 31 days (when controls were sacrificed; P < 0.01, Wilcoxon test). Control animals survived 31 days after the i.p. injections began, and AG3340 mice survived 71 days, representing a >2-fold increase in survival associated with tumor growth delay. Histological examination found that AG3340-treated tumors were smaller, had lower rates of proliferation, and significantly less invasion than control-treated tumors. Hepatic or pulmonary metastases were not seen in either group. In a separate experiment, the tumors were smaller and sampled after a shorter duration of treatment; the changes in proliferation were more marked and occurred earlier than differences in tumor invasion between the two groups. Furthermore, in vitro cell growth was not inhibited at AG3340 concentrations of <1 mM. AG3340 plasma concentrations in vivo, 1 h after administration, ranged from 67 to 365 nM. Thus, AG3340 produced a profound inhibition of glioma tumor growth and invasion. AG3340 markedly increased survival in this in vivo glioma model. Treatment with AG3340 may be potentially useful in patients with malignant gliomas.

The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus.

Protein HU is a ubiquitous prokaryotic protein which controls the architecture of genomic DNA. It binds DNA non-specifically and promotes the bending and supercoiling of the double helical structure. HU is involved in many DNA-associated cellular processes, including replication, transcription and the packaging of DNA into chromosome-like structures. Originally determined at medium resolution, the crystal structure of HU has now been refined at 2.0 A resolution. The high-resolution structure shows that the dimeric molecule is essentially a compact platform for two flexible and basic arms which wrap around the DNA molecule. To maximize the protein's stability, non-secondary structural regions are reduced to a minimum, there is an extensive aromatic hydrophobic core and several salt bridges and hydrogen-bonded water molecules knit together crucial regions. Based on the original medium-resolution structure of HU, several proposals were made concerning the structural basis of HU's ability to bind, bend and supercoil DNA. Each of these proposals is fully supported by the high-resolution structure. Most notably, the surfaces of the molecule which appear to mediate protein-DNA and protein-protein interactions have the ideal shapes and physicochemical properties to perform these functions.

Characterization and kinetic mechanism of catalytic domain of human vascular endothelial growth factor receptor-2 tyrosine kinase (VEGFR2 TK), a key enzyme in angiogenesis.

Vascular endothelial growth factor (VEGF) is a dimeric protein which induces formation of new blood vessels (angiogenesis) through binding to VEGF-receptor-2 tyrosine kinase (VEGFR2 TK) or KDR (kinase insert domain-containing receptor) on the surface of endothelial cells. Angiogenesis has been shown to be essential for malignancy of tumors; therefore, VEGFR2 TK is a potential therapeutic target for the treatment of cancer. Sequence homology studies indicate that VEGFR2 TK contains three domains: extracellular (ligand-binding domain), transmembrane, and intracellular (catalytic domain). In this work, the catalytic domain of VEGFR2 TK was cloned and expressed in a soluble active form using a baculovirus expression system. In the absence of ligand, the enzyme is shown to catalyze its autophosphorylation in a time-dependent and enzyme-concentration-dependent manner, consistent with a trans mechanism for this reaction. Mass spectrometry analysis revealed incorporation of 5.5 +/- 0.5 mol of phosphate/mole of enzyme (monomer). In addition, the enzyme was shown to catalyze phosphorylation of a synthetic peptide, poly(E4Y). Using poly(E4Y) as substrate, the kinetic constants of both native and phosphorylated enzyme were determined. Enzyme phosphorylation increased catalytic efficiency of the enzyme by at least an order of magnitude. Furthermore, the enzyme was shown to catalyze the reverse reaction using phospho-poly(E4Y) as substrate. Cd2+ was found to be an inhibitor of the enzyme. Kinetic studies revealed that inhibition by Cd2+ was competitive with respect to Mg2+ and noncompetitive with respect to MgATP. These results indicate that Cd2+ competes for a second metal-binding site. Therefore, the reaction catalyzed by this enzyme was treated as a terreactant system. The kinetic mechanism of VEGFR2 TK was elucidated through the use of steady-state kinetic studies. According to these studies, the enzyme binds Mg2+ and MgATP in a random fashion followed by ordered addition of the peptide substrate. The release of product is also ordered, with MgADP being released last. The order of substrate binding was confirmed by using AMP-PCP, a dead-end inhibitor.

Antitumor efficacy of AG3340 associated with maintenance of minimum effective plasma concentrations and not total daily dose, exposure or peak plasma concentrations.

Oral administration of AG3340, a novel metalloprotease (MMP) inhibitor, suppresses the growth of human colon adenocarcinoma (COLO-320DM) tumors in vivo (Proc Am Assoc Cancer Res 39: 2059, 1998). In this report, we tested the hypothesis that the growth inhibition of these tumors is associated with maintaining minimum effective plasma concentrations of AG3340. Nude mice were given a total oral daily dose of 25 or 200 mg/kg; 6.25 mg/kg was given four times per day (QID) (25 mg/kg/day), and 100 mg/kg was given in two daily doses (BID) (200 mg/kg/day). Peak plasma concentrations (Cmax) of 83 +/- 43 (mean +/- SD) and 1998 +/- 642 ng/ml were detected 30 min after a single dose with 6.25 mg/kg and 100 mg/kg AG3340, respectively. AUC(0-24 h) values estimated from dosing with 25 and 200 mg/kg/day AG3340 were 672 and 10882 ng*h/ml, respectively. Importantly, both regimen inhibited tumor growth equivalently (74 to 82%). Efficacy was also compared at a total daily dose of 25 mg/kg by giving AG3340: QID (6.25 mg/kg per dose), BID (12.5 mg/kg per dose), and once daily (25 mg/kg per dose). The Cmax of these regimens was 83 +/- 43, 287 +/- 175 and 462 +/- 495 ng/ml, respectively. AG3340 did not inhibit tumor growth with the latter two regimens. The efficacy of 6.25 mg/kg QID (25 mg/kg/day) was superior to the efficacy of 25 mg/kg BID (50 mg/kg/day), substantiating the independence of efficacy from the total daily dose and Cmax. Expectedly, peak to trough fluctuations were significantly smaller with the QID regimen than with BID and QD dosing. After 24 h, the trough was greater than 1 ng/ml with QID dosing but was less than 1 ng/ml after QD and BID dosing. These results suggest that the antitumor efficacy of AG3340 was associated with maintaining minimum effective plasma concentrations of AG3340 and demonstrate that the antitumor efficacy of AG3340 was independent of the total daily dose, peak plasma concentration, and drug exposure in this tumor model.

Viracept (nelfinavir mesylate, AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease.

Using a combination of iterative structure-based design and an analysis of oral pharmacokinetics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor (Ki = 2 nM) and antiviral agent (HIV-1 ED50 = 14 nM). An X-ray cocrystal structure of the enzyme-AG1343 complex reveals how the novel thiophenyl ether and phenol-amide substituents of the inhibitor interact with the S1 and S2 subsites of HIV-1 protease, respectively. In vivo studies indicate that AG1343 is well absorbed orally in a variety of species and possesses favorable pharmacokinetic properties in humans. AG1343 (Viracept) has recently been approved for marketing for the treatment of AIDS.

Rodent pharmacokinetic and anti-tumor efficacy studies with a series of synthetic inhibitors of matrix metalloproteinases.

Matrix metalloproteinases are a family of zinc-containing proteases that degrade extracellular matrix and basement membranes. These enzymes are thought to play a role in processes essential for tumor growth, invasion, and metastasis. Here we report pharmacokinetic and anti-tumor efficacy studies with a series of structurally related inhibitors of these enzymes that were synthesized at Agouron Pharmaceuticals using protein structure based drug design. The compounds studied were AG3287, AG3293, AG3294, AG3296, AG3319, and AG3340. Rat oral bioavailability ranged from 15 to 68%. Despite similar profiles of enzyme inhibition across the family of enzymes, and similar pharmacokinetics following i.p. administration to mice, efficacy against the Lewis lung carcinoma murine model varied from tumor growth enhancement, to significant reductions in the size of primary tumors and the number of lung metastases. AG3340 was the most efficacious compound against the Lewis lung carcinoma model, resulting in the complete cessation of primary tumor growth throughout the experiment in 4/6 mice treated with daily i.p. injections at a dose of 50 mg/kg. This treatment inhibited the formation of lung metastases greater than 5 mm in diameter by 90%.

Structure-based design and synthesis of substituted 2-butanols as nonpeptidic inhibitors of HIV protease: secondary amide series.

The design, synthesis, and crystallographic analysis of protein-inhibitor complexes is described for a novel series of nonpeptidic HIV protease (HIV Pr)inhibitors. Beginning with a cocrystal structure of a Phe-Pro peptidomimetic bound to the HIV Pr, design was initiated that resulted in the substituted 2-butanol compound 8 as the lead compound (Ki = 24.5 microM, racemic mixture). Modifications on the initial compound were then made on the basis of its cocrystal structure with HIV Pr and inhibition data, resulting in compounds with enhanced potency against the enzyme (compound 18, Ki = 0.48 microM). These inhibitors were found to bind to the enzyme essentially as predicted on the basis of the original design hypothesis. Stereospecific synthesis of individual enantiomers confirmed the prediction of a binding preference for the S alcohol stereochemistry. Modest antiviral activity was demonstrated for several of the more potent HIV Pr inhibitors in a HIV-1 infected CEM-SS cell line.