Argatroban therapy in pediatric patients requiring nonheparin anticoagulation: an open-label, safety, efficacy, and pharmacokinetic study.

BACKGROUND: An increasing number of pediatric patients suffer from thrombotic events necessitating anticoagulation therapy including heparins. Some such patients develop heparin-induced thrombocytopenia (HIT) and thus require alternative anticoagulation. As such, studies evaluating the safety, efficacy, and dosing of alternative anticoagulants are required. PROCEDURE: In this multicenter, single arm, open-label study, 18 patients ≤ 16 years old received argatroban for either a suspicion of or being at risk for HIT, or other conditions requiring nonheparin anticoagulation. Endpoints included thrombosis, thromboembolic complications, and bleeding. RESULTS: Patients (ages, 1.6 weeks to 16 years) received argatroban usually for continuous anticoagulation (n = 13) or cardiac catheterization (n = 4). One catheterization patient received a 250 µg/kg bolus only; 17 patients received argatroban continuous infusion (median (range)) 1.1 (0.3-12) µg/kg/min (of whom four received a bolus) for 3.0 (0.1-13.8) days. In patients without bolus dosing, typically argatroban 1 µg/kg/min was initiated, with therapeutic activated partial thromboplastin times (aPTTs) (1.5-3× baseline) achieved within 7 hr. Within 30 days, thrombosis occurred in five patients (two during therapy). No one required amputation or died due to thrombosis during therapy. Two patients had major bleeding. Pharmacometric analyses demonstrated the optimal initial argatroban dose to be 0.75 µg/kg/min (if normal hepatic function), with dose reduction necessary in hepatic impairment. CONCLUSIONS: In pediatric patients requiring nonheparin anticoagulation, argatroban rapidly provides adequate levels of anticoagulation and is generally well tolerated. For continuous anticoagulation, argatroban 0.75 µg/kg/min (0.2 µg/kg/min in hepatic impairment), adjusted to achieve therapeutic aPTTs, is recommended.

Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB.

Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

Expression, purification, and characterization of the Mycobacterium tuberculosis acyl carrier protein, AcpM.

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: the multifunctional polypeptide, FASI, in which the acyl carrier protein (ACP) domain forms an integral part of the polypeptide, and the dissociated FASII system, which is composed of monofunctional enzymes and a discrete ACP (AcpM). In order to characterize enzymes of the FASII system, large amounts of AcpM are required to generate substrates such as holo-AcpM, malonyl-AcpM and acyl-AcpM. The M. tuberculosis acpM gene was overexpressed in Escherichia coli and AcpM purified, yielding approximately 15-20 mg/l of culture. Analysis of AcpM by mass spectrometry, N-terminal sequencing, amino acid analysis, and gas chromatography indicated the presence of three species, apo-, holo-, and acyl-AcpM, the former comprising up to 65% of the total pool. The apo-AcpM was purified away from the in vivo generated holo- and acyl-forms, which were inseparable and heterogeneous with respect to acyl chain lengths. Once purified, we were able to convert apo-AcpM into holo- and acyl-forms. These procedures provide the means for the preparation of the large quantities of AcpM and derivatives needed for characterization of the purified enzymes of the mycobacterial FASII system.

Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae.

The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positive pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31% identity with the MurB protein from Escherichia coli, and contains the catalytic residues, substrate-binding residues and FAD-binding motif identified previously in the E. coli protein. The gene was cloned into the pET28a+ expression vector, and the 34.5 kDa protein that it encodes was overexpressed in E. coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insoluble. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavoprotein, like its E. coli homologue, with a characteristic absorption at 463 nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine enolpyruvate with the concomitant oxidation of NADPH, and was characterized kinetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen provides a good starting point for the discovery of antibacterial agents against MurB.

Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations.

The aroA gene (Escherichia coli nomenclature) encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the gram-positive pathogen Streptococcus pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate. Activation by univalent cations was observed in the forward reaction, with NH+4, Rb+ and K+ exerting the greatest effects. Km(PEP) was lowered by increasing [NH+4] and [K+], whereas Km(S3P) rose with increasing [K+], but fell with increasing [NH+4]. Increasing [NH+4] and [K+] resulted in an overall increase in kcat. Glyphosate (GLP) was found to be a competitive inhibitor with PEP, but the potency of inhibition was profoundly affected by [NH+4] and [K+]. For example, increasing [NH+4] and [K+] reduced Ki(GLP versus PEP) up to 600-fold. In the reverse reaction, the enzyme catalysis was less sensitive to univalent cations. Our analysis included univalent cation concentrations comparable with those found in bacterial cells. Therefore, the observed effects of these metal ions are more likely to reflect the physiological behavior of EPSP synthase and also add to our understanding of how to inhibit this enzyme in the host organism. As there is a much evidence to suggest that EPSP synthase is essential for bacterial survival, its discovery in the serious gram-positive pathogen S. pneumoniae and its inhibition by GLP indicate its potential as a broad-spectrum antibacterial target.

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma.

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.

Agonist potency at the cloned human beta-3 adrenoceptor depends on receptor expression level and nature of assay.

The cloned human beta-3 adrenoceptor was expressed in Chinese hamster ovary cells at three different levels (130, 400 and 3000 fmol/mg). The potency and intrinsic activity of a range of agonists in functional assays with these cell lines rose as a function of increasing receptor density. Operational analysis of concentration-response data allowed calculation of functional affinity and efficacy of agonists at the human beta-3 adrenoceptor. The data highlighted the low efficacy of BRL 37344 ¿(RR,SS)-(+/-)-4-[(2-(2-(3-chlorophenyl)-2-hydroxyethyl)amino)-propyl] phenoxyacetate¿ for the human beta-3 adrenoceptor, which may explain its lower potency at the human receptor despite its higher affinity relative to isoprenaline. The potency of catecholamines at the human beta-3 adrenoceptor was found to be 1 to 2 orders of magnitude higher when determined in an intact cell cAMP accumulation assay compared with a membrane-based adenylyl cyclase activation assay. The reason for this enhanced sensitivity is not clear, but the result is that the potency of the natural agonist noradrenaline in the intact cell is considerably higher than predicted either from its ligand binding affinity, or from its potency in membrane-based assays. Much smaller enhancements in sensitivity were observed for compounds of the aryloxypropanolamine class such as CGP 12177 [(+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one], with the result that the rank order of potency of such agonists at the beta-3 adrenoceptor was altered. In particular, CGP 12177 exhibited high relative potency in the cyclase assay, but low relative potency in intact cell assays. These findings highlight the importance of selecting appropriate expression levels and appropriate assay methodology when cloned receptors are used to characterize agonists.

Characterization of the helicase and ATPase activity of human papillomavirus type 6b E1 protein.

Human papillomavirus type 6b (HPV-6b) is one of the most common causes of human genital warts, an important sexually transmitted disease. Discovery of antiviral therapies for this condition has been hampered by the inability to propagate the virus using standard tissue culture techniques and through difficulties in expressing sufficient recombinant viral proteins in vitro. Replication of papillomavirus DNA requires two viral proteins, E1 and E2. In an effort to establish assays to discover compounds active against this virus, we have co-expressed HPV-6b E1 and E2 proteins in insect cells. We demonstrate that the two proteins form a heteromeric complex which can be purified by sequence-specific DNA affinity chromatography. We also demonstrate that the complex has both E1-associated ATPase and ATP-dependent DNA helicase activity and report further characterization of these functions.