Efficacy and safety of point-of-care ultrasound-guided intra-articular corticosteroid joint injections in patients with haemophilic arthropathy.

INTRODUCTION AND OBJECTIVES: Intra-articular corticosteroid injections are standard of care for managing joint pain secondary to osteoarthritis or rheumatoid arthritis but are rarely used in haemophilic arthropathy. We have introduced and evaluated the efficacy and safety of ultrasound-guided corticosteroid injections for pain relief in patients with haemophilic arthropathy. PATIENTS AND METHODS: Ultrasound-guided intra-articular injections performed on haemophilia patients at UCSD between March 2012 and January 2016 were analysed. Needle placement and injection (40 mg triamcinolone; 3-5 mL lidocaine) were performed with musculoskeletal ultrasound and Power Doppler. Analysis included patient demographics, joint-specific parameters such as tissue hypervascularity and effusions, pain relief, and procedure-associated complications. RESULTS: Forty-five injections (14 ankles, 13 elbows, 18 knees) were administered in 25 patients. Advanced arthropathy with hypervascularity and/or effusions was present in 91% and 61% of joints, respectively. Ninety-one per cent of injections resulted in pain relief which was significant in 84% (>30% reduction). Median pain score was reduced from 7 of 10 to 1 of 10 (P < 0.001), usually within 24 h. Median duration of pain relief was 8 weeks (range 1-16 weeks). Haemophilia B patients experienced longer periods of relief, and high Pettersson scores were associated with shorter duration of relief. There were no procedure-associated complications. Repeat ultrasound of eight joints within 4 weeks of injection demonstrated nearly complete resolution of hypervascularity. CONCLUSIONS: Point-of-care ultrasound enabled intra-articular corticosteroid injections that provided highly effective, safe, and relatively long-lasting pain relief in haemophilic arthropathy. This approach should be used to improve pain management in haemophilic arthropathy.

Modulation of the activated protein C pathway in severe haemophilia A patients: The effects of thrombomodulin and a factor V-stabilizing fab.

INTRODUCTION: The thrombomodulin (TM)/activated protein C (APC) system is a key regulator of haemostasis, limiting amplification and propagation of the formed blood clot to the injury site. Dampening APC's inhibition of factor V (FV) and factor VIII (FVIII) may be a future strategy in developing next-generation therapeutic targets for haemophilia treatment. AIMS: To determine ex vivo the respective concentration-dependent effects of TM and a FV-stabilizing Fab on the APC regulatory pathway in severe FVIII-deficient blood and plasma. METHODS: Ten severe haemophilia A subjects and one healthy control were enrolled. Blood was spiked with TM (0, 1, 2.5, 5, 10, 20.0 nmol/L) and FV-stabilizing Fab (0, 3, 15, 65, 300 nmol/L). The respective effects were compared to FVIII concentrations of 3- and 10% using rotational thromboelastometry clotting time (CT) and thrombin generation analysis (TGA). RESULTS: With 1 and 2.5 nmol/L TM, 5% FVIII resulted in CT similar to the absence of TM, suggesting it completely reversed the effect of APC. Increasing TM concentrations also reduced peak thrombin generation and ETP. The addition of 300 nmol/L FV-stabilizing Fab returned CT to nearly baseline, but for most subjects was less than the effects of 3- or 10% FVIII. The FV-stabilizing Fab produced similar or greater thrombin generation compared to samples with 3- or 10% FVIII. CONCLUSIONS: The FV-stabilizing Fab resulted in enhanced CT and TGA parameters consistent with FVIII levels of 3- and 10%. Additional studies need to further characterize how modulating the APC pathway may prove beneficial in developing new haemophilia drug targets.

Coated platelets and severe haemophilia A bleeding phenotype: Is there a connection?

INTRODUCTION: Coated platelets are a subpopulation of platelets that possess highly prothrombotic properties. Previous observational data suggest that bleeding phenotype in severe haemophilia A is associated with coated platelet levels. Haemophilia A patients with higher coated platelet levels may have a mild bleeding phenotype; those with lower levels may have a more severe bleeding phenotype. AIM: The aim of the study was to test the hypothesis that coated platelet levels are correlated with clinical bleeding phenotype. METHODS: This cross-sectional, observational study enrolled 20 severe haemophilia A patients, including 15 with severe and five with a mild bleeding phenotype, and a control group of 12 healthy volunteers. The haemophilia bleeding phenotype was determined by the patient's medical history and haemophilia treatment centre records. Blood was obtained from each patient by venipuncture and platelets were analysed by flow cytometry. RESULTS: Patients categorized as having a severe bleeding phenotype experienced a median eight bleeds per year compared to one bleed annually in the mild bleeding phenotype group. Both groups had similar total platelet counts and fibrinogen levels. There was no difference in coated platelet percentage between severe and mild bleeding phenotype (17 and 16% respectively), however, both groups had significantly lower % coated platelets compared to controls (44%, P < 0.0001). CONCLUSION: Coated platelet levels were not associated with bleeding phenotype in this study; however, these data may suggest coated platelet levels are lower in haemophilia patients relative to healthy volunteers.

The brain in three crustaceans from cavernous darkness.

BACKGROUND: While a number of neuroanatomical studies in other malacostracan taxa have recently contributed to the reconstruction of the malacostracan ground pattern, little is known about the nervous system in the three enigmatic blind groups of peracarids from relict habitats, Thermosbaenacea, Spelaeogriphacea, and Mictocarididae. This first detailed description of the brain in a representative of each taxon is largely based on a combination of serial semi-thin sectioning and computer-aided 3D-reconstructions. In addition, the mictocaridid Mictocaris halope was studied with a combination of immunolabeling (tubulin, nuclear counter-stains) and confocal laser scanning microscopy, addressing also the ventral nerve cord. RESULTS: Adjacent to the terminal medulla, all three representatives exhibit a distal protocerebral neuropil, which is reminiscent of the lobula in other Malacostraca, but also allows for an alternative interpretation in M. halope and the thermosbaenacean Tethysbaena argentarii. A central complex occurs in all three taxa, most distinctively in the spelaeogriphacean Spelaeogriphus lepidops. The deutocerebral olfactory lobe in M. halope and S. lepidops is large. The comparably smaller olfactory lobe in T. argentarii appears to be associated with a unique additional deutocerebral neuropil. A small hemiellipsoid body exists only in the protocerebrum of T. argentarii. Distinctive mechanosensory neuropils corresponding to other malacostracans are missing. CONCLUSIONS: The considerable reduction of the optic lobe in the studied taxa is higher than in any other blind malacostracan. The large size of deutocerebral olfactory centers implies an important role of the olfactory sense. The presence of a distinctive central complex in the blind S. lepidops adds further support to a central-coordinating over a visual function of this structure. The lack of a hemiellipsoid body in M. halope and S. lepidops suggests that their terminal medulla takes over the function of a second order olfactory center completely, as in some other peracarids. The reduction of the optic lobe and hemiellipsoid body is suggested to have occurred several times independently within Peracarida. The missing optic sense in the studied taxa is not correlated with an emphasized mechanosense.

Is some better than none: are TEG and TGA profiles different in severe FVIII-deficient patients with inhibitors?

Severe factor VIII (FVIII)-deficient patients with and without FVIII inhibitors cannot be distinguished using FVIII levels. The FVIII assay is sensitive to detect factor levels below 1%. While severe FVIII-deficient, non-inhibitor patients have FVIII < 1%, they may retain unmeasurable residual factor activity. In contrast, inhibitor patients have a FVIII antibody that presumably fully eliminates FVIII activity. It is unknown whether thromboelastography (TEG) and thrombin generation assay (TGA) can differentiate between patients with FVIII < 1% with and without the presence of FVIII inhibitors. The primary objective was to discern whether TEG and TGA could differentiate between severe FVIII-deficient patients with and without the presence of FVIII inhibitors. A secondary objective was to correlate TEG and TGA to annualized bleeding rates. This observational study performed TEG and TGA in healthy volunteers (N = 15), severe FVIII-deficient (N = 15) and severe FVIII-deficient patients with inhibitors (N = 15). Kaolin-activated TEG was better at differentiating reaction time (31.3 vs. 120 min respectively, P = 0.004) and kinetics time (6.1 vs. 23.1 min respectively, P = 0.028) between the non-inhibitor and inhibitor patients. TEG activated by tissue factor in plasma-containing corn trypsin inhibitor failed to differentiate groups. The TGA failed to differentiate peak thrombin, endogenous thrombin potential and lag time between groups. There was no correlation between TEG and TGA with annualized bleeding rates. Kaolin-activated TEG, but not TGA, differentiated between severe FVIII-deficient patients with and without inhibitors. These assays did not find a correlation to annualized bleeding rate.

Monitoring rFVIII prophylaxis dosing using global haemostasis assays.

Secondary factor VIII (FVIII) prophylaxis converts severe haemophiliacs (FVIII:C < 1 IU dL(-1)) to a moderate phenotype (FVIII:C ≥ 1 IU dL(-1)), however, plasma FVIII:C is a poor predictor of bleeding risk. This study used thromboelastography (TEG) and thrombin generation assay (TGA) to quantify coagulation across a 48 h rFVIII prophylaxis period. 10 severe haemophiliacs with varying clinical bleeding phenotypes received their standard rFVIII prophylaxis dose and blood samples were obtained over 48 h. Measured parameters included FVIII:C, TEG and TGA at each time point. FVIII:C pharmacokinetics (PK) and correlation between global assay parameters was performed. The FVIII:C PK parameters were consistent with previous literature. There was significant correlation between FVIII:C and TEG R-time and aPTT (both P < 0.001). Significant correlations existed between FVIII:C and TGA peak, ETP and velocity parameters (all P < 0.001). At 24 h the TEG parameters were sub-therapeutic despite median FVIII:C of 13.0 IU dL(-1). TGA was sensitive to FVIII:C below 1 IU dL(-1). Those with the severest bleeding phenotype had the lowest TGA parameters. There was significant correlation between FVIII:C and TEG and TGA. TEG lost sensitivity at 48 h, but not TGA. Prospective studies are needed to determine whether these data can be used to design individualized rFVIII prophylaxis regimens.

Monitoring rFVIIa 90 µg kg⁻¹ dosing in haemophiliacs: comparing laboratory response using various whole blood assays over 6 h.

Recombinant FVIIa is a haemostatic agent administered to patients with severe FVIII or FIX deficiency with inhibitors. Although rFVIIa is effective at stopping bleeding, a reliable assay to monitor its effect is lacking. To characterize the pharmacokinetics and global coagulation effects of rFVIIa for 6 h following a IV dose of 90 µg kg⁻¹. Ten non-bleeding subjects with severe FVIII or FIX deficiency were infused with a single-dose of rFVIIa 90 µg k⁻¹ body weight and blood was collected before and at 0.5, 1, 2, 4 and 6 h postdose. Global haemostasis was characterized throughout the study utilizing whole blood analyses (Hemodyne HAS, TEG, ROTEM). The clearance and half-life of factor FVII:C was estimated as 39.0 ± 8.8 mL h⁻¹ kg⁻¹ and 2.1 ± 0.2 h respectively. There was good inter-assay agreement with respect to clot initiation parameters (R, CT and FOT) and these parameters all fell to a mean of approximately 9 min following rFVIIa dosing. The platelet contractile force (PCF) and clot elastic modulus (CEM) were positively correlated to FVII:C (P < 0.0001), and these parameters were dynamic throughout the 6-h period. The MA and MCF did not correlate to FVII:C nor did they significantly change during the study. Prothrombin F1 + 2 significantly increased following rFVIIa dosing (P < 0.001), but remained steady throughout the study. There was no change in D-dimer concentrations over time. The FOT, R and CT characterized clot initiation following rFVIIa dosing. The PCF and CEM were correlated to FVII:C and characterized the dynamics of platelet function and clot strength over the rFVIIa dosing interval. The clinical significance of these findings needs additional study.

Thrombin generation time is a novel parameter for monitoring enoxaparin therapy in patients with end-stage renal disease.

BACKGROUND: Patients with end-stage renal disease (ESRD) who receive enoxaparin are at increased risk for adverse bleeding episodes. This phenomenon appears to occur despite judicious monitoring of antifactor Xa (aFXa) activity. Better monitoring parameters are needed to quantify the anticoagulant effects of enoxaparin in the ESRD population. OBJECTIVES: The objective of this study was to determine the utility of using thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM) to monitor the degree of anticoagulation in ESRD subjects, and to compare these results to aFXa activity, the current gold-standard monitoring parameter. METHODS: Eight healthy volunteers without renal dysfunction and eight ESRD subjects were enrolled into this study. Subjects received a single dose of enoxaparin 1 mg kg(-1) subcutaneously, and blood samples were obtained for the determination of aFXa activity, TGT, PCF and CEM at baseline, 4, 8, and 12 h postdose. RESULTS: Baseline, 4, 8, and 12-h aFXa activity concentrations were not different between groups. However, the corresponding TGT at 8 and 12 h was significantly prolonged in the ESRD group (P = 0.04, and P = 0.008, respectively). The 4-h peak TGT trended toward significance (P = 0.06). There were no differences in PCF or CEM across time. CONCLUSIONS: These data suggest that the parameter aFXa activity is a poor predictor of the anticoagulant effect of enoxaparin in patients with ESRD. Thrombin generation time appears to be more sensitive to the antithrombotic effects of enoxaparin in this population. Further large-scale trials are needed to corroborate these data.

Rauwolfia use and breast cancer: a case-control study.

Breast cancer risk among 1,362 cases and 1,250 controls participating in a large multicenter screening program was examined in relation to hypertension and the use of rauwolfia derivatives. A previous diagnosis of hypertension, reported by 22% of the cases and 23% of the controls, was not associated with an increased risk of breast cancer [odds ratio (OR) = 0.9]; nor was there any excess risk for long-term hypertensives. In addition, there was no significant increase in risk associated with use of either rauwolfia derivatives (OR = 1.2), thiazide preparations (OR = 1.2), or methyldopa (OR = 1.1). However, there were significant excess risks among long-term users and those with extended intervals since first use of rauwolfia. Rauwolfia users of 10 or more years' duration or those whose initial use occurred greater than or equal to 10 years before diagnosis had risk ratios of 4.5 (95% Cl, 1.2-19.8) and 3.8 (95% Cl, 2.3-11.6), respectively. These results suggest that women exposed to long-term rauwolfia use have an elevated risk of developing breast cancer, although the results fail to support previous observations of a generalized adverse effect.

Allosterism-based simultaneous, dual anticoagulant and antiplatelet action: allosteric inhibitor targeting the glycoprotein Ibα-binding and heparin-binding site of thrombin.

BACKGROUND: Allosteric inhibition is a promising approach for developing a new group of anticoagulants with potentially reduced bleeding consequences. Recently, we designed sulfated ß-O4 lignin (SbO4L) as an allosteric inhibitor that targets exosite 2 of thrombin to reduce fibrinogen cleavage through allostery and compete with glycoprotein Ibα to reduce platelet activation. OBJECTIVE: To assess: (i) the antithrombotic potential of a novel approach of simultaneous exosite 2-dependent allosteric inhibition of thrombin and competitive inhibition of platelet activation; and (ii) the promise of SbO4L as the first-in-class antithrombotic agent. METHODS: A combination of whole blood thromboelastography, hemostasis analysis, mouse arterial thrombosis models and mouse tail bleeding studies were used to assess antithrombotic potential. RESULTS AND CONCLUSIONS: SbO4L extended the clot initiation time, and reduced maximal clot strength, platelet contractile force, and the clot elastic modulus, suggesting dual anticoagulant and antiplatelet effects. These effects were comparable to those observed with enoxaparin. A dose of 1 mg of SbO4L per mouse prevented occlusion in 100% of arteries, and lower doses resulted in a proportionally reduced response. Likewise, the time to occlusion increased by ~ 70% with a 0.5-mg dose in the mouse Rose Bengal thrombosis model. Finally, tail bleeding studies demonstrated that SbO4L does not increase bleeding propensity. In comparison, a 0.3-mg dose of enoxaparin increased the bleeding time and blood volume loss. Overall, this study highlights the promise of the allosteric inhibition approach, and presents SbO4L as a novel anticoagulant with potentially reduced bleeding side effects.

Computational approaches for combinatorial library design and molecular diversity analysis.

New approaches for combinatorial library design and molecular diversity analysis have been developed by extending previous work from the fields of quantitative structure-activity relationship, computational chemistry, and chemical information. Recent work has begun to address design efficiency and validation of descriptors for combinatorial library design.

Early hemostatic responses to trauma identified with hierarchical clustering analysis.

BACKGROUND: Trauma-induced coagulopathy is a complex multifactorial hemostatic response that is poorly understood. OBJECTIVES: To identify distinct hemostatic responses to trauma and identify key components of the hemostatic system that vary between responses. PATIENTS/METHODS: A cross-sectional observational study of adult trauma patients at an urban level I trauma center emergency department was performed. Hierarchical clustering analysis was used to identify distinct clusters of similar subjects according to vital signs, injury/shock severity, and comprehensive assessment of coagulation, clot formation, platelet function, and thrombin generation. RESULTS: Among 84 total trauma patients included in the model, three distinct trauma clusters were identified. Cluster 1 (N = 57) showed platelet activation, preserved peak thrombin generation, plasma coagulation dysfunction, a moderately decreased fibrinogen concentration and normal clot formation relative to healthy controls. Cluster 2 (N = 18) showed platelet activation, preserved peak thrombin generation, and a preserved fibrinogen concentration with normal clot formation. Cluster 3 (N = 9) was the most severely injured and shocked, and showed a strong inflammatory and bleeding phenotype. Platelet dysfunction, thrombin inhibition, plasma coagulation dysfunction and a decreased fibrinogen concentration were present in this cluster. Fibrinolytic activation was present in all clusters, but was particularly increased in cluster 3. Trauma clusters were most noticeably different in their relative fibrinogen concentration, peak thrombin generation, and platelet-induced clot contraction. CONCLUSIONS: Hierarchical clustering analysis identified three distinct hemostatic responses to trauma. Further insights into the underlying hemostatic mechanisms responsible for these responses are needed.

Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons.

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH2-terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large "dense" organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons.

Batroxobin-induced clots exhibit delayed and reduced platelet contractile force in some patients with clotting factor deficiencies.

Thrombin causes platelet activation via multiple pathways, and deficient thrombin generation reduces platelet contractile force (PCF) during clot retraction. We hypothesized that PCF in blood samples from clotting factor-deficient patients would be diminished due to delayed or deficient thrombin generation. Blood samples from patients with fibrinogen, and factor V, VII, VIII, IX, X, XI and XIII deficiencies were compared to samples from normal controls. PCF in patient blood clotted with thrombin (1 NIH UmL(-1)) was compared to PCF in clots formed with batroxobin (0.25 micro g mL(-1)). PCF in the former should be normal, but PCF in the latter is dependent on thrombin generation within the sample and might be deficient. In factor VII-(n = 2, P < 0.05), factor VIII-(n = 6, P < 0.005) and factor XI-(n = 2, P < 0.05) deficient platelet-rich plasmas, PCF in batroxobin-induced clots was significantly lower than in thrombin-induced clots. In factor IX deficiency (n = 2), one patient had a dramatic reduction in PCF while a second patient had increased PCF. PCF was insignificantly (P = 0.346) reduced in two patients with factor X deficiency, and was normal in one patient with factor V deficiency. The factor X result is consistent with work in model systems, which indicates that as little as 1-3% factor X activity is sufficient to restore thrombin generation to normal. The factor V result probably indicates that the deficiency is incomplete. PCF in thrombin-induced clots was normal in all of these patients. Low fibrinogen and factor XIII deficiency reduced PCF in both thrombin- and batroxobin-induced clots. These results indicate that PCF is reduced, probably due to delayed thrombin generation, in some factor-deficient platelet-rich plasma samples.

Onset of force development as a marker of thrombin generation in whole blood: the thrombin generation time (TGT).

Prothrombin activation requires the direct interplay of activated platelets and plasma clotting factors. Once formed, thrombin causes profound, irreversible activation of platelets and reinforces the platelet plug via fibrin formation. Delayed or deficient thrombin production increases bleeding risk. Commonly employed coagulation assays, the prothrombin and partial thromboplastin times, use clot formation as a surrogate marker of thrombin generation. These assays routinely utilize platelet-poor plasma and completely miss the effects of platelets. Other markers of thrombin generation, prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex, are typically measured after the fact. We report a simple assay, which employs the onset of platelet contractile force (PCF) as a surrogate marker of thrombin generation. PCF generation occurs concomitant with the burst of F1 + 2 release. The time between assay start and PCF onset is termed the thrombin generation time (TGT). TGT is prolonged in clotting factor deficiencies and in the presence of direct and indirect thrombin inhibitors. TGT shortens to normal with clotting factor replacement and shortens with administration of recombinant factor VIIa. TGT is short in thrombophilic states such as coronary artery disease, diabetes and thromboangiitis obliterans and prolongs toward normal with oral and intravenous anticoagulants.

Antifactor Xa activity correlates to thrombin generation time, platelet contractile force and clot elastic modulus following ex vivo enoxaparin exposure in patients with and without renal dysfunction.

Antifactor Xa activity is the gold standard monitoring parameter for low molecular weight heparin (LMWH) derivatives. It is frequently measured in high-risk populations, such as patients with renal dysfunction. Despite antifactor Xa monitoring, however, bleeding in renal dysfunction patients receiving LMWH remains a problem. This study determined the relationship between antifactor Xa activity and three novel coagulation monitoring parameters: thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM). This study also assessed the effect of renal dysfunction on these relationships. This was an ex vivo pharmacodynamic study of the relationship between antifactor Xa activity and TGT, PCF and CEM in subjects both with and without renal dysfunction. Thirty subjects completed this study (10 controls, 10 chronic kidney disease subjects, and 10 end-stage renal disease subjects receiving hemodialysis). Blood samples obtained from participants were spiked with increasing enoxaparin concentrations (0.25, 0.5, 1.0 and 3.0 IU mL(-1)). Samples were analyzed for TGT, PCF and CEM. The relationship between antifactor Xa activity and TGT, PCF and CEM was determined by Pearson's correlation. The effect of renal dysfunction on the relationship between antifactor Xa activity and TGT, PCF and CEM was determined by analysis of covariance. There is strong correlation between antifactor Xa activity and TGT, CEM and PCF. The presence of renal dysfunction significantly prolongs the TGT, and decreases the CEM relative to controls. These results suggest that patients with renal dysfunction have a greater pharmacodynamic response to LMWH, independent of the pharmacokinetics of LMWH.

3-D pharmacophores in drug discovery.

In this chapter we review the use of 3-D pharmacophores in drug discovery. Recent advances are highlighted, including the application of pharmacophore descriptors generated both from ligands and protein binding sites. The application of 3-D pharmacophore fingerprints as molecular descriptors for similarity and diversity applications such as virtual screening, library design and QSAR is discussed. In addition, we highlight the quantification of structure-based diversity using site-derived fingerprints, and review virtual screening methods using both single refined hypotheses and the fingerprints of multiple potential hypotheses. Further, we discuss methods that take protein flexibility and molecular shape-into account. Each of the above techniques are reviewed with particular reference to the recent advances, advantages and challenges of each methodology.

Measuring diversity: experimental design of combinatorial libraries for drug discovery.

Screening synthetic combinatorial libraries, such as mixtures of oligo(N-substituted)glycines, facilitates rapid drug lead discovery and optimization by vastly increasing the number of candidate molecules made and tested. Discovery efficiency and productivity can be further improved by using experimental design to maximize molecular diversity for a given library size or to bias the library with key features for a specific receptor. We describe new methods to quantify molecular diversity using descriptors that characterize lipophilicity, shape and branching, chemical functionality, and specific binding features. Experimental design methods select sets of side chains that are diverse in these properties, and "flower plots" allow the diversity to be graphically compared. We also quantify the overall diversity accessible to different families of combinatorial chemistry.

Discovery of nanomolar ligands for 7-transmembrane G-protein-coupled receptors from a diverse N-(substituted)glycine peptoid library.

Screening a diverse, combinatorial library of ca. 5000 synthetic dimer and trimer N-(substituted)glycine "peptides" yielded novel, high-affinity ligands for 7-transmembrane G-protein-coupled receptors. The peptoid library was efficiently assembled using readily available chemical building blocks. The choice of side chains was biased to resemble known ligands to 7-transmembrane G-protein-coupled receptors. All peptides were screened in solution-phase, competitive radioligand-binding assays. Peptoid trimer CHIR 2279 binds to the alpha 1-adrenergic receptor with a Ki of 5 nM, and trimer CHIR 4531 binds to the mu-opiate receptor with a Ki of 6 nM. This represents the first example of the discovery of high-affinity receptor ligands from a combinatorial library of non-natural chemical entities.