Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth.

Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti-PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8+ T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase.

Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the Escherichia coli ortholog (EcTP). However, bacterial enzymes are counter-indicated for human therapeutic applications because they are recognized as foreign by the human immune system, thereby eliciting adverse immune responses and raising significant safety and efficacy risks. Thus, it is critical to utilize the HsTP enzyme as starting scaffold for pre-clinical drug development, thus de-risking the safety concerns associated with the use of bacterial enzymes. However, HsTP expresses very poorly in E. coli, whereas its PEGylation, a crucial chemical modification for achieving long serum persistence of therapeutic enzymes, is highly inefficient and negatively affects its catalytic activity. Here we focused on the engineering of the recombinant expression profile of HsTP in E. coli cells, as well as on the optimization of its PEGylation efficiency aiming at the development of an alternative therapeutic approach for MNGIE. We show that phylogenetic and structural analysis of proteins can provide important insights for the rational design of N'-terminus-truncation constructs which exhibit significantly improved recombinant expression levels. In addition, we developed and implemented a criteria-driven rational surface engineering strategy for the substitution of arginine-to-lysine and lysine-to-arginine residues to achieve more efficient, homogeneous and reproducible PEGylation without negatively affecting the enzymatic catalytic activity upon PEGylation. Collectively, our proposed strategies provide an effective way to optimize enzyme PEGylation and E. coli recombinant expression and are likely applicable for other proteins and enzymes.

Arginine depletion as a therapeutic approach for patients with COVID-19.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a source of significant morbidity and death worldwide, and effective treatments are urgently needed. Clinical trials have focused largely on direct antiviral therapies or on immunomodulation in patients with severe manifestations of COVID-19. One therapeutic approach that remains to be clinically investigated is disruption of the host-virus relationship through amino acid restriction, a strategy used successfully in the setting of cancer treatment. Arginine is an amino acid that has been shown in nonclinical studies to be essential in the life cycle of many viruses. Therefore, arginine depletion may be an effective therapeutic approach against SARS-CoV-2. Several arginine-metabolizing enzymes in clinical development may be a viable approach to induce a low arginine environment to treat COVID-19 and other viral diseases. Herein, we explore the rationale for arginine depletion as a therapeutic approach for COVID-19.

Preclinical safety and antitumor activity of the arginine-degrading therapeutic enzyme pegzilarginase, a PEGylated, cobalt-substituted recombinant human arginase 1.

Metabolic remodeling contributes to the development and progression of some cancers and exposes them to vulnerabilities, including specific nutrient dependencies that can be targeted therapeutically. Arginine is a semiessential amino acid, and several cancers are unable to endogenously synthesize sufficient levels of arginine for survival and proliferation, most commonly due to reduced expression of argininosuccinate synthase (ASS1). Such cancers are dependent on arginine and they can be targeted via enzyme-mediated depletion of systemic arginine. We report the preclinical safety, antitumor efficacy, and immune-potentiating effects of pegzilarginase, a highly potent human arginine-degrading enzyme. Toxicology studies showed that pegzilarginase-mediated arginine depletion is well tolerated at therapeutic levels that elicit an antitumor growth effect. To determine which tumor types are best suited for clinical development, we profiled clinical tumor samples for ASS1 expression, which correlated with pegzilarginase sensitivity in vivo in patient-derived xenograft (PDx) models. Among the histologies tested, malignant melanoma, small cell lung cancer and Merkel cell carcinoma had the highest prevalence of low ASS1 expression, the highest proportion of PDx models responding to pegzilarginase, and the strongest correlation between low or no ASS1 expression and sensitivity to pegzilarginase. In an immune-competent syngeneic mouse model, pegzilarginase slowed tumor growth and promoted the recruitment of CD8+ tumor infiltrating lymphocytes. This is consistent with the known autophagy-inducing effects of arginine depletion, and the link between autophagy and major histocompatibility complex antigen presentation to T cells. Our work supports the ongoing clinical investigations of pegzilarginase in solid tumors and clinical combination of pegzilarginase with immune checkpoint inhibitors.

Targeting chemoresistant colorectal cancer via systemic administration of a BMP7 variant.

Despite intense research and clinical efforts, patients affected by advanced colorectal cancer (CRC) have still a poor prognosis. The discovery of colorectal (CR) cancer stem cell (CSC) as the cell compartment responsible for tumor initiation and propagation may provide new opportunities for the development of new therapeutic strategies. Given the reduced sensitivity of CR-CSCs to chemotherapy and the ability of bone morphogenetic proteins (BMP) to promote colonic stem cell differentiation, we aimed to investigate whether an enhanced variant of BMP7 (BMP7v) could sensitize to chemotherapy-resistant CRC cells and tumors. Thirty-five primary human cultures enriched in CR-CSCs, including four from chemoresistant metastatic lesions, were used for in vitro studies and to generate CR-CSC-based mouse avatars to evaluate tumor growth and progression upon treatment with BMP7v alone or in combination with standard therapy or PI3K inhibitors. BMP7v treatment promotes CR-CSC differentiation and recapitulates the cell differentiation-related gene expression profile by suppressing Wnt pathway activity and reducing mesenchymal traits and survival of CR-CSCs. Moreover, in CR-CSC-based mouse avatars, BMP7v exerts an antiangiogenic effect and sensitizes tumor cells to standard chemotherapy regardless of the mutational, MSI, and CMS profiles. Of note, tumor harboring PIK3CA mutations were affected to a lower extent by the combination of BMP7v and chemotherapy. However, the addition of a PI3K inhibitor to the BMP7v-based combination potentiates PIK3CA-mutant tumor drug response and reduces the metastatic lesion size. These data suggest that BMP7v treatment may represent a useful antiangiogenic and prodifferentiation agent, which renders CSCs sensitive to both standard and targeted therapies.

Oncogene-Selective Sensitivity to Synchronous Cell Death following Modulation of the Amino Acid Nutrient Cystine.

Cancer cells reprogram their metabolism, altering both uptake and utilization of extracellular nutrients. We individually depleted amino acid nutrients from isogenic cells expressing commonly activated oncogenes to identify correspondences between nutrient supply and viability. In HME (human mammary epithelial) cells, deprivation of cystine led to increased cell death in cells expressing an activated epidermal growth factor receptor (EGFR) mutant. Cell death occurred via synchronous ferroptosis, with generation of reactive oxygen species (ROS). Hydrogen peroxide promoted cell death, as both catalase and inhibition of NADPH oxidase 4 (NOX4) blocked ferroptosis. Blockade of EGFR or mitogen-activated protein kinase (MAPK) signaling similarly protected cells from ferroptosis, whereas treatment of xenografts derived from EGFR mutant non-small-cell lung cancer (NSCLC) with a cystine-depleting enzyme inhibited tumor growth in mice. Collectively, our results identify a potentially exploitable sensitization of some EGFR/MAPK-driven tumors to ferroptosis following cystine depletion.

Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth.

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53-/- mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.

Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency.

Arginase deficiency is caused by deficiency of arginase 1 (ARG1), a urea cycle enzyme that converts arginine to ornithine. Clinical features of arginase deficiency include elevated plasma arginine levels, spastic diplegia, intellectual disability, seizures and growth deficiency. Unlike other urea cycle disorders, recurrent hyperammonemia is typically less severe in this disorder. Normalization of plasma arginine levels is the consensus treatment goal, because elevations of arginine and its metabolites are suspected to contribute to the neurologic features. Using data from patients enrolled in a natural history study conducted by the Urea Cycle Disorders Consortium, we found that 97% of plasma arginine levels in subjects with arginase deficiency were above the normal range despite conventional treatment. Recently, arginine-degrading enzymes have been used to deplete arginine as a therapeutic strategy in cancer. We tested whether one of these enzymes, a pegylated human recombinant arginase 1 (AEB1102), reduces plasma arginine in murine models of arginase deficiency. In neonatal and adult mice with arginase deficiency, AEB1102 reduced the plasma arginine after single and repeated doses. However, survival did not improve likely, because this pegylated enzyme does not enter hepatocytes and does not improve hyperammonemia that accounts for lethality. Although murine models required dosing every 48 h, studies in cynomolgus monkeys indicate that less frequent dosing may be possible in patients. Given that elevated plasma arginine rather than hyperammonemia is the major treatment challenge, we propose that AEB1102 may have therapeutic potential as an arginine-reducing agent in patients with arginase deficiency.

A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

Bone morphogenetic proteins (BMPs), members of the TGF-ß superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

Action at a distance: mutations of peripheral residues transform rapid reversible inhibitors to slow, tight binders of cyclooxygenase-2.

Cyclooxygenase enzymes (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin G2. The inhibitory activity of rapid, reversible COX inhibitors (ibuprofen, naproxen, mefenamic acid, and lumiracoxib) demonstrated a significant increase in potency and time dependence of inhibition against double tryptophan murine COX-2 mutants at the 89/90 and 89/119 positions. In contrast, the slow, time-dependent COX inhibitors (diclofenac, indomethacin, and flurbiprofen) were unaffected by those mutations. Further mutagenesis studies suggested that mutation at position 89 was principally responsible for the changes in inhibitory potency of rapid, reversible inhibitors, whereas mutation at position 90 may exert some effect on the potency of COX-2-selective diarylheterocycle inhibitors; no effect was observed with mutation at position 119. Several crystal structures with or without NSAIDs indicated that placement of a bulky residue at position 89 caused a closure of a gap at the lobby, and alteration of histidine to tryptophan at position 90 changed the electrostatic profile of the side pocket of COX-2. Thus, these two residues, especially Val-89 at the lobby region, are crucial for the entrance and exit of some NSAIDs from the COX active site.

Knockdown of cancer testis antigens modulates neural stem cell marker expression in glioblastoma tumor stem cells.

The cancer stem cell hypothesis posits that a subpopulation of cancer stem cells is frequently responsible for a tumor's progression and resistance to treatment. The differential cellular morphology and gene expression between cancer stem cells and the majority of the tumor is becoming a point of attack for research into the next generation of therapeutic agents that may work through an induction of differentiation rather than apoptosis. Advances in the field of high-content imaging (HCI), combined with modern shRNA technology and subpopulation analysis tools, have created an ideal screening system to detect these morphological changes in a subset of cells upon gene knockdown. The authors examined several glioblastoma stem cell isolates pre- and postdifferentiation to elucidate the phenotypic effects caused by both serum differentiation and gene knockdown. Neural markers were first characterized in these cells at varying states of differentiation using HCI and immunoblots. The authors then chose one of these isolates, in both the pre- and postdifferentiated forms, for further analysis and screened for morphological changes upon shRNA knockdown of a panel of cancer testis antigens (CTAs). CTAs are a family of proteins that are normally expressed in male germ cells as well as heterogeneously expressed in some metastatic tumors. This gene family has also been implicated in the differentiation of normal human stem cells, therefore making it an ideal candidate for modulation in tumor stem cells. Using their approach, the authors identified the differential effects of gene knockdown in both cell types leading to either changes in neural stem cell marker expression or a decreased cell density likely due to growth arrest or cell death. The resolution that HCI brings to a screen at the subpopulation level makes it an excellent tool for the analysis of phenotypic changes induced by shRNA knockdown in a variety of tumor stem cells.

An agonist-induced conformational change in the growth hormone receptor determines the choice of signalling pathway.

The growth and metabolic actions of growth hormone (GH) are believed to be mediated through the GH receptor (GHR) by JAK2 activation. The GHR exists as a constitutive homodimer, with signal transduction by ligand-induced realignment of receptor subunits. Based on the crystal structures, we identify a conformational change in the F'G' loop of the lower cytokine module, which results from binding of hGH but not G120R hGH antagonist. Mutations disabling this conformational change cause impairment of ERK but not JAK2 and STAT5 activation by the GHR in FDC-P1 cells. This results from the use of two associated tyrosine kinases by the GHR, with JAK2 activating STAT5, and Lyn activating ERK1/2. We provide evidence that Lyn signals through phospholipase C gamma, leading to activation of Ras. Accordingly, mice with mutations in the JAK2 association motif respond to GH with activation of hepatic Src and ERK1/2, but not JAK2/STAT5. We suggest that F'G' loop movement alters the signalling choice between JAK2 and a Src family kinase by regulating TMD realignment. Our findings could explain debilitated ERK but not STAT5 signalling in some GH-resistant dwarfs and suggest pathway-specific cytokine agonists.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4.

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.

A novel mechanism of cyclooxygenase-2 inhibition involving interactions with Ser-530 and Tyr-385.

A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G2 by the cyclooxygenase (COX) activity of prostaglandin endoperoxide synthases. Several modes of inhibitor binding in the COX active site have been described including ion pairing of carboxylic acid containing inhibitors with Arg-120 of COX-1 and COX-2 and insertion of arylsulfonamides and sulfones into the COX-2 side pocket. Recent crystallographic evidence suggests that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin. We tested the generality of this binding mode by analyzing the action of a series of COX inhibitors against site-directed mutants of COX-2 bearing changes in Arg-120, Tyr-355, Tyr-348, and Ser-530. Interestingly, diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation. Determination of the crystal structure of a complex of diclofenac with murine COX-2 demonstrates that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530. This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120. Mutagenesis experiments suggest Ser-530 is also important in time-dependent inhibition by nimesulide and piroxicam.

Selective enhancement of multipotential hematopoietic progenitors in vitro and in vivo by IL-20.

In a search for novel growth factors, we discovered that human interleukin-20 (IL-20) enhanced colony formation by CD34+ multipotential progenitors. IL-20 had no effect on erythroid, granulocyte-macrophage, or megakaryocyte progenitors. IL-20 transgenic mice increased the numbers and cell cycling of multipotential but not other progenitors. IL-20 administration to normal mice significantly increased only multipotential progenitor cells, demonstrating that IL-20 significantly influences hematopoiesis, with specificity toward multipotential progenitors. This is the first cytokine with such specificity identified.

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide.

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.

Functional analysis of the molecular determinants of cyclooxygenase-2 acetylation by 2-acetoxyphenylhept-2-ynyl sulfide.

Selective inhibition of cyclooxygenase-2 (COX-2) leads to relief of pain and inflammation with reduced gastrointestinal side effects relative to nonsteroidal anti-inflammatory drugs. 2-Acetoxyphenylhept-2-ynyl sulfide (APHS) is a selective COX-2 inhibitor that covalently modifies the protein by acetylating Ser-530. We utilized site-directed mutants in the COX-2 active site to probe the molecular determinants of APHS acetylation of COX-2. Incorporation of acetyl groups into Ser-530 was monitored by HPLC and mass spectrometry. Mutations that introduce steric bulk into a channel at the top of the active site (e.g., G533A, G533V) lead to a significant reduction in APHS acetylation. Reduction in acetylation is also observed by mutation of the active-site tyrosine (Tyr-385) to phenylalanine. Mutations in the side-pocket region, into which diarylheterocycle inhibitors insert, do not affect the ability of APHS to acetylate COX-2. Surprisingly, mutation of Arg-120, which is located on the floor of the active site, strongly reduces acetylation. Based on these results, we propose that the heptynyl side chain of APHS inserts into the top channel and acetylates Ser-530 with the assistance of hydrogen bonding from Tyr-385. Arg-120 is proposed to fix the conformation of the active site to one that favors acetylation.

Enantiospecific, selective cyclooxygenase-2 inhibitors.

Cyclooxygenase inhibition studies with novel indomethacin alkanolamides demonstrate the potential for dramatic differences in inhibitor properties conferred by subtle structural modifications. The transformation of non-selective alpha-(S)-substituted indomethacin ethanolamides to potent, COX-2 selective inhibitors by simple stereocenter inversion highlights this property.

A novel bioassay for human somatogenic activity in serum samples supports the clinical reliability of immunoassays.

OBJECTIVE: Because there is discordance between different immunoassay values for serum hGH, and because clinical state may not correlate with immunoreactive hGH, we have developed an assay to accurately measure serum hGH somatogenic bioactivity. The results of this assay were compared with the Elegance two-site ELISA assay across 135 patient samples in a variety of clinical states. DESIGN: The somatogenic assay was based on stable expression of hGH receptor in the murine BaF line, allowing these cells to proliferate in response to hGH. To eliminate interference by other growth factors in serum, we created a specific antagonist of the hGH receptor (similar to Trovert or Pegvisomant) which allowed us to obtain a true measure of hGH somatogenic activity by subtraction of the activity in the presence of the antagonist. The assay was carried out in microtiter plates over 24 h, with oxidation of a chromogenic tetrazolium salt (MTT) as the endpoint. PATIENTS: These encompassed a number of different clinical conditions related to short stature, including idiopathic short stature, neurosecretory dysfunction and renal failure, as well as obese patients on dietary restriction and normal volunteers. MEASUREMENTS: In addition to the colourimetric (MTT) response to hGH, we measured free hGH by stripping out GHBP-bound hGH using beads coupled to a monoclonal antibody to the GHBP (GH binding protein). All samples were measured in both bioassay and ELISA assay. RESULTS: This bioassay was sensitive (5 mU/l or 2 microg/l) and precise, and not subject to interference by the GHBP. There was a good correlation (r = 0.95) between bioactivity and immunoactivity across clinical states. There was, however, an increased bioactivity during secretory peaks (over 25 mU/l), which has been reported previously for the Nb2 bioassay. Free hGH did not correlate with clinical state. CONCLUSIONS: Because the results of the Elegance ELISA and the bioassay correlate well, even though there is greater bioactivity at higher hormone concentrations, it is evident that an appropriate immunoassay is able to act as a reliable indicator for clinical assessment. In those rare cases where bio-inactive GH exists, our bioassay should provide an appropriate means to demonstrate this.

Amide derivatives of meclofenamic acid as selective cyclooxygenase-2 inhibitors.

This paper describes SAR studies involved in the transformation of the NSAID meclofenamic acid into potent and selective cyclooxygenase-2 (COX-2) inhibitors via neutralization of the carboxylate moiety in this nonselective COX inhibitor.