Avelumab or talazoparib in combination with binimetinib in metastatic pancreatic ductal adenocarcinoma: dose-finding results from phase Ib of the JAVELIN PARP MEKi trial.

BACKGROUND: Combinations of avelumab [anti-programmed death-ligand 1 (anti-PD-L1)] or talazoparib [poly(adenosine diphosphate ribose) polymerase (PARP) inhibitor] with binimetinib (MEK inhibitor) were expected to result in additive or synergistic antitumor activity relative to each drug administered alone. Here, we report phase Ib results from JAVELIN PARP MEKi, which investigated avelumab or talazoparib combined with binimetinib in metastatic pancreatic ductal adenocarcinoma (mPDAC). PATIENTS AND METHODS: Patients with mPDAC that had progressed with prior treatment received avelumab 800 mg every 2 weeks plus binimetinib 45 mg or 30 mg two times daily (continuous), or talazoparib 0.75 mg daily plus binimetinib 45 mg or 30 mg two times daily (7 days on/7 days off). The primary endpoint was dose-limiting toxicity (DLT). RESULTS: A total of 22 patients received avelumab plus binimetinib 45 mg (n = 12) or 30 mg (n = 10). Among DLT-evaluable patients, DLT occurred in five of 11 patients (45.5%) at the 45-mg dose, necessitating de-escalation to 30 mg; DLT occurred in three of 10 patients (30.0%) at the 30-mg dose. Among patients treated at the 45-mg dose, one (8.3%) had a best overall response of partial response. Thirteen patients received talazoparib plus binimetinib 45 mg (n = 6) or 30 mg (n = 7). Among DLT-evaluable patients, DLT occurred in two of five patients (40.0%) at the 45-mg dose, necessitating de-escalation to 30 mg; DLT occurred in two of six patients (33.3%) at the 30-mg dose. No objective responses were observed. CONCLUSIONS: Combinations of avelumab or talazoparib plus binimetinib resulted in higher-than-expected DLT rates. However, most DLTs were single occurrences, and the overall safety profiles were generally consistent with those reported for the single agents. CLINICAL TRIAL REGISTRATION: ClinicalTrials.govNCT03637491; https://clinicaltrials.gov/ct2/show/NCT03637491.

The role for interleukin-12 therapy of cutaneous T cell lymphoma.

Recent phase I and phase II trials using recombinant human interleukin-12 (rhIL-12) for cutaneous T cell lymphoma (CTCL) have been completed. Observations on 32 evaluable patients revealed an overall response rate approaching 50 percent. Biopsy of regressing lesions revealed an increase in numbers of CD8+ and/or TIA-1+ T cells. These results suggest that rhIL-12 may induce lesion regression by augmenting antitumor cytotoxic T cell responses. Future trials will be focused on strategies for further immune enhancement by the concomitant use of additional immune augmenting cytokines with rhIL-12.

Identification and characterization of a novel class of interleukin-1 post-translational processing inhibitors.

Lipopolysaccharide (LPS)-activated monocytes and macrophages produce large quantities of pro-interleukin (IL)-1beta but externalize little mature cytokine. Efficient post-translational processing of the procytokine occurs in vitro when these cells encounter a secretion stimulus such as ATP, cytolytic T cells, or hypotonic stress. Each of these stimuli promotes rapid conversion of 31-kDa pro-IL-1beta to its mature 17-kDa species and release of the 17-kDa cytokine. In this study, two novel pharmacological agents, CP-424,174 and CP-412,245, are identified as potent inhibitors of stimulus-coupled IL-1beta post-translational processing. These agents, both diarylsulfonylureas, block formation of mature IL-1beta without increasing the amount of procytokine that is released extracellularly, and they inhibit independently of the secretion stimulus used. Conditioned medium derived from LPS-activated/ATP-treated human monocytes maintained in the absence and presence of CP-424,174 contained comparable quantities of IL-6, tumor necrosis factor-alpha (TNFalpha), and IL-1RA, but 30-fold less IL-1beta was generated in the test agent's presence. As a result of this decrease, monocyte conditioned medium prepared in the presence of CP-424,174 demonstrated a greatly diminished capacity to promote an IL-1-dependent response (induction of serum amyloid A synthesis by Hep3B cells). Oral administration of CP-424,174 to mice resulted in inhibition of IL-1 in the absence of an effect on IL-6 and TNFalpha. These novel agents, therefore, act as selective cytokine release inhibitors and define a new therapeutic approach for controlling IL-1 production in inflammatory diseases.

Decreased capacity of asthmatic bronchial fibroblasts to degrade collagen.

The mechanisms of fibrillar collagen accumulation in asthmatic bronchi remain unclear, an imbalance between synthesis and degradation of collagen may be implicated in this process. The aim of this study was to compare the capacities of normal (BNF) and asthmatic (BAF) bronchial fibroblasts to degrade collagen. Metalloproteinases and their inhibitors were measured by ELISA, types I and III procollagen synthesis was determined by liquid RIA and, finally, zymography was used to assess the presence of active and latent forms of MMPs. The capacity of fibroblasts to degrade collagen coated onto latex beads was evaluated by flow cytometry. Our results showed that MMP-2 secretion was significantly higher in BNF when compared to BAF and this was confirmed by gelatin zymography. In BNF culture, TIMP-1 and MMP-1 secretions positively correlated with types I and III procollagen synthesis. However, in BAF, this correlation was negative. This suggests that a balance exists between collagen synthesis and degradation in BNF and that this balance is compromised in BAF. On the other hand, BAF did show significantly reduced capacity to degrade collagen when compared to that of BNF. This reduced phagocytic activity was not associated with a decrease in collagen receptor expression. This study establishes for the first time that a relationship exists between metalloproteinases enzyme dysregulation and the reduced capacity of asthmatic bronchial fibroblast to degrade collagen. These events may shed light on why accumulation of collagen can be observed in asthmatic airways.

ATP acts as an agonist to promote stimulus-induced secretion of IL-1 beta and IL-18 in human blood.

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1beta, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1beta, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X(7) receptor involvement. Quantities of IL-1beta generated by an individual donor's blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-alpha levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1beta and IL-18, and suggest that circulating blood monocytes constitutively express a P2X(7)-like receptor.

ATP treatment of human monocytes promotes caspase-1 maturation and externalization.

Mechanisms that regulate conversion of prointerleukin-1beta (pro-IL-1beta) to its mature form by the cysteine protease caspase-1 are not well understood. In this study, we demonstrate that mature caspase-1 subunits are produced when human monocytes are treated with ATP and, like mature IL-1beta, are released extracellularly. Characterization of the pharmacological sensitivity of this stimulus-coupled response revealed that some caspase-1 inhibitors allow pro-IL-1beta secretion, whereas others do not. Two nonselective alkylating agents, N-ethylmaleimide and phenylarsine oxide, also blocked maturation and release of pro-IL-1beta. Two inhibitors of anion transport, glyburide and ethacrynic acid, blocked maturation of both caspase-1 and pro-IL-1beta and prevented release of the propolypeptides. Procaspase-3 was detected in monocyte extracts, but its proteolytic activation was not efficient in the presence of ATP. Maturation of procaspase-1 and release of the mature enzyme subunits therefore accompany stimulus-coupled human monocyte IL-1 post-translational processing. Agents that appear to selectively inhibit mature caspase-1 do not prevent ATP-treated cells from releasing their cytosolic components. On the other hand, anion transport inhibitors and alkylating agents arrest ATP-treated monocytes in a state where membrane latency is maintained. The data provided support the hypothesis that stimulus-coupled IL-1 post-translational processing involves a commitment to cell death.

Human monocyte ATP-induced IL-1 beta posttranslational processing is a dynamic process dependent on in vitro growth conditions.

Despite a large production capacity, freshly isolated lipopolysaccharide (LPS)-activated human monocytes release only a small percentage of their newly synthesized interleukin (IL)-1 beta into the medium. Extracellular ATP, acting via surface P2z-type purino-receptors, increases cytokine posttranslational processing. To explore whether this ATP response was affected by culture conditions, monocytes were maintained for different time periods in the absence and presence of various media components including fetal bovine and human sera and recombinant human cytokines. The ability of monocytes to produce radiolabeled pro-IL-1 beta in response to LPS and to posttranslationally process the procytokine after ATP stimulation was affected both by time in culture and by the presence of specific media components. These observations indicate that ATP's ability to promote human monocyte IL-1 beta posttranslational processing is a dynamic process that is subject to regulation by cytokines and/or growth factors. Changes in monocyte/macrophage ATP responsiveness may provide an important regulatory mechanism for the control of IL-1 biological activity in vivo.

Human monocyte interleukin-1beta posttranslational processing. Evidence of a volume-regulated response.

Interleukin (IL)-1beta produced by monocytes and macrophages is not released via the normal secretory apparatus, and prior to its release, this cytokine must be proteolytically processed to generate a mature biologically active species. Biochemical mechanisms that regulate these posttranslational steps are not well understood. Lipopolysaccharide (LPS) is a poor activator of IL-1 posttranslational processing despite serving as a potent inducer of IL-1 synthesis. For example, freshly isolated human monocytes treated with LPS released <30% of their newly synthesized IL-1beta as the mature 17-kDa cytokine species, and monocytes that were aged overnight in culture prior to LPS treatment released no 17-kDa cytokine. In contrast, addition of extracellular ATP promoted IL-1beta posttranslational processing from both monocyte populations. Previous studies indicated that ATP, acting via surface P2Z-type receptors, promoted major intracellular ionic changes. To explore whether these ionic changes were required for cytokine posttranslational processing, LPS-stimulated human monocytes were maintained in ionically altered media. Hypotonic conditions promoted an efficient and selective release of mature 17-kDa IL-1beta from LPS-activated monocytes in the absence of ATP. In contrast, hypertonic conditions blocked the ATP-induced posttranslational processing reactions. Both hypotonic stress- and ATP-induced processing were blocked when NaI was substituted for NaCl within the medium; substitution with NaSCN or NaNO3 also blocked the ATP response, but these salts were less inhibitory against the hypotonic stimulus. Sodium glucuronate substitution did not inhibit cytokine processing induced by either stimulus. Removal of divalent cations from the medium did not affect the ATP response, but pretreatment of monocytes with the phosphatase inhibitor okadaic acid dose-dependently suppressed ATP-induced IL-1beta posttranslational processing. A volume-induced change to the intracellular ionic environment, therefore, may represent a key element of the mechanism by which IL-1beta posttranslational processing is initiated. The strong dependence of this cytokine release mechanism on chloride anions suggests that selective anion transporters function as important components of this response.

Lower airways remodeling in nonasthmatic subjects with allergic rhinitis.

We analyzed by immunohistochemistry the distribution of types I, II, III, IV, V, and VII collagens, laminin, and fibronectin in the bronchial biopsy specimens of nonasthmatic subjects with seasonal allergic rhinitis (n = 8) and compared these results with those found in mild stable allergic asthmatics (n = 6) and normal controls (n = 5). The content of type I and III collagens was increased in rhinitic subjects compared with controls. These collagens were focally deposited in the reticular basement membrane area. Three subjects with allergic rhinitis had no fibronectin deposition in their basement membrane, as in controls, whereas the other five had a focal fibronectin deposition. In asthmatic patients, type I and III collagens and fibronectin were more abundant and more uniformly distributed underneath the basement membrane than they were in rhinitic subjects. Expression of type II, IV, V, and VII collagens and laminin were similar in the three groups. Electron microscopic and immunohistochemical analyses of bronchial mucosa showed a network of myofibroblasts beneath the epithelium in rhinitis as in asthma subjects. These data show that the irregularly distributed subepithelial fibrosis observed in subjects with allergic rhinitis results from the deposition of type I and III collagens and fibronectin, probably produced by bronchial myofibroblasts. These results suggest the presence of an active structural remodeling in the lower airways of allergic rhinitic subjects that is similar in nature to that seen in asthma, although less marked.

Inhibition of cytokine activation processes in vitro by tenidap, a novel anti-inflammatory agent.

Tenidap is a novel anti-inflammatory and anti-arthritic agent that lowers intracellular pH and suppresses anion transport when applied to cells in vitro. Both of these parameters are known to influence pro-inflammatory cell function. To investigate whether tenidap can modulate cellular responses to cytokine stimulation, several in vitro cytokine-driven assays were characterized with respect to their tenidap sensitivity. Human monocytes treated with granulocyte-macrophage colony stimulating factor (GM-CSF) demonstrated an increased production of IL-6 as well as an increased total translational activity. Tenidap dose-dependently inhibited both cytokine-induced responses; the effect on IL-6, however, occurred at lower tenidap concentrations than those required to prevent the increase in total translational activity. In contrast, the known translational inhibitor cycloheximide did not demonstrate selectivity for IL-6; this agent decreased the GM-CSF-induced increase in total translational activity in parallel with its effects on IL-6. GM-CSF-treated monocytes also produced greater amounts of IL-1 beta in response to LPS stimulation than did non-GM-CSF-treated cells, and tenidap again suppressed this cytokine-induced activation. Human Hep3B cells treated with a combination of interleukin (IL)-1 beta and IL-6 demonstrated an acute phase-type of response. These hepatoma cells increased production of the positive acute phase protein serum amyloid A (SAA) while they decreased production of a negative acute phase protein human serum albumin (HSA). Tenidap dose-dependently inhibited the cytokine-induced increase in SAA production without effecting synthesis of HSA or total TCA-precipitable macromolecules. Importantly, the ability of tenidap to alter these various cytokine responses was not shared with piroxicam, a potent cyclooxygenase inhibitor. Finally, human neutrophils treated with either GM-CSF or tumor necrosis factor (TNF)-alpha demonstrated an increased chloride conductance as measured by the loss of radioactive chloride from 36Cl-loaded cells. When tenidap was included within the medium during cytokine stimulation, loss of radioactive chloride was prevented. Thus, tenidap inhibited the cytokine-induced increase in anion transport. Together, these results indicate that tenidap can suppress cellular activation processes induced by a variety of cytokines. This functional antagonism is not dependent on cyclooxygenase inhibition but, rather, appears to link to tenidap's unique ability to alter ionic homeostasis. These in vitro observations, therefore, may help to explain how this novel anti-inflammatory agent acts to lower acute phase proteins and IL-6 levels in man.

Tenidap modulates cytoplasmic pH and inhibits anion transport in vitro. II. Inhibition of IL-1 beta production from ATP-treated monocytes and macrophages.

IL-1 beta is an important inflammatory mediator produced by monocytes and macrophages after LPS stimulation. In the absence of a secondary stimulus, however, little IL-1 beta is released into the medium. Previously, ATP was shown to promote the release and proteolytic maturation of IL-1 beta from LPS-stimulated murine peritoneal macrophages. Tenidap, a new anti-inflammatory and antiarthritic agent, inhibited the release and maturation of IL-1 beta induced in vitro by ATP treatment of murine peritoneal macrophages. Tenidap's inhibitory activity was mimicked by other agents that blocked anion transport, such as UK5099 and DIDS. In contrast, cyclooxygenase-inhibiting nonsteroidal anti-inflammatory drugs, such as piroxicam and naproxen, did not impair ATP-induced post-translational processing. Human monocytes responded to LPS to produce IL-1 beta, but externalized little of their newly synthesized cytokine. ATP at concentrations > or = 2 mM promoted IL-1 beta release from these cells. The degree to which the released cytokine was proteolytically processed to its biologically active 17-kDa species, however, depended on the pH of the medium; a greater processing efficiency was observed at slightly acidic (pH 6.9) values. Tenidap and other anion transport inhibitors effectively prevented the ATP response of cultured human monocytes. Likewise, LPS-stimulated human alveolar macrophages responded to ATP by releasing 17-kDa IL-1 beta, and tenidap inhibited this response. The ATP-induced release and maturation of IL-1 beta from human monocytes and macrophages, therefore, was suppressed by anion transport inhibitors, suggesting that anion conductance is a necessary component of the ATP-promoted externalization mechanism. In view of IL-1's importance as an inflammatory mediator, tenidap may demonstrate novel anti-inflammatory activities by virtue of its inhibition of the post-translational release and maturation of this cytokine.

Effect of mitochondrial viability and metabolism on technetium-99m-sestamibi myocardial retention.

This study investigated the mechanism of myocardial retention of technetium-99m-sestamibi. 99mTc-sestamibi was injected intravenously into guinea pigs, and the myocardium was homogenized and fractionated by differential centrifugation. More than 90% of myocardial 99mTc-sestamibi was localized within the mitochondrial fraction. Calcium was found to release 99mTc-sestamibi from the mitochondrial fraction, with an IC50 of 2.54 +/- 0.98 mM. This effect was potentiated by NaCl, and inhibited by the mitochondrial calcium channel blocker ruthenium red. In vitro uptake of 99mTc-sestamibi was found to increase from 10.5% +/- 3.0% to 61.2% +/- 0.2% with the addition of 10 mM succinate, indicating that respiration is involved. Since irreversible ischemia results in cellular and mitochondrial calcium "overload" and loss of mitochondrial metabolic function, 99mTc-sestamibi should not be retained in necrotic or irreversibly ischemic myocardium, and could potentially act as a sensitive indicator of myocardial cell viability.

Nomifensine in the treatment of depressed geriatric patients.

In a randomized double-blind study, the efficacy and safety of nomifensine and amitriptyline were compared in 33 geriatric patients with endogenous and reactive depression. Significant improvement was noted over the 4-week study period for all groups on the Clinical Global Impression, Hamilton, and Plutchik rating scales. There was significantly more improvement among the patients with reactive depression treated with nomifensine and among the patients with endogenous depression treated with amitriptyline, as assessed by the Plutchik Geriatric Rating Scale.

Ineffectiveness of pyridoxine (B6) to alter secretion of growth hormone and prolactin and absence of therapeutic effects on galactorrhea-amenorrhea syndromes.

The acute effect of pyridoxine (B6) on serum GH and PRL levels and its chronic effects on galactorrhea in nine subjects (group I, n=4, idiopathic galactorrhea with normal PRL levels and normal menses; Group II, n=5, galactorrhea-amenorrhea with increased PRL levels) have been studied. Pyridoxine did not acutely alter GH or PRL levels. There was no decrease in galactorrhea, no resumption of menses and no decrease in PRL following tow months of B6 therapy. In contrast, bromocriptine was effective in suppressing galactorrhea and restoring normal menses in group II subjects and remains the therapy of choice for this purpose.

The influence of fasting on the disposition of warfarin in rats.

A study was undertaken to investigate the influence of fasting on the disposition of warfarin in rats. Fasting consisted of withholding solid food, but not water, immediately following warfarin (3 or 10 mg/kg s.c.) administration until sacrifice at different time intervals (3, 6, 12, 24 and 30 hours). Control animals were fed ad libitum. Total and unbound warfarin concentrations were measured in plasma and liver supernatant as well as unchanged warfarin and its metabolites in urine. The disposition of unbound warfarin was found to be markedly affected by fasting, especially at the 3 mg/kg dose: the disappearance rate of unbound warfarin from plasma was accelerated in fasted animals in contrast to that of total warfarin. In addition, unbound warfarin was cleared from plasma at a more rapid rate than total warfarin in both control and fasted animals. At the 10 mg/kg dose, the disposition of total and unbound warfarin was little affected by fasting. The concentration of unbound warfarin in the liver supernatant of fasted rats given warfarin, 3 mg/kg, was significantly increased at 6 and 24 hours of fasting. Plasma free fatty acids were significantly elevated starting at 6 hours, but no such difference was noticed with liver homogenate, except at 24 hours. The 24-hour urinary excretion of unchanged warfarin was higher in fasted rats, but fasting failed to produce any change in the excretion of warfarin metabolites. The results of the present investigation indicate that short periods of fasting influence the disposition of unbound warfarin without apparently modifying its biotransformation and further show the importance of plasma protein binding on the pharmacokinetics of warfarin.