Pulmonary insulin delivery by means of the Technosphere™ drug carrier mechanism

Technosphere?/Insulin (TI) is a formulation of regular human insulin designed for efficient transport across the respiratory epithelium into the circulation. The drug carrier mechanism achieves a fast systemic insulin uptake (maximum time ～ 15 – 20 min), a fast onset of action (maximum activity ～ 25 – 30 min) and a short duration of action (～ 2 h). Bioavailability, relative to subcutaneous injection, was established to be between 30 and 50% with a linear dose–response relationship and low variability. In all published short-term studies, TI was well tolerated. Provided a reliable long-term safety profile, TI may become a suitable alternative to subcutaneous injection for prandial insulin delivery. TI offers the possibility of new treatment regimens, especially in patients with Type 2 diabetes.     

Inactivation gating determines drug potency：a common mechanism for drug blockade of HERG channels

INTRODUCTIONThe K channel encoded by HERG (the humanether-a-go-go related gene) is well accepted as anequivalent of the native delayed rectifier K channel IKrin cardiac myocytes. Indeed, when expressed in eithermammalian cells or Xenopus oocytes, the HERG chan-nel current is virtually identical to IKr in terms of thebiophysical properties and pharmacological sensitivity.This channel provides a mechanistic link between theacquired and the inherited long Q-T syndrome[1]. Thepharm…     

Why does the inner-helix mutation A413C double the stoichiometry of Kv1.3 channel block by emopamil but not by verapamil?

hKv1.3 channels in lymphocytes are targets for the chemotherapy treatment of autoimmune diseases. Phenylalkylamines block Kv1.3 channels by poorly understood mechanisms. In the inactivation-reduced mutant H399T, the second mutation A413C in S6 substantially decreases the potency of phenylalkylamines with a para-methoxy group at the phenylethylamine end, whereas potency of phenylalkylamines lacking this group is less affected. Intriguingly, completely demethoxylated emopamil blocks mutant H399T/A413C with a 2:1 stoichiometry. Here, we generated a triple mutant, H399T/C412A/A413C, and found that its emopamil-binding properties are similar to those of the double mutant. These data rule out disulfide bonding Cys412-Cys413, which would substantially deform the inner helix, suggest a clash of Cys413 with the para-methoxy group, and provide a distance constraint to dock phenylalkylamines in a Kv1.2-based homology model. Monte Carlo minimizations predict that the verapamil ammonium group donates an H-bond to the backbone carbonyl of Thr391 at the P-loop turn, the pentanenitrilephenyl moiety occludes the pore, whereas the phenylethylamine meta- and para-methoxy substituents approach, respectively, the side chains of Met390 and Ala413. In the double-mutant model, the Cys413 side chains accept H-bonds from two emopamil molecules whose phenyl rings fit in the hydrophobic intersubunit interfaces, whereas the pentanenitrilephenyl moieties occlude the pore. Because these interfaces are unattractive for a methoxylated phenyl ring, the ammonium group of respective phenylalkylamines cannot approach the Cys413 side chain and binds at the focus of P-helices, whereas the para-methoxy group clashes with Cys413. Our study proposes an atomistic mechanism of Kv1.3 block by phenylalkylamines and highlights the intra- and intersubunit interfaces as ligand binding loci.     

Molecular mechanism of positive allosteric modulation of the metabotropic glutamate receptor 2 by JNJ‐46281222

Background and Purpose

Allosteric modulation of the mGlu₂ receptor is a potential strategy for treatment of various neurological and psychiatric disorders. Here, we describe the in vitro characterization of the mGlu₂ positive allosteric modulator (PAM) JNJ‐46281222 and its radiolabelled counterpart [³H]‐JNJ‐46281222. Using this novel tool, we also describe the allosteric effect of orthosteric glutamate binding and the presence of a bound G protein on PAM binding and use computational approaches to further investigate the binding mode.

Experimental Approach

We have used radioligand binding studies, functional assays, site‐directed mutagenesis, homology modelling and molecular dynamics to study the binding of JNJ‐46281222.

Key Results

JNJ‐46281222 is an mGlu₂‐selective, highly potent PAM with nanomolar affinity (K _D = 1.7 nM). Binding of [³H]‐JNJ‐46281222 was increased by the presence of glutamate and greatly reduced by the presence of GTP, indicating the preference for a G protein bound state of the receptor for PAM binding. Its allosteric binding site was visualized and analysed by a computational docking and molecular dynamics study. The simulations revealed amino acid movements in regions expected to be important for activation. The binding mode was supported by [³H]‐JNJ‐46281222 binding experiments on mutant receptors.

Conclusion and Implications

Our results obtained with JNJ‐46281222 in unlabelled and tritiated form further contribute to our understanding of mGlu₂ allosteric modulation. The computational simulations and mutagenesis provide a plausible binding mode with indications of how the ligand permits allosteric activation. This study is therefore of interest for mGlu₂ and class C receptor drug discovery.

Abbreviations

JNJ‐46281222

3‐(Cyclopropylmethyl)‐7‐[(4‐phenyl‐1‐piperidinyl)methyl]‐8‐(trifluoromethyl)‐1,2,4‐triazolo[4,3‐a]pyridine

NAM

negative allosteric modulator

PAM

positive allosteric modulator

VFT

Venus Flytrap domain

     

A comparison of medical symptoms reported by cocaine‐, opiate‐, and alcohol‐dependent patients

Substance abuse is frequently associated with adverse medical consequences. The differences in medical symptoms reported by 101 alcohol‐, 113 cocaine‐, and 107 opiate‐dependent individuals receiving outpatient treatment were studied using a 134‐item questionnaire (MILCOM). Data analysis revealed interesting and unexpected findings, with cocaine patients reporting the fewest total symptoms among the three groups. Moreover, cocaine patients reported significantly fewer CNS and musculo‐skeletal symptoms compared to both alcohol and opiate patients and significantly fewer GI and urinary symptoms than the alcohol but not the opiate patients. In addition, there were sex‐ and race‐related differences in the pattern of symptoms reported. Women reported significantly more CVS, mood, nose/throat, CNS, skin, and GI symptoms than men. Similarly, Caucasians reported significantly more mood, CNS, nose/throat, head/neck, musculoskeletal, and GI symptoms than African‐Americans. The study highlights the influence of drug of choice, gender, and race on medical needs of substance‐abusing persons.     

Metered dose inhaler add-on devices: is the inhaled mass of drug dependent on the size of the infant?

Limited cooperation and low tidal volumes in infants make aerosol therapy difficult. We measured the amount of drug delivered from two baby spacer devices especially developed for use in infants. Designed as a randomized crossover study, aerolized budesonide from a pressurized metered dose inhaler (pMDI) was collected in the inspiratory filter interposed between the face mask and the spacer in 13 infants aged from 2 to 19 months old. The study was performed in connection with pulmonary function testing with a plethysmograph, and the children were sedated with cloral hydrate. Two small-volume baby spacer devices were used: a Babyhaler spacer (GlaxoWellcome, Hertfordshire, UK) made of polycarbonate with a volume of 350 mL and a built-in dead space of 40 mL and a NebuChamber spacer (AstraZeneca, Lund, Sweden) made of stainless steel with a volume of 250 mL and no dead space. Budesonide delivery from the NebuChamber was significantly higher than from the Babyhaler: 38.2% (range, 28.3%-47.5%) of the nominal dose versus 12% (range, 3.3%-21.25%) of the nominal dose of 400 micrograms of budesonide (P = 0.002). The inhaled mass of budesonide from the Babyhaler correlated significantly with skin surface area (r = 0.68, P = 0.018), weight (r = 0.66, P = 0.019), height (r = 0.69; P = 0.017), tidal volume (r = 0.82; P = 0.004), and minute volume (r = 0.67; P = 0.019). No correlations were found between these variables and the inhaled mass of budesonide from the NebuChamber. The results indicate that the design of the NebuChamber spacer affords stable drug delivery in infants and that a large variability in the inhaled mass of drug may be found when infants are inhaling from different baby spacers.     

Is the onset of psychoactive drug effects compatible with a protein-synthesis mechanism?

While many have suggested that protein synthesis may mediate the action of antipsychotic drugs, it is difficult to test. In this math modeling study it is found that the time course of action of the drugs are compatible with a protein-synthesis model and, furthermore, that the half-lives required by the model are indeed found in relevant proteins in the brain.     

SP protects cerebellar granule cells against β-amyloid-induced apoptosis by down-regulation and reduced activity of Kv4 potassium channels

The tachykinin endecapeptide substance P (SP) has been demonstrated to exert a functional role in neurodegenerative disorders, including Alzheimer's disease (AD). Aim of the present study was to evaluate the SP neuroprotective potential against apoptosis induced by the neurotoxic beta-amyloid peptide (Aβ) in cultured rat cerebellar granule cells (CGCs). We found that SP protects CGCs against both Aβ_25-35- and Aβ_1-42-induced apoptotic CGCs death as revealed by live/dead cell assay, Hoechst staining and caspase(s)-induced PARP-1 cleavage, through an Akt-dependent mechanism.Since in CGCs the fast inactivating or A-type K⁺ current (I_KA) was potentiated by Aβ treatment through up-regulation of Kv4 subunits, we investigated whether I_KA and the related potassium channel subunits could be involved in the SP anti-apoptotic activity.Patch-clamp experiments showed that the Aβ-induced increase of I_KA current amplitude was reversed by SP treatment. In addition, as revealed by Western blot analysis and immunofluorescence studies, SP prevented the up-regulation of Kv4.2 and Kv4.3 channel subunits expression.These results indicate that SP plays a role in the regulation of voltage-gated potassium channels in Aβ-mediated neuronal death and may represent a new approach in the understanding and treatment of AD.     

Role of endothelial TRPV4 channels in vascular actions of the endocannabinoid, 2‐arachidonoylglycerol

Background and Purpose

Metabolites of the endocannabinoid, 2‐arachidonoylglycerol (2‐AG) have been postulated to act as endogenous activators of TRPV4, a Ca²⁺‐permeable cation channel that plays a critical role in endothelium‐dependent relaxation. However, it is unclear if TRPV4 contributes to the vascular actions of 2‐AG.

Experimental Approach

Isometric tension recording of rat small mesenteric arteries and aortae were used to assess the effect of 2‐AG and the synthetic TRPV4 activator, GSK1016790A (GSK) on vascular reactivity. Changes in intracellular Ca²⁺ concentration and single‐channel currents were measured in TRPV4‐expressing human coronary endothelial cells.

Key Results

In mesenteric arteries, endothelium‐dependent relaxation to both 2‐AG and GSK was attenuated by structurally distinct TRPV4 antagonists, HC067047, RN1734 and ruthenium red. The responses were inhibited by K_Ca inhibitors (apamin + charybdotoxin) and a gap junction inhibitor (18α‐glycyrrhetinic acid). In contrast to GSK, 2‐AG elicited considerable relaxation independently of the endothelium or TRPV4. Inhibition of 2‐AG metabolism via monoacylglycerol lipase and COX (by MAFP and indomethacin) caused potentiation, while cytochrome P450 and lipoxygenase inhibitors had no effect on 2‐AG relaxation. In coronary endothelial cells, 2‐AG (with and without MAFP) induced HC067047‐sensitive increases in intracellular Ca²⁺ concentration. 2‐AG also increased TRPV4 channel opening in inside‐out patches. However, in aortae, GSK induced a relaxation sensitive to HC067047 and ruthenium red, whereas 2‐AG induced contractions.

Conclusions and Implications

These data suggest that 2‐AG can directly activate endothelial TRPV4, which partly contributes to the relaxant response to 2‐AG. However, the functional role of TRPV4 is highly dependent on the vascular region.

Abbreviations

4α‐PDD

4α‐phorbol‐12,13‐didecanoate

GSK

GSK1016790A

JTE907

N‐(1,3‐benzodioxol‐5‐ylmethyl)‐1,2‐dihydro‐7‐methoxy‐2‐oxo‐8‐(pentyloxy)‐3‐quinolinecarboxamide

SKF525A

α‐phenyl‐α‐propylbenzeneacetic acid 2‐(diethylamino)ethyl ester N,N‐diethylaminoethyl 2,2‐diphenylvalerate

     

Medication control in hospitals. A practical approach to the problem of drug‐drug interactions.

An important task of pharmacists is medication control by screening the medication of an individual patient. Many computerized drug interaction screening programs are available, but they all have their drawbacks. Screening without a computerized program is possible, but very timeconsuming. In contrast to daily living at home, the patient in a hospital setting is carefully monitored and relevant biochemical parameters are regularly checked. Many potential drug interactions are countered immediately by changing (dosage of) medication. The aim of our study was to determine the number of drugdrug interactions and (pseudo)double medications on the internal, pulmonary and cardiological ward in order to discuss them with the physicians. During this discussion the clinical relevance of interactions was determined. We conclude that the number of clinically relevant interactions and (pseudo)double medication is limited, but that the role of the pharmacist is an important one, especially with regard to medication, that is not regularly used on a ward. Potential drug interactions should be predicted and dealt with by close teamwork of physician and pharmacist at the moment medication is prescribed.     

Sensitization of nociceptive ion channels by inhaled anesthetics--a pain in the gas?

A remarkable new article in this issue of Molecular Pharmacology (p. 1261) shows that the capsaicin-sensitive ion channel TRPV1 is sensitized to activation by chemical and physical stimuli in the presence of inhaled general anesthetics. This finding provides another example of an ion channel in which the anesthetic acts to modify channel gating. This may have important clinical implications in view of the role of TRPV1 in nociception.     

The effect of postdetoxification drug‐free residential living on birth outcome in the pregnant drug abuser 1

The birth outcome for pregnant drug abusers is complicated by polydrug use, neonatal withdrawal symptoms, and low birth weight. To improve birth outcome, in‐hospital detoxification and stabilization from drug use followed by drug‐free residential living with prenatal care were provided for patients committed to cessation of illicit drug use. Pregnant heroin or cocaine abusers with significant drug use were eligible for the study. The birth outcome for the 30 term singletons born to the mothers in the program was statistically compared with respect to low birth weight, mean birth weight and length of stay in the nursery to that for 44 term singleton infants of a similar group of drug‐abusing ambulatory pregnant women in our aftercare clinic, not in the residential program. Twenty percent of 152 patients evaluated were found suitable for admission to the program. Numerical data for term singletons were as follows: weights (g ± SD), 3218 ± 596 (30) vs control 2806 ± 317 (44); low birth weights; 1/30 vs control 12/44; and length of stay in the nursery (days ± SD), 3 ± 0.5 (30) vs control 5.5 ± 2.5 (44). For the pregnant drug abuser committed to cessation of drug use, detoxification followed by drug free living leads to a statistically significant improvement in birth outcome.     

Stabilized Amorphous State of Ibuprofen by Co‐Spray Drying With Mesoporous SBA‐15 to Enhance Dissolution Properties

A novel formulation process via co‐spray drying ibuprofen (IBU) with mesoporous SBA‐15 submicron particles exhibited excellence in production of stable amorphous IBU with significantly enhanced dissolution rate. With drug loading of IBU/SBA‐15 ratio being 50:50 (w/w) or below, most drug molecules were entrapped inside the straight mesoporous channels via the co‐spray drying and the morphology of SBA‐15 submicron particles remained unchanged. IBU confined inside the mesoporous structure was in the amorphous state shown by PXRD and DSC measurements. The amorphous state of IBU in the solid dispersion showed remarkable stability when subject to stress test condition of 40°C/75% RH in open pans for 12 months. The uniform pore walls were believed to prevent the re‐crystallization of the homogeneously dispersed drug molecules inside the mesoporous channels with confined nanospace. The dissolution rate of IBU from the co‐spray‐dried solid dispersion was significantly enhanced to achieve a rapid release. Even after the accelerated stability test, the rapid drug release property was well preserved. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1997–2007, 2010     

In vitro assessment of the roles of drug transporters in the disposition and drug–drug interaction potential of olaparib

1.?In vitro assessments were conducted to examine interactions between olaparib (a potent oral inhibitor of poly[ADP-ribose] polymerase) and drug transporters.

2.?Olaparib showed inhibition of the hepatic drug uptake transporters OATP1B1 (IC₅₀ values of 20.3?μM and 27.1?μM) and OCT1 (IC₅₀ 37.9?μM), but limited inhibition of OATP1B3 (25% at 100?μM); inhibition of the renal uptake transporters OCT2 (IC₅₀ 19.9?μM) and OAT3 (IC₅₀ 18.4?μM), but limited inhibition of OAT1 (13.5% at 100?μM); inhibition of the renal efflux transporters MATE1 and MATE2K (IC₅₀s 5.50?μM and 47.1?μM, respectively); inhibition of the efflux transporter MDR1 (IC₅₀ 76.0?μM), but limited inhibition of BCRP (47% at 100?μM) and no inhibition of MRP2. At clinically relevant exposures, olaparib has the potential to cause pharmacokinetic interactions via inhibition of OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K in the liver and kidney, as well as MDR1 in the liver and GI tract. Olaparib was found to be a substrate of MDR1 but not of several other transporters.

3.?Our assessments indicate that olaparib is a substrate of MDR1 and may cause clinically meaningful inhibition of MDR1, OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K.     

Depression of drug metabolizing activity in the human liver by interferon-α

Summary The depressant effect of interferon- on drug metabolizing activity in the liver has been investigated in 12 patients with chronic active hepatitis B. 7-methoxy-coumarin (7-MC) O-demethylase and 7-ethoxycoumarin (7-EC) O-deethylase, in specimens obtained by liver biopsy, were measured before and after interferon treatment. 7-MC and 7-EC O-dealkylase activity were significantly reduced after interferon treatment, from 13.4 to 9.24 nmol·g^–1 liver·min^–1, and from 3.22 to 2.16 nmol·g^–1 liver·min^–1, respectively. The magnitude of the fall varied widely between individual patients. The study provides the first direct evidence that interferon- can impair the activity of drug metabolizing enzymes in the human liver.     

Co-expression of NaVβ subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins

Background and Purpose

Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing α-subunit (Na_V1) and one or two accessory β-subunits (Na_Vβs). Neurons in mammals can express one or more of seven isoforms of Na_V1 and one or more of four isoforms of Na_Vβ. The peptide μ-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in Na_V1. Hitherto, the effects of Na_Vβ-subunit co-expression on the activity of these toxins have not been comprehensively assessed.

Experimental Approach

Four μ-conotoxins (μ-TIIIA, μ-PIIIA, μ-SmIIIA and μ-KIIIA), TTX and STX were tested against Na_V1.1, 1.2, 1.6 or 1.7, each co-expressed in Xenopus laevis oocytes with one of Na_Vβ1, β2, β3 or β4 and, for Na_V1.7, binary combinations of thereof.

Key Results

Co-expression of Na_Vβ-subunits modifies the block by μ-conotoxins: in general, Na_Vβ1 or β3 co-expression tended to increase k_on (in the most extreme instance by ninefold), whereas Na_Vβ2 or β4 co-expression decreased k_on (in the most extreme instance by 240-fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by Na_Vβ-subunit co-expression. Tests of Na_Vβ1 : β2 chimeras co-expressed with Na_V1.7 suggest that the extracellular portion of the Na_Vβ subunit is largely responsible for altering μ-conotoxin kinetics.

Conclusions and Implications

These results are the first indication that Na_Vβ subunit co-expression can markedly influence μ-conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. μ-Conotoxins could, in principle, be used to pharmacologically probe the Na_Vβ subunit composition of endogenously expressed VGSCs.     

Schedule dependent potentiation of antitumor drug effects by α-difluoromethylornithine in human gastric carcinoma cells in vitro

A clone of human gastric cancer cells (AGS-6) and the parental line (AGS-P) from which it was isolated were used in cell survival studies to determine whether pretreatment for 24, 48 or 72h with -difluoromethylornithine (DFMO, 5mM) would increase the cell's sensitivity to 5-Fluorouracil (5FU), Adriamycin (Adria), 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU), or Bleomycin (Bleo). Generally, the AGS parental cells were most sensitive to the anticancer agents after exposures to DFMO. However, there was no way to predict in advance from DFMO-induced changes in ornithine decarboxylase (ODC), polyamine or cell kinetics values, how long an exposure to DFMO was required before sensitization to an anticancer agent occurred. The degree of potentiation for a single drug was variable from time to time during exposure to DFMO, and broad differences in the sensitizations were demonstrated among the four anticancer drugs. The AGS-6 clone exhibited little or no increased sensitivity as a result of pretreatment with DFMO, even though the DFMO-induced reductions in ODC and polyamine values in these cells were similar to those produced in the more sensitive parental line.     

Curcumin improves the antitumor effect of X-ray irradiation by blocking the NF-κB pathway: an in-vitro study of lymphoma

Curcumin, a phenolic compound from the rhizomes of Curcuma longa, inhibits the growth of a variety of malignant cell types including lymphoma cells. We investigated the role of curcumin in modulating the response of Burkitt's lymphoma cells to ionizing radiation (IR) in vitro and explored the mechanisms that mediated this effect. We treated three Burkitt's lymphoma cell lines with vehicle, curcumin, IR, and curcumin in combination with IR. Cell viability, apoptosis, and cell cycle distribution were determined to ascertain the radiosensitization effect of curcumin. Nuclear factor-kappa B (NF-κB) activation was assessed by nuclear translocation of p65. Apoptosis-related proteins were monitored by western blot assay and real-time RT-PCR. Pretreatment of curcumin sensitized lymphoma cells to IR-induced apoptosis and increased G2/M phase arrest in the cell cycle distribution. Accordingly, the antiapoptotic Bcl-xL protein, cell cycle modulating protein CDC2, and cyclin B1 were downregulated by the curcumin treatment. IR activated NF-κB as evidenced by an increased nuclear p65 translocation and cytoplasmic IκBα expression. However, pretreatment with curcumin significantly decreased the nuclear translocation of p65 and cytoplasmic IκBα degradation. Survivin and hexokinase II, downstream effectors of NF-κB that mediate the antiapoptotic effect of NF-κB, were suppressed by the pretreatment of curcumin. These observations suggest that the activated NF-κB pathway plays a prosurvival role in Burkitt's lymphoma in response to IR. Curcumin blocks this pathway and has therapeutic potential for improving the antitumor effects of radiotherapy.