Application of Polyglycerol Coating to Plasmid DNA Lipoplex for the Evasion of the Accelerated Blood Clearance Phenomenon in Nucleic Acid Delivery

Cationic liposomes (CLs) have shown promise as nonviral delivery systems. To achieve in vivo stability and long circulation, most liposomes are modified with hydrophilic polymer polyethylene glycol (PEG). However, we have reported that repeated administration of PEG-coated CLs containing plasmid DNA (pDNA; PEGylated lipoplexes) induces what is referred to as “the accelerated blood clearance (ABC) phenomenon” and, consequently, subsequently administered lipoplexes lose their prolonged circulation characteristics. Anti-PEG IgM produced in response to the first dose of PEG-coated pDNA–lipoplexes (PEG–DCL) has proven to be a major cause of the ABC phenomenon. In this study, to evade and/or attenuate this unexpected immune response, we modified the surface of a lipoplex with polyglycerol (PG)-derived lipid. The PG-coated pDNA–lipoplex (PG–DCL) attenuated the production of anti-polymer IgM, whereas PEG-coated pDNA–lipoplex (PEG–DCL) did not. In addition, a second dose of PG–DCL maintained the accumulation level in the tumor tissue of a tumor-bearing mouse model, comparable to that of the first dose, whereas the tumor accumulation level of a second dose of PEG–DCL was significantly compromised, compared with the first dose of PEG–DCL. Our results indicate that surface modification of lipoplex with PG represents a viable means for the attenuation, and/or evasion, of the ABC phenomenon that is encountered upon repeated administrations of nucleic acids containing PEG-coated nanocarriers.     

Generation,characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs

PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs — HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG₂₀₀₀ conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH₂CH₂) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics.     

Development of a liposomal formulation of the natural flavonoid fisetin

The natural flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) has been shown to possess antiangiogenic and anticancer properties. Because of the limited water solubility of fisetin, our aim was to design and optimize a liposomal formulation that could facilitate its in vivo administration, taking into account the availability and cost of the various components. Several methods were evaluated such as probe sonication, homogeneization, film hydration and lipid cake formation. A selection of lipid and lipid-PEG was also performed via their incorporation in different formulations based on the size of the liposomes, their polydispersity index (PDI) and the fisetin encapsulation yield. An optimal liposomal formulation was developed with P90G and DODA-GLY-PEG2000, possessing a diameter in the nanometer scale (175 nm), a high homogeneity (PDI 0.12) and a high fisetin encapsulation (73%). Fisetin liposomes were stable over 59 days for their particle diameter and still retained 80% of their original fisetin content on day 32. Moreover, liposomal fisetin retained the cytotoxicity and typical morphological effect of free fisetin in different tumour and endothelial cell lines. In conclusion, based on its physico-chemical properties and retention of fisetin biological effects, the developed liposomal fisetin preparation is therefore suitable for in vivo administration.     

Time Interval of Two Injections and First-Dose Dependent of Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomal Gambogenic Acid: The Contribution of PEG-Specific IgM

Repeated injection of PEGylated liposomes can cause the disappearance of long circulating property because of the induction of anti-PEG IgM antibody referred to as “accelerated blood clearance (ABC) phenomenon.” Although ABC phenomenon typically occurs when entrapped drugs are chemotherapeutic agent with low cytotoxic, there is little evidence of accelerated blood clearance of PEGylated herbal-derived compound on repeated injection. Herein, we investigated the blood concentration of PEGylated liposomal gambogenic acid (PEG-GEA-L), a model PEGylated liposomal herbal extract, on its repeated injection to rats. We found time interval between injections had considerable impact on the magnitude of ABC phenomenon induced by PEG-GEA-L. When time interval was prolonged from 3 days to 7 days, ABC phenomenon could be attenuated. Furthermore, its magnitude was enhanced accompanied by a marked rise in the accumulation of PEG-GEA-L in the liver and spleen in a first-dose–dependent manner. Consistently, the level of anti-PEG IgM significantly increased with the first dose of PEG-GEA-L and decreased with the extended time interval between injections, which implies anti-PEG IgM is a major contributor to the ABC phenomenon. Notably, the increased expression of liver anti-PEG IgM was accompanied by an increased expression of efflux transporters in the induction process of the ABC phenomenon.     

Enhanced Encapsulation of Amphotericin B into Liposomes by Complex Formation with Polyethylene Glycol Derivatives

Purpose. A highly efficient method was developed for the encapsulation of amphotericin B (AmB) in liposomes, and the mechanism involved was characterized. Methods. AmB was encapsulated in dipalmitoylphosphatidylcholine/cholesterol (DPPC/CH, 2:1) liposomes after complex formation with distearoyl-N-(monomethoxy poly(ethylene glycol) succinyl) phosphatidylethanolamine (DSPE-PEG). Hydration of lipids was done with 9% sucrose solution. Results. The encapsulated amount of AmB was 111 g/mg lipid, which was much higher than that obtained by the same method without DSPE-PEG (14 g/mg lipid). The amount encapsulated increased with amount of DSPE-PEG used and with PEG molecular weight. Encapsulation efficacy was also influenced by the type of PEG derivatives used and by the modification of AmB, suggesting the involvement of complex formation between AmB and DSPE-PEG. Absorption and ³¹P-NMR spectral analyses indicated that interactions between the amino and phosphate groups and between the polyene and PEG moieties in AmB and DSPE-PEG, respectively, play an important role in the complex formation. Conclusions. Complex formation of AmB with DSPE-PEG allows the highly efficient encapsulation of the drug in liposomes. This simple technique should be applicable to other hydrophobic drugs.     

The accelerated blood clearance phenomenon of PEGylated nanoemulsion upon cross administration with nanoemulsions modified with polyglycerin

For investigating the accelerated blood clearance (ABC) phenomenon of polyglycerin modified nanoemulsions upon cross administration with polyethylene glycol (PEG) covered nanoemulsion, we used the 1,2-distea-royl-sn-glycero-3-phosphoethanolamine-n-polyglycerine-610 and the 1,2-distearoyl-n-glycero-3-phosphoethanolamine-n-[me-thoxy(polyethylene glycol)-2000] as modify materials, the dialkylcarbocyanines as fluorescence indicator. Exhausted macrophages rat model was established and new material containing polycarboxyl structure was synthesized. The microplate reader and the in vivo optical imaging system were applied to measure the concentration of nanoemulsions in tissues. The results show that the first dose of polyglycerin modified nanoemulsion can induce the ABC phenomenon of the second dose of PEGylated nanoemulsion. With the increase in the amount of the surface polyglycerin, the extent of the ABC phenomenon decreases. Liver accumulation has positive relationship with the ABC phenomenon. Furthermore, kupffer cells in liver can get more immune information from polyhydroxy structure than polycarboxyl group in the modify compound. The results of our work imply that the polycarboxyl structure has advantages to eliminate the ABC phenomenon.     

Repeated injection of pegylated liposomal antitumour drugs induces the disappearance of the rapid distribution phase

Upon repeated administration, empty pegylated liposomes lose their long‐circulating characteristics, referred to as the accelerated blood clearance (ABC) phenomenon. To investigate whether cytotoxic drug‐containing pegylated liposomes could also elicit a similar phenomenon, two pegylated liposomal antitumour drugs (doxorubicin and mitoxantrone) were prepared, and they were administrated twice in the same animals with a 10‐day interval at a dose level of 8 mg kg^?1 (pegylated liposomal doxorubicin) and 4 mg kg^?1 (pegylated liposomal mitoxantrone). By comparing the overall pharmacokinetics after a single‐dose injection with that in animals treated with two doses, it was surprising to find that repeated administration of pegylated liposomal antitumour drugs caused the disappearance of rapid distribution phase instead of the ABC phenomenon, resulting in the conversion of a two‐compartment model to a one‐compartment model. Further investigation revealed that repeated injection induced the decreased uptake of liposomal antitumour drugs by the spleen at the early time point of 0.5–8 h after injection. In contrast, the deposition of liposomal antitumour drugs into liver was not affected. Therefore, the disappearance of the rapid distribution phase might be related to the reduced spleen uptake at the early time point.     

Interaction of resveratrol with membrane glycerophospholipids in model system in vitro

Resveratrol is a polyphenol that among other sources occurs in grapes and for this reason, red wines also contain considerable amounts of this compound. Interactions of resveratrol with pure molecular species of phosphatidylcholine (PC), phosphatidylethanoloamine (PE) and phosphatidylserine (PS) were studied with the Langmuir technique on monolayers and with differential scanning calorimetry on unilamellar liposomes. Resveratrol caused a modest increase in the mean molecular area (MMA) of dipalmitoyl-PC (DPCC) and PE (DPPE) monolayers, but profoundly increased the MMA of dipalmitoyl-PS (DPPS). The resveratrol-induced increase in MMA was enlarged in PS species containing stearolyl and oleoyl acyls suggesting that increase in the acyl chain length and unsaturation enhanced the resveratrol–phospholipid interaction. In liposomes resveratrol lowered T_m (main transition temperature) and increased T_C1/2 (transition interval) in DPPE, DPPS and 1-palmitoyl-2-oleoyl-PS (POPS), suggesting that resveratrol causes an increase in the anisotropy of these liposomes. In DPPE and POPS liposomes resveratrol caused a lowering of ΔH, further substantiating a pure enhancement of anisotropy for these molecular species. However, resveratrol caused a marked increase of ΔH in DPPS liposomes, indicating that, in addition to increase anisotropy, DPPS and resveratrol attracted each other specifically. This study has clearly shown interactions between resveratrol and glycerophospholipids on a molecular level and that these interactions are influenced by the acyl chain length, degree of unsaturation and head group of the lipids.     

Formulation and in vitro/in vivo evaluation of levodopa transdermal delivery systems

This study aims to investigate the feasibility of Levodopa transdermal delivery systems (TDSs). Levodopa TDSs were formulated using various vehicles and permeation enhancers, and in vitro permeation and in vivo pharmacokinetic studies were carried out. In the in vitro study, ester-type vehicles showed relatively high enhancing effects; propylene glycol monocaprylate and propylene glycol monolaurate showed the highest permeation fluxes from both solution and pressure sensitive adhesive (PSA) TDS formulations. Lag time was dramatically shortened with PSA TDS formulations as compared with solution formulations. In the in vivo study, the addition of fatty acids increased blood drug concentrations regardless of the kind or concentration of fatty acid; the AUC_inf increased up to 8.7 times as compared with propylene glycol (PG) alone. PSA TDS containing 10% linoleic acid exhibited prolonged T_max as compared with oral form. Total clearance of L-dopa from PSA TDSs was significantly lower than from oral form (up to 86.8 times). Especially, PSA TDS containing 10% linoleic acid (LOA) revealed 76.2 fold higher AUC_inf than oral administration. Based on our results, the L-dopa PSA TDS containing PG with 10% LOA could be used as a good adjuvant therapy for Parkinson's disease patients who experience symptom fluctuation by L-dopa oral administration.     

Quantitative relationship between guanine O-alkyl lesions produced by Onrigin™ and tumor resistance by O-alkylguanine-DNA alkyltransferase

O⁶-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O⁶-chloroethyl (Onrigin™ and carmustine) and O⁶-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O⁶-methylguanines/cell and ∼300 O⁶-chloroethylguanines/cell, respectively. AGT repaired O⁶-methylguanines until the AGT pool was exhausted, while its repair of O⁶-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O⁶-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O⁶-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.     

Accelerated Blood Clearance of PEGylated PLGA Nanoparticles Following Repeated Injections: Effects of Polymer Dose, PEG Coating, and Encapsulated Anticancer Drug

Purpose

To investigate accelerated blood clearance (ABC) induction upon repeated injections of PLGA-PEG nanoparticles as a commonly used polymeric drug carrier.

Methods

Etoposide-loaded PLGA-PEG NPs were developed and administered as the test dose to rats pre-injected with various NP treatments at certain time intervals. Pharmacokinetic parameters of etoposide and production of anti-PEG IgM antibody were evaluated.

Results

A notable ABC effect was induced by a wide range of polymer doses (0.1 to 20 mg) of empty NPs, accompanied by IgM secretion. However, a further increase in polymer dose resulted not only in the abrogation of the observed ABC induction but also in distinctly a higher value for AUC of the NPs relative to the control. The data from the PEG-negative group verified the fundamental role of PEG for ABC induction. The first injection of etoposide-containing PEGylated nanoparticles (a cell cycle phase-specific drug) produced a strong ABC phenomenon. Three sequential administrations of etoposide-loaded NPs abolished ABC, although a high level of IgM was still detected, which suggests saturation with insignificant poisoning of immune cells.

Conclusion

The presented results demonstrate the importance of clinical evaluations for PLGA-PEG nanocarriers that consider the administration schedule in multiple drug delivery, particularly in cancer chemotherapy.     

Influence of Cocaine History on the Behavioral Effects of Dopamine D3 Receptor-Selective Compounds in Monkeys

Although dopamine D₃ receptors have been associated with cocaine abuse, little is known about the consequences of chronic cocaine on functional activity of D₃ receptor-preferring compounds. This study examined the behavioral effects of D₃ receptor-selective 4-phenylpiperazines with differing in vitro functional profiles in adult male rhesus monkeys with a history of cocaine self-administration and controls. In vitro assays found that PG 619 (N-(3-hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-(pyridin-2-yl)benzamide HCl) was a potent D₃ antagonist in the mitogenesis assay, but a fully efficacious agonist in the adenylyl cyclase assay, NGB 2904 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide HCl) was a selective D₃ antagonist, whereas CJB 090 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridin-2-yl)benzamide HCl) exhibited a partial agonist profile in both in vitro assays. In behavioral studies, the D₃ preferential agonist quinpirole (0.03–1.0 mg/kg, i.v.) dose-dependently elicited yawns in both groups of monkeys. PG 619 and CJB 090 elicited yawns only in monkeys with an extensive history of cocaine, whereas NGB 2904 did not elicit yawns, but did antagonize quinpirole and PG 619-elicited yawning in cocaine-history monkeys. In another experiment, doses of PG 619 that elicited yawns did not alter response rates in monkeys self-administering cocaine (0.03–0.3 mg/kg per injection). Following saline extinction, cocaine (0.1 mg/kg) and quinpirole (0.1 mg/kg), but not PG 619 (0.1 mg/kg), reinstated cocaine-seeking behavior. When given before a cocaine prime, PG 619 decreased cocaine-elicited reinstatement. These findings suggest that (1) an incongruence between in vitro and in vivo assays, and (2) a history of cocaine self-administration can affect in vivo efficacy of D₃ receptor-preferring compounds PG 619 and CJB 090, which appear to be dependent on the behavioral assay.     

In vivo pharmacokinetics, tissue distribution and underlying mechanisms of various PEI(-PEG)/siRNA complexes

Background

RNA interference (RNAi) represents a novel therapeutic strategy allowing the knockdown of any pathologically relevant target gene. Since it relies on the action of small interfering RNAs (siRNAs), the in vivo delivery of siRNAs is instrumental. Polyethylenimines (PEIs) and PEGylated PEIs have been shown previously to complex siRNAs, thus mediating siRNA protection against nucleolytic degradation, cellular uptake and intracellular release.

Purpose

The present study determines in vivo pharmacokinetics, tissue distribution/efficacy of siRNA delivery and adverse effects of a broad panel of PEI(-PEG)-based siRNA complexes. The aim is to systematically evaluate the effects of different degrees and patterns of PEGylation in PEI-PEG copolymers on the in vivo behavior of PEI(-PEG)/siRNA complexes in mice.

Results

Upon i.v. injection of radioactively labeled, PEI(-PEG) complexed siRNAs, marked differences in the pharmacokinetics and biodistribution of the complexes are observed, with the fate of the PEI(-PEG)/siRNA complexes being mainly dependent on the degree of uptake in liver, spleen, lung and kidney. Thus, the role of these tissues is investigated in greater detail using representative PEI(-PEG)/siRNA complexes. The induction of erythrocyte aggregation and hemorrhage is dependent on the degree and pattern of PEGylation as well as on the PEI/siRNA (N/P) ratio, and represents one important effect in the lung. Furthermore, siRNA uptake in liver and spleen, but not in lung or kidney, is mediated by macrophage and is dependent on macrophage activity. In the kidney PEI(-PEG)/siRNA uptake is mostly passive and reflects the total stability of the complexes.

Conclusion

Liver, lung, spleen and kidney are the major players determining the in vivo biodistribution of PEI(-PEG)/siRNA complexes. Beyond their physicochemical and in vitro bioactivity characteristics, PEI(-PEG)/siRNA complexes show marked differences in vivo which can be explained by distinct effects in different tissues. Based on these data, our study also identifies which PEGylated PEIs are promising tools for in vivo siRNA delivery in future therapeutic studies and which major determinants require further investigation.     

Analysis of the actions of the novel dopamine receptor-directed compounds (S)-OSU6162 and ACR16 at the D2 dopamine receptor

BACKGROUND AND PURPOSE

The two phenylpiperidines, OSU6162 and ACR16, have been proposed as novel drugs for the treatment of brain disorders, including schizophrenia and Huntington''s disease, because of their putative dopamine stabilizing effects. Here we evaluated the activities of these compounds in a range of assays for the D₂ dopamine receptor in vitro.

EXPERIMENTAL APPROACH

The affinities of these compounds for the D₂ dopamine receptor were evaluated in competition with [³H]spiperone and [³H]NPA. Agonist activity of these compounds was evaluated in terms of their ability to stimulate [³⁵S]GTPγS binding.

KEY RESULTS

Both compounds had low affinities for inhibition of [³H]spiperone binding (pK_i vs. [³H]spiperone, ACR16: <5, OSU6162: 5.36). Neither compound was able to stimulate [³⁵S]GTPγS binding when assayed in the presence of Na⁺ ions, but if the Na⁺ ions were removed, both compounds were low-affinity, partial agonists (E_max relative to dopamine: ACR16: 10.2%, OSU6162:54.3%). Schild analysis of the effects of OSU6162 to inhibit dopamine-stimulated [³⁵S]GTPγS binding indicated Schild slopes of ∼0.9, suggesting little deviation from competitive inhibition. OSU6162 was, however, able to accelerate [³H]NPA dissociation from D₂ dopamine receptors, indicating some allosteric effects of this compound.

CONCLUSIONS AND IMPLICATIONS

The two phenylpiperidines were low-affinity, low-efficacy partial agonists at the D₂ dopamine receptor in vitro, possibly exhibiting some allosteric effects. Comparing their in vitro and in vivo effects, the in vitro affinities were a reasonable guide to potencies in vivo. However, the lack of in vitro–in vivo correlation for agonist efficacy needs to be further addressed.     

Search for Compounds with Hypoglycemic Activity in the Series of 1-[2-(1H-Tetrazol-5-yl)-R1-phenyl]-3-R2-phenyl(ethyl)ureas and R1-Tetrazolo[1,5-c]quinazolin-5(6H)-ones

Methods of 1-[2-(1H-tetrazol-5-yl)-R¹-phenyl]-3-R²-phenyl(ethyl)ureas and R¹-tetrazolo[1,5-c]quinazolin-5(6H)-ones synthesis were designed. IR, LC-MS, ¹H NMR, and elemental analysis data evaluated the structure and purity of the obtained compounds. Different products, depending on the reaction conditions, were distinguished and discussed. The preliminary hypoglycemic activity of 36 synthesized compounds was revealed. Docking studies to 11β-hydroxysteroid dehydrogenase 1, γ-peroxisome proliferator-activated receptor, and dipeptidyl peptidase-4 were conducted. Eight of these substances were further tested on glucocorticoid-induced insulin resistance models, namely glucose tolerance, oral rapid insulin, and adrenalin tests. One of the most active compounds turned out to be tetrazolo[1,5-c]quinazolin-5(6H)-one 3.1, exceeding the reference drugs Metformin (50 and 200 mg/kg) and Gliclazide (50 mg/kg).     

Volume to dissolve applied dose (VDAD) and apparent dissolution rate (ADR): Tools to predict in vivo bioavailability from orally applied drug suspensions

Low solubility of drug candidates generated in research contributes to their elimination during subsequent development due to insufficient oral bioavailability (BA) of crystalline compound. Therefore, the purpose of the study was to identify critical in vitro solubility and dissolution parameter that would predict critical in vivo dissolution by means of in vitro-in vivo correlation. Thermodynamic solubility and apparent dissolution rate (ADR) were determined using the shake-flask method and mini-flow-through-cell, respectively. Oral BA studies in rats and humans were conducted from drug solution and suspension/tablets. Relative BA was calculated using F_rel [%] = AUC_suspension/AUC_solution * 100, representing a measure of in vivo dissolution. Roughly, F_rel rat >50% translates into F_rel human of >90%. Both, ADR and log volume to dissolve applied dose (VDAD), when plotted against F_rel rat, revealed certain threshold levels, (ADR, ∼150-200 μg of compound dissolved under respective assay conditions; VDAD, ∼100-500 ml/kg) which translate into F_rel in rats of >50%.Thus, assuming that F_rel > 50% in rats is indicative of sufficient in vivo dissolution in humans after oral application, drugs should exhibit a VDAD of ∼100-500 ml/kg or less in aqueous media to avoid insufficient or varying drug absorption.     

The plasma–occupancy relationship of the novel GABAA receptor benzodiazepine site ligand, α5IA,is similar in rats and primates

Background and purpose:

α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is a triazolophthalazine with subnanomolar affinity for α1-, α2-, α3- and α5-containing GABA_A receptors. Here we have evaluated the relationship between plasma α5IA concentrations and benzodiazepine binding site occupancy in rodents and primates (rhesus monkey).

Experimental approach:

In awake rats, occupancy was measured at various times after oral dosing with α5IA (0.03–30 mg·kg⁻¹) using an in vivo {[³H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} binding assay. In anaesthetized rhesus monkeys, occupancy was measured using {[¹²³I]iomazenil (ethyl 5,6-dihydro-7-iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} γ-scintigraphy and a bolus/infusion paradigm. In both rat and rhesus monkey, the plasma drug concentration corresponding to 50% occupancy (EC₅₀) was calculated.

Key results:

In rats, α5IA occupancy was dose- and time-dependent with maximum occupancy occurring within the first 2 h. However, rat plasma EC₅₀ was time-independent, ranging from 42 to 67 ng·mL⁻¹ over a 24 h time course with the average being 52 ng·mL⁻¹ (i.e. occupancy decreased as plasma drug concentrations fell). In rhesus monkeys, the EC₅₀ for α5IA displacing steady-state [¹²³I]iomazenil binding was 57 ng·mL⁻¹.

Conclusions and implications:

Rat plasma EC₅₀ values did not vary as a function of time indicating that α5IA dissociates readily for the GABA_A receptor in vivo. These data also suggest that despite the different assays used (terminal assays of [³H]flumazenil in vivo binding in rats and [¹²³I]iomazenil γ-scintigraphy in anaesthetized rhesus monkeys), these techniques produced similar plasma α5IA EC₅₀ values (52 and 57 ng·mL⁻¹ respectively) and that the plasma–occupancy relationship for α5IA translates across these two species.     

A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes

Estragole has been shown to be hepatocarcinogenic in rodent species at high-dose levels. Translation of these results into the likelihood of formation of DNA adducts, mutation, and ultimately cancer upon more realistic low-dose exposures remains a challenge. Recently we have developed physiologically based biokinetic (PBBK) models for rat and human predicting bioactivation of estragole. These PBBK models, however, predict only kinetic characteristics. The present study describes the extension of the PBBK model to a so-called physiologically based biodynamic (PBBD) model predicting in vivo DNA adduct formation of estragole in rat liver. This PBBD model was developed using in vitro data on DNA adduct formation in rat primary hepatocytes exposed to 1′-hydroxyestragole. The model was extended by linking the area under the curve for 1′-hydroxyestragole formation predicted by the PBBK model to the area under the curve for 1′-hydroxyestragole in the in vitro experiments. The outcome of the PBBD model revealed a linear increase in DNA adduct formation with increasing estragole doses up to 100 mg/kg bw. Although DNA adduct formation of genotoxic carcinogens is generally seen as a biomarker of exposure rather than a biomarker of response, the PBBD model now developed is one step closer to the ultimate toxic effect of estragole than the PBBK model described previously. Comparison of the PBBD model outcome to available data showed that the model adequately predicts the dose-dependent level of DNA adduct formation. The PBBD model predicts DNA adduct formation at low levels of exposure up to a dose level showing to cause cancer in rodent bioassays, providing a proof of principle for modeling a toxicodynamic in vivo endpoint on the basis of solely in vitro experimental data.     

Loading 3-deazaneplanocin A into pegylated unilamellar liposomes by forming transient phenylboronic acid-drug complex and its pharmacokinetic features in Sprague-Dawley rats

3-Deazaneplanocin A (DZNep) is an attractive epigenetic anticancer agent through the inhibition of the cellular enhancer of zeste homolog 2 (EZH2) protein. The purpose of this study was to improve the pharmacokinetic characteristics of DZNep in vivo through developing a unilamellar pegylated liposomal formulation encapsulating DZNep (L-DZNep). A remote-loading method in the presence of phenylboronic acid (R-w-PBA) was developed to stably encapsulating DZNep inside liposomes (encapsulation efficiency = 50.7% at molar ratio of 1:10 of drug to lipids) through forming a transient PBA-DZNep complex. The pharmacokinetics of L-DZNep was investigated in Sprague-Dawley rats. In comparison with free drug, encapsulation of the DZNep in pegylated liposomes resulted in 99.3% reduction of the plasma clearance, whereas it increased the elimination half-life from 1.1 h to 8.0 h and the area under the plasma concentration curve by 138-fold. These findings demonstrate a novel approach (R-w-PBA method) through the development of L-DZNep, which may be extensively applied for the encapsulation of hydrophilic nucleoside analogs containing vicinal hydroxyl groups and protonable amino in the pegylated liposomes. Additionally, the pegylated liposomes could effectively prolong the retention of DZNep in the systemic circulation and therefore is highly likely to increase the DZNep’s tumor localization.     

Use of the H3 receptor antagonist radioligand [3H]-A-349821 to reveal in vivo receptor occupancy of cognition enhancing H3 receptor antagonists

Background and purpose:

The histamine H₃ receptor antagonist radioligand [³H]-A-349821 was characterized as a radiotracer for assessing in vivo receptor occupancy by H₃ receptor antagonists that affect behaviour. This model was established as an alternative to ex vivo binding methods, for relating antagonist H₃ receptor occupancy to blood levels and efficacy in preclinical models.

Experimental approach:

In vivo cerebral cortical H₃ receptor occupancy by [³H]-A-349821 was determined in rats from differences in [³H]-A-349821 levels in the isolated cortex and cerebellum, a brain region with low levels of H₃ receptors. Comparisons were made to relate antagonist H₃ receptor occupancy to blood levels and efficacy in a preclinical model of cognition, the five-trial inhibitory avoidance response in rat pups.

Key results:

In adult rats, [³H]-A-349821, 1.5 µg·kg⁻¹, penetrated into the brain and cleared more rapidly from cerebellum than cortex; optimally, [³H]-A-349821 levels were twofold higher in the latter. With increasing [³H]-A-349821 doses, cortical H₃ receptor occupancy was saturable with a binding capacity consistent with in vitro binding in cortex membranes. In studies using tracer [³H]-A-349821 doses, ABT-239 and other H₃ receptor antagonists inhibited H₃ receptor occupancy by [³H]-A-349821 in a dose-dependent manner. Blood levels of the antagonists corresponding to H₃ receptor occupancy were consistent with blood levels associated with efficacy in the five-trial inhibitory avoidance response.

Conclusions and implications:

When employed as an occupancy radiotracer, [³H]-A-349821 provided valid measurements of in vivo H₃ receptor occupancy, which may be helpful in guiding and interpreting clinical studies of H₃ receptor antagonists.