Developmental alterations in anxiety and cognitive behavior in serotonin transporter mutant mice

Rationale

A promoter variant of the serotonin transporter (SERT) gene is known to affect emotional and cognitive regulation. In particular, the “short” allelic variant is implicated in the etiology of multiple neuropsychiatric disorders. Heterozygous (SERT^+/?) and homozygous (SERT^?/?) SERT mutant mice are valuable tools for understanding the mechanisms of altered SERT levels. Although these genetic effects are well investigated in adulthood, the developmental trajectory of altered SERT levels for behavior has not been investigated.

Objectives

We assessed anxiety-like and cognitive behaviors in SERT mutant mice in early adolescence and adulthood to examine the developmental consequences of reduced SERT levels. Spine density of pyramidal neurons was also measured in corticolimbic brain regions.

Results

Adult SERT^?/? mice exhibited increased anxiety-like behavior, but these differences were not observed in early adolescent SERT^?/? mice. Conversely, SERT^+/? and SERT^?/? mice did display higher spontaneous alternation during early adolescence and adulthood. SERT^+/? and SERT^?/? also exhibited greater neuronal spine densities in the orbitofrontal but not the medial prefrontal cortices. Adult SERT^?/? mice also showed an increased spine density in the basolateral amygdala.

Conclusions

Developmental alterations of the serotonergic system caused by genetic inactivation of SERT can have different influences on anxiety-like and cognitive behaviors through early adolescence into adulthood, which may be associated with changes of spine density in the prefrontal cortex and amygdala. The altered maturation of serotonergic systems may lead to specific age-related vulnerabilities to psychopathologies that develop during adolescence.     

The effect of Eudragit type on BSA-loaded PLGA nanoparticles

Poly(d,l-lactide-co-glycolide) nanoparticle (PLGA NP) have been broadly studied as a carrier for drug delivery system of peptides and proteins. However, negative surface charge of PLGA NP using only PLGA decreases bioavailability under oral administration. In this study, novel carriers for oral delivery system through an additional bioadhesive polymer, Eudragit was introduced. Our purpose is to prepare PLGA NP using bovine serum albumin (BSA) as a model drug and Eudragit and evaluate their physiochemical characteristics, eventually expand to peptide and protein drug such as insulin or exenatide. In this study, PLGA NP were spherical and the size was around 400–500 nm. The encapsulation efficiency (EE) of PLGA NP when prepared with only PLGA was the highest, approximately 95.3 %. The polydispersity index values were low approximately 0.1, which meant their size was regular. In mucoadhesion test, we knew PLGA NP prepared by using Eudragit RS or Eudragit RL had a high affinity to mucin particles through zeta-potential change of mucin particle to cover their surface. Also, PLGA NP did not show cytotoxicity against Caco-2 cells. Especially, BSA-loaded PLGA NP using Eudragit RS 100 prepared had high EE, low polydispersity index, spherical shape having a smooth surface, sustained release profile, non-cytotoxicity and bioadhesive effect.     

MDMA administration during adolescence exacerbates MPTP-induced cognitive impairment and neuroinflammation in the hippocampus and prefrontal cortex

Rationale

We have recently shown that chronic exposure to 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) of adolescent mice exacerbates dopamine neurotoxicity and neuroinflammatory effects elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the substantia nigra and striatum at adulthood.

Objectives

The present study investigated whether the amplification of MPTP effects by previous treatment with MDMA extends to the limbic and cortical regions and consequently affects cognitive performance.

Methods

Mice received MDMA (10 mg/kg, twice a day/twice a week) for 9 weeks, followed by MPTP (20 mg/kg?×?4 administrations), starting 2 weeks after MDMA discontinuation. Complement type 3 receptor (CD11b) and glial fibrillary acidic protein (GFAP) were evaluated by immunohistochemistry in both the hippocampus and the medial prefrontal cortex (mPFC) to measure microglia and astroglia activation. These neurochemical evaluations were paired with an assessment of cognitive performance by means of the novel object recognition (NOR) and spontaneous alternation tasks.

Results

MPTP administration to MDMA-pretreated mice elicited a stronger activation of CD11b and GFAP in both the hippocampus and the mPFC compared with either substance administered alone. Furthermore, NOR performance was lower in MDMA-pretreated mice administered MPTP compared with mice that received either substance alone.

Conclusions

These results demonstrate that MDMA–MPTP negative interactions extend to the limbic and cortical regions and may result in cognitive impairment, providing further evidence that exposure to MDMA may amplify the effects of later neurotoxic insults.     

MDMA alters emotional processing and facilitates positive social interaction

Background

±3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) produces “prosocial” effects, such as feelings of empathy and closeness, thought to be important to its abuse and its value in psychotherapy. However, it is not fully understood how MDMA alters basic emotional processes to produce these effects, or whether it produces corresponding changes in actual social behavior. Here, we examined how MDMA affects perceptions of and responses to emotional expressions, and tested its effects on behavior during a social interaction. We also examined whether MDMA’s prosocial effects related to a measure of abuse liability.

Methods

Over three sessions, 36 healthy volunteers with previous ecstasy use received MDMA (0.75, 1.5 mg/kg) and placebo under double-blind conditions. We measured (i) mood and cardiovascular effects, (ii) perception of and psychophysiological responses to emotional expressions, (iii) use of positive and negative words in a social interaction, and (iv) perceptions of an interaction partner. We then tested whether these effects predicted desire to take the drug again.

Results

MDMA slowed perception of angry expressions, increased psychophysiological responses to happy expressions, and increased positive word use and perceptions of partner empathy and regard in a social interaction. These effects were not strongly related to desire to take the drug again.

Conclusions

MDMA alters basic emotional processes by slowing identification of negative emotions and increasing responses to positive emotions in others. Further, it positively affects behavior and perceptions during actual social interaction. These effects may contribute to the efficacy of MDMA in psychotherapy, but appear less closely related to its abuse potential.     

PACE: Pharmacists use the power of communication in paediatric asthma

Background Paediatric asthma is a public health burden in Australia despite the availability of national asthma guidelines. Community pharmacy interventions focusing on paediatric asthma are scarce. Practitioner Asthma Communication and Education (PACE) is an evidence-based program, developed in the USA for general practice physicians, aimed at addressing the issues of poor clinician-patient communication in the management of paediatric asthma. This program has been shown to improve paediatric asthma management practices of general practitioners in the USA and Australia. The development of a PACE program for community pharmacists will fill a void in the current armamentarium for pharmacist-patient care. Objectives To adapt the educational program, PACE, to the community pharmacy setting. To test the feasibility of the new program for pharmacy and to explore its potential impact on pharmacists’ communication skills and asthma related practices. Setting Community pharmacies located within the Sydney metropolitan. Method The PACE framework was reviewed by the research team and amended in order to ensure its relevance within the pharmacy context, thereby developing PACE for Pharmacy. Forty-four pharmacists were recruited and trained in small groups in the PACE for Pharmacy workshops. Pharmacists’ satisfaction and acceptability of the workshops, confidence in using communication strategies pre- and post-workshop and self-reported behaviour change post workshop were evaluated. Main Outcome Measure Pharmacist self-reported changes in communication and teaching behaviours during a paediatric asthma consultation. Results All 44 pharmacists attended both workshops, completed pre- and post-workshop questionnaires and provided feedback on the workshops (100 % retention). The participants reported a high level of satisfaction and valued the interactive nature of the workshops. Following the PACE for Pharmacy program, pharmacists reported significantly higher levels in using the communication strategies, confidence in their application and their helpfulness. Pharmacists checked for written asthma self-management plan possession and inhaler device technique more regularly, and provided verbal instructions more frequently to paediatric asthma patients/carers at the initiation of a new medication. Conclusion This study provides preliminary evidence that the PACE program can be translated into community pharmacy. PACE for Pharmacy positively affected self-reported communication and education behaviours of pharmacists. The high response rate shows that pharmacists are eager to expand on their clinical role in primary healthcare.     

ABC Transporters in Multi-Drug Resistance and ADME-Tox of Small Molecule Tyrosine Kinase Inhibitors

The past decade has seen tremendous efforts in the research and development of new chemotherapeutic drugs using target-based approaches. These efforts have led to the discovery of small molecule tyrosine kinase inhibitors (TKIs). Following the initial approval of imatinib by the US FDA in 2001, more than 15 TKIs targeting different tyrosine kinases have been approved, and numerous others are in various phases of clinical evaluation. Unlike conventional chemotherapy that can cause non-discriminating damage to both normal and cancerous cells, TKIs attack cancer-specific targets and therefore have a more favorable safety profile. However, although TKIs have had outstanding success in cancer therapy, there has been increasing evidence of resistance to TKIs. The enhanced efflux of TKIs by ATP-binding cassette (ABC) transporters over-expressed in cancer cells has been found to be one such important resistance mechanism. Another major drawback of TKI therapies that has been increasingly recognized is the extensive inter-individual pharmacokinetic variability, in which ABC transporters seem to play a major role as well. This review covers recent findings on the interactions of small molecule TKIs with ABC transporters. The effects of ABC transporters on anticancer efficacy and the absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) of the small molecule TKIs are summarized in detail. Since TKIs have been found to not only serve as substrates of ABC transporters, but also as modulators of these proteins via inhibition or induction, their influence upon ABC transporters and potential role on TKI-drug interactions are discussed as well.     

The role of ovarian hormone-derived neurosteroids on the regulation of GABAA receptors in affective disorders

Rationale

Neuroactive derivatives of steroid hormones, neurosteroids, can act on GABA_A receptors (GABA_ARs) to potentiate the effects of GABA on these receptors. Neurosteroids become elevated to physiologically relevant levels under conditions characterized by increased steroid hormones. There is considerable evidence for plasticity of GABA_ARs associated with altered levels of neurosteroids which may counteract the fluctuations in the levels of these allosteric modulators.

Objectives

The objective of this review is to summarize the current literature on GABA_AR plasticity under conditions characterized by alterations in neurosteroid levels, such as over the estrous cycle, during puberty, and throughout pregnancy and the postpartum period.

Results

The expression of specific GABA_AR subunits is altered over the estrous cycle, at puberty, and throughout pregnancy and the postpartum period. Inability to regulate δ subunit-containing GABA_ARs throughout pregnancy and the postpartum period is associated with depression-like behavior restricted to the postpartum period.

Conclusions

GABA_AR plasticity associated with alterations in neurosteroid levels represents a homeostatic compensatory mechanism to maintain an ideal level of inhibition to offset the potentiating effects of neurosteroids on GABAergic inhibition. Failure to properly regulate GABA_ARs under conditions of altered neurosteroid levels may increase vulnerability to mood disorders, such as premenstrual syndrome (PMS), premenstrual dysphoric disorder (PMDD), and postpartum depression.     

Characterization of Endogenous G-CSF and the Inverse Correlation to Chemotherapy-Induced Neutropenia in Patients with Breast Cancer Using Population Modeling

Purpose

Neutropenia is a severe adverse-event of chemotherapeutics. Neutrophils (ANC) are mainly regulated by granulocyte colony stimulating factor (G-CSF). The aim was to characterize the dynamics between endogenous G-CSF and ANC over time following chemotherapy.

Methods

Endogenous G-CSF and ANC were monitored in forty-nine breast cancer patients treated with sequential adjuvant 5-fluorouracil–epirubicin–cyclophosphamide and docetaxel.

Results

During treatment courses ANC was transiently decreased and was reflected in an endogenous G-CSF increase, which was well described by a semi-mechanistic model including control mechanisms; when G-CSF concentrations increased the proliferation rate increased and the bone maturation time reduced for ANC. Subsequently, ANC in the circulation increased leading to increased elimination of G-CSF. Additionally, a non-specific elimination for G-CSF was quantified. The ANC-dependent elimination contributed to 97% at baseline and 49% at an ANC of 0.1?·?10⁹/L to the total G-CSF elimination.

Conclusion

The integrated G-CSF–myelosuppression model captured the initial rise in endogenous G-CSF following chemotherapy-induced neutropenia and the return to baseline of G-CSF and ANC. The model supported the self-regulatory properties of the system and may be a useful tool for further characterization of the biological system and in optimization of chemotherapy treatment.     

Potential role of nitric oxide synthase isoforms in pathophysiology of neuropathic pain

Neuropathic pain triggers a cascade of events in the sensory neurons. It is the main complication of diabetes after cardiovascular disease. Nitric oxide (NO) produced from nitric oxide synthases (NOS) is an important signaling molecule which is crucial for many physiological processes such as synaptic plasticity, neuronal survival, vasodilation, vascular homeostasis, immune regulation. Overproduction of NO due to changes in NOS isoforms level involves pathological processes such as neurotoxicity, septic shock and neuropathic pain. All three isoforms of NOS as well as their end product, NO have modulatory effect on neuropathic pain. Overactivation of the N-Methyl-d-Aspartate receptor and peroxynitrite formation results in high levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) which suggest that nNOS and eNOS are critical for pain hypersensitivity. Inducible NOS induced in glia by inflammation due to activation of Tumor Necrosis Factor α, Calcitonin Gene Regulating Peptide, Mitogen Activated Protein Kinases, Extracellular signal Regulated Kinase, c-Jun N-terminal kinases can induce neuronal death. This review focuses on different nitric oxide synthases and their role in pathophysiology of neuropathic pain considering NOS as an important therapeutic target.     

The self-administration of rapidly delivered cocaine promotes increased motivation to take the drug: contributions of prior levels of operant responding and cocaine intake

Rationale

Rapid drug delivery to the brain might increase the risk for developing addiction. In rats, increasing the speed of intravenous cocaine delivery (5 vs. 90 s) increases drug intake and the subsequent motivation to self-administer cocaine. Increased motivation for cocaine could result not only from more extensive prior drug intake and operant responding for drug, but also from neuroplasticity evoked by rapid drug uptake.

Objective

We determined the contributions of prior drug intake and operant responding to the increased motivation for cocaine evoked by rapid delivery. We also investigated the effects of cocaine delivery speed on corticostriatal expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) mRNA.

Methods

Rats self-administered cocaine (0.25 mg/kg/infusion) delivered over 5 or 90 s during short-access (1 h/session; ShA) or long-access (6 h; LgA) sessions. Motivation for cocaine was then assessed by measuring responding under a progressive ratio schedule of reinforcement. Next, BDNF and TrkB mRNA levels were measured in 5- and 90-s rats.

Results

Five-second ShA and 5-s-LgA rats were more motivated for cocaine than their 90-s counterparts. This effect was dissociable from previous levels of drug intake or of operant responding for cocaine. In parallel, only rats self-administering rapid cocaine injections had altered BDNF and TrkB mRNA levels in corticostriatal regions.

Conclusions

Rapid drug delivery augments the motivation for cocaine independently of effects on the levels of drug intake or operant responding for drug. We suggest that rapid delivery might increase the motivation for drug by promoting neuroplasticity within reward pathways. This neuroplasticity could involve increased regulation of BDNF/TrkB.     

Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function

Rationale

Neurosteroids and likely other lipid modulators access transmembrane sites on the GABA_A receptor (GABA_AR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABA_ARs.

Objectives

In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine.

Methods

We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABA_ARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion.

Results

We found no evidence that PIP2 depletion affected modulation of GABA_ARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity.

Conclusions

We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABA_ARs by neurosteroids or related compounds.     

Development of High Drug Loaded and Customizing Novel Nanoparticles for Modulated and Controlled Release of Paclitaxel

Purpose

The objective of this study was to develop a custom-tailored polymeric drug delivery system for paclitaxel, employing a novel biodegradable block co-polymer (P), intended to be intravenously administered, capable of improving therapeutic index of the drug and devoid of the adverse effect of an uncontrolled release.

Methods

Paclitaxel loaded nanoparticles (PTX-NPs) were prepared by a modified nanoprecipitation method and emulsification-solvent evaporation method. Our approach involves a focusing on the formulation parameters that can be modified in order to obtain completely customized NPs in terms of size, zeta-potential, drug content and release profile. The biocompatibility and anti-proliferative efficiency of PTX-NPs against glioblastoma cell line were evaluated in vitro by MTS.

Results

All formulations showed spherical nanometric (<200 nm), monodisperse (~0.1), Poly (Ethylene Glycol) (PEG)-coated and negatively charged particles. Selected NPs revealed higher PTX content (up to 24%) in comparison with polyester-based NPs. The release behaviour of PTX from the developed NPs exhibited an approximately first-order profile, without initial burst and characterized by a slow and constant release. Hydrophobic character of the NPs can be set in order to achieve a slower and more controlled release for a prolonged period of time. PTX-NPs were hemocompatible and had significant in vitro anti-tumoral activity against human primary glioblastoma cell line (U-87 MG); cytotoxicity was in time- and drug concentration- dependent manner.

Conclusions

The developed drug delivery system proved to be suitable for intravenous administration. NPs characteristics can be customized to obtain high PTX loaded NPs that can improve therapeutic index and avoid an uncontrolled release.     

Lithium reduces the effects of rotenone-induced complex I dysfunction on DNA methylation and hydroxymethylation in rat cortical primary neurons

Rationale

Mitochondrial complex I dysfunction and alterations in DNA methylation levels are consistently reported in bipolar disorder (BD) and are regulated by lithium. One of the mechanisms by which lithium may exert its effects in BD is by improving mitochondrial complex I function. Therefore, we examined whether complex I dysfunction induces methylation and hydroxymethylation of DNA and whether lithium alters these effects in rat primary cortical neurons.

Methods

Rotenone was used to induce mitochondrial complex I dysfunction. Cell viability was measured by MTT assay, and ATP levels were assessed by Cell-Titer-Glo®. Complex I activity was measured using an ELISA-based assay. Apoptosis, DNA methylation, and hydroxymethylation levels were measured by immunocytochemistry.

Results

Rotenone decreased complex I activity and ATP production, but increased cell death and apoptosis. Rotenone treatment increased levels of 5-methylcytosine (5mc) and hydroxymethylcytosine (5hmc), suggesting a possible association between complex I dysfunction and DNA alterations. Lithium prevented rotenone-induced changes in mitochondrial complex I function, cell death and changes to DNA methylation and hydroxymethylation.

Conclusions

These findings suggest that decreased mitochondrial complex I activity may increase DNA methylation and hydroxymethylation in rat primary cortical neurons and that lithium may prevent these effects.     

3,4-Methylenedioxymethamphetamine (MDMA – Ecstasy) Decreases Neutrophil Activity Through the Glucocorticoid Pathway and Impairs Host Resistance to Listeria Monocytogenes Infection in Mice

Ecstasy is the popular name of the abuse drug 3,4-methylenedioxymethamphetamine (MDMA) that decreases immunity in animals. The mechanisms that generate such alterations are still controversial. Seven independent pharmacological approaches were performed in mice to identify the possible mechanisms underlying the decrease of neutrophil activity induced by MDMA and the possible effects of MDMA on host resistance to Listeria monocytogenes. Our data showed that MDMA (10 mg kg^?1) administration decreases NFκB expression in circulating neutrophils. Metyrapone or RU-486 administration prior to MDMA treatment abrogated MDMA effects on neutrophil activity and NFκB expression, while 6-OHDA or ICI-118,551 administration did not. As MDMA treatment increased the plasmatic levels of adrenaline and noradrenaline, propranolol pre-treatment effects were also evaluated. Propranolol suppressed both MDMA-induced increase in corticosterone serum levels and its effects on neutrophil activity. In a L. monocytogenes experimental infection context, we showed that MDMA: induced myelosuppression by decreasing granulocyte-macrophage hematopoietic progenitors (CFU-GM) in the bone marrow but increased CFU-GM in the spleen; decreased circulating leukocytes and bone marrow cellularity and increased spleen cellularity; decreased pro-inflammatory cytokine (IL-12p70, TNF, IFN-γ, IL-6) and chemokine (MCP-1) production 24 h after the infection; increased the production of pro-inflammatory cytokines and chemokines 72 h after infection and decreased IL-10 levels at all time points analyzed. It was proposed that MDMA immunosuppressive effects on neutrophil activity and host resistance to L monocytogenes rely on NFκB signaling, being mediated by HPA axis activity and corticosterone.     

Sex differences in neurosteroid and hormonal responses to metyrapone in posttraumatic stress disorder

Rationale

Mechanisms contributing to sex differences in the regulation of acute stress responsivity and their effect on the increased incidence of posttraumatic stress disorder (PTSD) in women are poorly understood. The reproductive hormone, progesterone, through conversion to allopregnanolone (ALLO), suppresses the hypothalamic pituitary adrenal (HPA) axis and has potent anxiolytic effects. The potential that progesterone and allopregnanolone reactivity modulate HPA axis responses and account for sex differences in PTSD has not been previously examined.

Objective

The present study examined the effects of sex and PTSD on adrenocorticotropic hormone (ACTH), progesterone, and allopregnanolone responses to metyrapone and whether progesterone and allopregnanolone reactivity could affect the ACTH response in PTSD.

Methods

Healthy medication-free male and premenopausal follicular phase female participants with chronic PTSD (n?=?43; 49 % female) and controls (n?=?42; 50 % female) completed an overnight metyrapone challenge and ACTH, progesterone, and allopregnanolone were obtained by repeated blood sampling.

Results

The increase in ACTH response to metyrapone was higher in PTSD subjects compared to controls and in women compared to men. Contrary to our initial prediction of an inverse relationship, progesterone and allopregnanolone were positively associated with ACTH. Progesterone and allopregnanolone partially mediated the relationship between PTSD and ACTH.

Conclusions

Our findings of increased ACTH to metyrapone in PTSD and in women may reflect heightened hypothalamic CRF hypersecretion. Progesterone and allopregnanolone partially mediated the ACTH response in PTSD. Further characterizing sex differences in these processes will advance our understanding of the pathophysiology of PTSD, and may ultimately lead to better-targeted, more effective treatment.     

TCDD-induced chick cardiotoxicity is abolished by a selective cyclooxygenase-2 (COX-2) inhibitor NS398

Halogenated aromatic hydrocarbons, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are known to cause severe heart defects in avian species. However, the mechanism of TCDD-induced chick cardiovascular toxicity is unclear. In this study, we investigated cyclooxygenase-2 (COX-2) as a possible mechanism of TCDD-induced cardiotoxicity. Fertile chicken eggs were injected with TCDD and a COX-2 selective inhibitor, NS398, and we investigated chick heart failure on day 10. We found that the chick heart to body weight ratio and atrial natriuretic factor mRNA expression were increased, but this increase was abolished with treatment of NS398. In addition, the morphological abnormality of an enlarged ventricle resulting from TCDD exposure was also abolished with co-treatment of TCDD and NS398. Our results suggested that TCDD-induced chick heart defects are mediated via the nongenomic pathway and that they do not require the genomic pathway.     

Dual and/or selective DNA-PK,PI3K inhibition and isoform selectivity of some new and known 2-amino-substituted-1,3-benzoxazines and substituted-1,3-naphthoxazines

The 2-morpholino-substituted-benzoxazines 7a and 7b were used in the synthesis of 2-morpholino-di-O-benzyl, O-pyridin-2yl, 3-yl and 4yl-methoxy)-1,3-benzoxazines 8a–8d, and N-(2-morpholino-4-oxo-4H-benz[e][1,3]oxazin-7-yl)-N-(pyridin-2-and-3-ylmethyl)acetamides 8e and 8f. The DNA-dependent protein kinase (DNA-PK) and phosphatidylinositol 3-kinase (PI3K) α, β, γ, and δ isoforms were studied for the new compounds 8a–8f and PI3K for the 18 previously synthesized compounds 9–26. The most active DNA-PK inhibitors were the 2-morpholino-O-substituted linear or angular naphthoxazine compounds 18–20 and 21–22 which showed potent and selective DNA-PK activity (IC₅₀ from 0.01 to 2.43 µM) over PI3K. 8-(2-(4-Methylpiperazin-1-yl)ethoxy)-2-morpholino compound 13, and 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy) compound 25 showed selective DNA-PK inhibition. 2-morpholino-8-substituted-benzoxazine 9 (8-ph) and 10–12 (8-(pyridine-2-, 3-, or 4-ylmethoxy) showed high-to-moderate inhibition of PI3K and DNA-PK. A similar pattern for DNA-PK nonselectivity over PI3K was observed for compounds with 7,8-O-bis-substituted 8a, 8c, and 8d. No DNA-PK selectivity over PI3K was observed regardless whether the substitution was phenyl, pyridin-2-ylmethoxy, pyridin-3-ylmethoxy, and pyridin-4-ylmethoxy.