From Silos to Solidarity: Case Study of a Patient-Centered, Integrative Approach to Opioid Tapering and Chronic Pain Mitigation in a Multidisciplinary AIDS Clinic.

BACKGROUND: People with HIV (PWH) are at a disproportionate risk for experiencing both chronic pain and opioid use disorder (OUD). Prescription opioid tapering is typically addressed within the "silo model" of medical care, whereby attention is focused solely on opioid addiction rather than also addressing chronic pain management, and limited communication occurs between patient and providers. OBJECTIVE: This descriptive case study examined an integrative, collaborative care model consisting of Provider, Physical Therapist (PT), and Patient aimed at decreasing chronic pain and opioid use within a multidisciplinary HIV/AIDS clinic. METHOD: A physical-therapy based model of chronic pain mitigation and physician-driven opioid tapering was implemented. The Provider, PT, and Patient worked collaboratively to address physiological pain, pain coping skills and opioid tapering. A patient case example was used to illustrate the implementation of the model for a future, larger study in the same patient population. RESULTS: This model was feasible in this case example in terms of clinic workflow and acceptability to both the Patient and Providers in this clinic. After the intervention, the Patient's pain was fully eliminated, and he had ceased all opioid use. CONCLUSION: Results of this case study suggest that utilizing an integrative, patient-centered approach to both chronic pain management and opioid tapering may be feasible within the context of a multidisciplinary HIV/AIDS clinic. Generalizability is limited by case study model; however, this gives insight into the value of a collaborative alternative compared to a "silo" model of opioid tapering and chronic pain management in preparation for a larger study.

Direct evidence of catalyst reduction on dye and catalyst co-sensitized NiO photocathodes by mid-infrared transient absorption spectroscopy.

Co-sensitization of molecular dyes and catalysts on semiconductor surfaces is a promising strategy to build photoelectrodes for solar fuel production. In such a photoelectrode, understanding the charge transfer reactions between the molecular dye, catalyst and semiconductor material is key to guide further improvement of their photocatalytic performance. Herein, femtosecond mid-infrared transient absorption spectroscopy is used, for the first time, to probe charge transfer reactions leading to catalyst reduction on co-sensitized nickel oxide (NiO) photocathodes. The NiO films were co-sensitized with a molecular dye and a proton reducing catalyst from the family of [FeFe](bdt)(CO)6 (bdt = benzene-1,2-dithiolate) complexes. Two dyes were used: an organic push-pull dye denoted E2 with a triarylamine-oligothiophene-dicyanovinyl structure and a coumarin 343 dye. Upon photo-excitation of the dye, a clear spectroscopic signature of the reduced catalyst is observed a few picoseconds after excitation in all co-sensitized NiO films. However, kinetic analysis of the transient absorption signals of the dye and reduced catalyst reveal important mechanistic differences in the first reduction of the catalyst depending on the co-sensitized molecular dye (E2 or C343). While catalyst reduction is preceded by hole injection in NiO in C343-sensitized NiO films, the singly reduced catalyst is formed by direct electron transfer from the excited dye E2* to the catalyst in E2-sensitized NiO films. This change in mechanism also impacts the lifetime of the reduced catalyst, which is only ca. 50 ps in E2-sensitized NiO films but is >5 ns in C343-sensitized NiO films. Finally, the implication of this mechanistic study for the development of better co-sensitized photocathodes is discussed.

Are short (blue) wavelengths necessary for light treatment of seasonal affective disorder?

Despite widely published speculation regarding a potential potency advantage of short-wavelength (blue-appearing) light for Seasonal Affective Disorder (SAD) treatment, there have been few systematic studies. Those comparing short-wavelength to broad-wavelength (white) light under actual clinical conditions suggest equivalent effectiveness. This multicenter, parallel-group design trial was undertaken to compare the effects of light therapy on SAD using blue (~465 nm) versus blue-free (595-612 nm) LED lights. Fifty-six medication-free subjects aged 21-64 years who met DSM-IV-TR criteria for recurrent major depression with winter-type seasonal pattern were enrolled in this blinded study at five participating centers between January and March 2012. Thirty-five subjects met the criteria for randomization to 30 min of either blue (~465 nm) or blue-free (595-612 nm) daily morning light therapy. Twenty-nine subjects completed the study; three subjects withdrew due to treatment-related adverse events, including migraines, and three withdrew for non-study-related reasons. The primary effectiveness variable was depression score (SIGH-ADS) after six weeks of daily light treatment. Secondary effectiveness variables included quality-of-life (QoL) and suicidality ratings. Using an intent-to-treat analysis, mean depression scores were different at baseline for the blue group (29 ± 5 versus 26 ± 5, p = 0.05 blue versus blue-free, respectively), and the initial score was used as a covariate. Baseline scores were not significantly different between treatment groups among those who completed the study, and no significant differences in depression scores were observed after 6 weeks (mean ± SD scores at 6 weeks: 5.6 ± 6.1 versus 4.5 ± 5.3, p = 0.74, blue versus blue-free, respectively). In addition, the proportion of subjects who met remission criteria, defined as a depression score ≤8, was not significantly different between the two groups (p = 0.41); among the 29 subjects who completed the study, 76% of subjects experienced remission by the end of the trial, which coincided with the beginning of spring. The QoL and suicidality ratings were also significantly improved from pre- to post-treatment, with no significant difference between treatments. No subject experienced worsening or non-improved symptoms over the 6-week trial. The main finding of this study is that subjects treated with blue light did not improve more than subjects treated with blue-free light; both showed substantial improvement on multiple measures. Failure to find differences may have resulted from methodological constraints, including a small sample size. Recruitment began mid-winter during an unusually mild season, and the trial was terminated earlier than planned by the study sponsor due to a failure to detect a difference. However, if confirmed in a larger randomized sample, these results suggest that blue wavelengths are not necessary for successful SAD treatment.

Neuropsychiatric Outcome of an Adolescent Who Received Deep Brain Stimulation for Tourette's Syndrome.

This case study followed one adolescent patient who underwent bilateral deep brain stimulation of the centromedian parafascicular complex (CM-Pf) for debilitating, treatment refractory Tourette's syndrome for a period of 1.5 years. Neurocognitive testing showed no significant changes between baseline and follow-up assessments. Psychiatric assessment revealed positive outcomes in overall adaptive functioning and reduction in psychotropic medication load in this patient. Furthermore, despite significant baseline psychiatric comorbidity, this patient reported no suicidal ideation following electrode implantation. Deep brain stimulation is increasingly being used in children and adolescents. This case reports on the positive neurologic and neuropsychiatric outcome of an adolescent male with bilateral CM-Pf stimulation.

2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT.

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.

Comparison of aromatic and tertiary amine N-oxides of acridine DNA intercalators as bioreductive drugs. Cytotoxicity, DNA binding, cellular uptake, and metabolism.

Some N-oxide derivatives of DNA intercalators are bioreductive prodrugs that are selectively toxic under hypoxic conditions. The hypoxic selectivity is considered to result from an increase in DNA binding affinity when the N-oxide moiety is reduced. This study investigated whether differences in DNA binding affinity between N-oxides and their corresponding amines, measured by equilibrium dialysis, can account for the hypoxic cytotoxicity ratios (HCR) of tertiary amine N-oxide (-tO) and aromatic N-oxide (-aO) derivatives of the 1-nitroacridine nitracrine (NC) and its non-nitro analogue 9-[3-(N,N-dimethylamino)propylamino]acridine (DAPA). Cytotoxicity was measured in aerobic and hypoxic suspensions of Chinese hamster ovary (CHO) AA8 cells by clonogenic assay. HCR were much greater for NC-tO (820-fold) than for NC (5-fold) or NC-aO (4-fold), whereas DAPA and its N-oxides lacked hypoxic selectivity (1-fold). DNA binding measurements demonstrated that binding affinity is lowered more by aromatic than tertiary amine (side-chain) N-oxides, an observation that does not correlate with HCR. Compounds were accumulated in cells to high concentrations (C(i)/C(e) approximately 10-200), with the exception of the tertiary amine N-oxides, for which the ratio of intracellular to extracellular drug was less than unity. For NC-tO this probably resulted from low pK(a) values for both the acridine chromophore and the side-chain, whereas DAPA-tO may be too hydrophilic for efficient membrane permeation. Bioreductive drug metabolism, assessed by HPLC, was faster for the NC than the DAPA N-oxides. The high HCR of NC-tO relative to NC-aO is ascribed to the rapid and selective reduction of its N-oxide moiety, followed by activation of the NC intermediate by O(2)-sensitive reduction of its 1-nitro group to the corresponding 1-amine. The metabolism studies suggest that unmasking of DNA binding affinity by reductive removal of the N-oxide moiety, although not the only determinant, is important and needs to occur before nitroreduction for optimal effect.

Preliminary observations on sexual behavior and the mating system in free-ranging lesser galagos. (Galago moholi).

Sexual and associated patterns of behavior of lesser galagos (Galago moholi) were recorded during an 18-month study conducted at the Nylsvley Nature Reserve in South Africa. Animals were trapped and fitted with radio transmitter belts in order to monitor nocturnal activities during twice-yearly mating seasons. Most copulations occurred during the last week in May, while a subsidiary (post-partum) mating season occurred in late September-early October. Females came into estrus sequentially during the May season. Adult males exhibited increase in body weight and testes volume during the mating season, changes which were most pronounced among the larger males (> 226 g). Larger males also had the greatest mating success, initiating 88% of observed copulations. Sixty-seven percent of matings involved more than one male copulating with the same female during her estrus, which lasted 1-3 days. Mounts were prolonged (range 2-53 min, mean 9.0 min) and males copulated repeatedly (2-5 times) with the same partner during a single night. These observations of sexual behavior and of large relative testes size in free-ranging lesser galagos are consistent with the occurrence of a dispersed mating system involving sperm competition in this nocturnal prosimian species.

High-affinity interactions of tumor necrosis factor receptor-associated factors (TRAFs) and CD40 require TRAF trimerization and CD40 multimerization.

Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TRAFs) that interact with receptor cytoplasmic domains following ligand-induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that directly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domains of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-terminal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences necessary for trimer formation and indicated that the coiled coil TRAF-N region is required for trimerization. Consistent with the idea that TRAF trimerization is required for high-affinity interactions with CD40, monomeric TRAF-C domains bound to CD40 significantly weaker than trimeric TRAFs. In surface plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2 > TRAF3 >> TRAF1 and TRAF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-kappaB and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary for maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomerized receptors as well as different requirements for degree of receptor multimerization appear to contribute to the selectivity of TRAF recruitment to receptor cytoplasmic domains.

Crystallographic analysis of CD40 recognition and signaling by human TRAF2.

Tumor necrosis factor receptor superfamily members convey signals that promote diverse cellular responses. Receptor trimerization by extracellular ligands initiates signaling by recruiting members of the tumor necrosis factor receptor-associated factor (TRAF) family of adapter proteins to the receptor cytoplasmic domains. We report the 2.4-A crystal structure of a 22-kDa, receptor-binding fragment of TRAF2 complexed with a functionally defined peptide from the cytoplasmic domain of the CD40 receptor. TRAF2 forms a mushroom-shaped trimer consisting of a coiled coil and a unique beta-sandwich domain. Both domains mediate trimerization. The CD40 peptide binds in an extended conformation with every side chain in contact with a complementary groove on the rim of each TRAF monomer. The spacing between the CD40 binding sites on TRAF2 supports an elegant signaling mechanism in which trimeric, extracellular ligands preorganize the receptors to simultaneously recognize three sites on the TRAF trimer.

CD40 signaling through tumor necrosis factor receptor-associated factors (TRAFs). Binding site specificity and activation of downstream pathways by distinct TRAFs.

Tumor necrosis factor receptor-associated factors (TRAFs) associate with the CD40 cytoplasmic domain and initiate signaling after CD40 receptor multimerization by its ligand. We used saturating peptide-based mutational analyses of the TRAF1/TRAF2/TRAF3 and TRAF6 binding sequences in CD40 to finely map residues involved in CD40-TRAF interactions. The core binding site for TRAF1, TRAF2, and TRAF3 in CD40 could be minimally substituted. The TRAF6 binding site demonstrated more amino acid sequence flexibility and could be optimized. Point mutations that eliminated or enhanced binding of TRAFs to one or both sites were made in CD40 and tested in quantitative CD40-TRAF binding assays. Sequences flanking the core TRAF binding sites were found to modulate TRAF binding, and the two TRAF binding sites were not independent. Cloned stable transfectants of human embryonic kidney 293 cells that expressed wild type CD40 or individual CD40 mutations were used to demonstrate that both TRAF binding sites were required for optimal NF-kappaB and c-Jun N-terminal kinase activation. In contrast, p38 mitogen-activated protein kinase activation was primarily dependent upon TRAF6 binding. These studies suggest a role in CD40 signaling for competitive TRAF binding and imply that CD40 responses reflect an integration of signals from individual TRAFs.

Determinants of psychotropic drug usage in a general intensive care unit.

During a 3-month period, determinants of psychotropic drug utilization (sex, age, length of stay, reason for admission, disease severity) and data on psychotropic consumption (type of medication--antidepressants, benzodiazepines and antipsychotics--dosage and length of treatment) were retrospectively collected in a general intensive care unit of a Dutch university hospital. Daily exposure to psychotropics was standardized in number of Defined Daily Doses (DDD). Benzodiazepines were used by 35.8% of all patients (137) during their stay in the ICU whereas 17.5% of all patients used a neuroleptic agent. Antidepressants were hardly prescribed. High doses of benzodiazepines (9.9 DDDs) and low doses of antipsychotics (0.5 DDDs) were prescribed, which probably reflect the unusual nature of this critically ill group of patients compared with the reference group for DDD's. Clear patterns of determinants of psychotropic drug use in ICU patients were found and both benzodiazepines, antipsychotics and combined use of these agents could be associated with the determinants assessed. The time patterns we found in terms of length of stay give clues for further investigations in order to rationalize psychotropic drug use in the management of severely ill and complex patients.

Recombinant IkappaB kinases alpha and beta are direct kinases of Ikappa Balpha.

Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha and IKKbeta, were overexpressed in insect cells and purified to homogeneity. Both purified IKKalpha and IKKbeta specifically catalyzed the phosphorylation of the regulatory serine residues of Ikappa Balpha. Hence, IKKalpha and IKKbeta were functional catalytic subunits of the IKK complex. Purified IKKalpha and IKKbeta also preferentially phosphorylated serine as opposed to threonine residues of Ikappa Balpha, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKalpha and IKKbeta revealed that the kinase activity of IKKbeta on Ikappa Balpha is 50-60-fold higher than that of IKKalpha. The primary difference between the two activities is the Km for Ikappa Balpha. The kinetics of both IKKalpha and IKKbeta followed a sequential Bi Bi mechanism. No synergistic effects on Ikappa Balpha phosphorylation were detected between IKKalpha and IKKbeta. Thus, in vitro, IKKalpha and IKKbeta are two independent kinases of Ikappa Balpha.

CD40-tumor necrosis factor receptor-associated factor (TRAF) interactions: regulation of CD40 signaling through multiple TRAF binding sites and TRAF hetero-oligomerization.

CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers. TRAF3-TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region. The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40 binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.

DNA-dependent transregulation by IE1 of Autographa californica nuclear polyhedrosis virus: IE1 domains required for transactivation and DNA binding.

IE1 is the principal early transregulator of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). The 582-residue protein stimulates viral transcription and binds as a dimer to 28-bp palindromic repeats (28-mers) comprising the AcMNPV homologous region (hr) transcription enhancers. To define IE1 domains responsible for hr-dependent transactivation, we first constructed a series of IE1 fusions to the DNA binding domain of the yeast GAL4 transactivator. In transfection assays, GAL4-IE1 fusions stimulated transcription from a TATA-containing AcMNPV promoter only upon cis linkage to GAL4 DNA binding sites. IE1 N-terminal residues 8 to 118 were sufficient for GAL4-binding-site-dependent transactivation. To identify IE1 residues required for hr interaction, we tested a series of IE1 mutations for 28-mer binding by using electrophoretic mobility shift assays. Deletion of IE1 residues other than the N-terminal transactivation domain eliminated 28-mer binding. Of 14 insertion mutations, only IE1(I425) and IE1(I553) failed to bind the 28-mer either as homodimers or as heterodimers with functional IE1. In contrast to insertion IE1(I425), IE1(I553) also failed to compete with wild-type IE1 for DNA binding and suggested a defect in oligomerization. Consistent with loss of oligomerization, substitutions within a hydrophobic repeat (residues 543 to 568) at the IE1 C terminus abolished 28-mer binding and demonstrated that this helix-loop-helix-like domain is required for DNA interaction. These data confirm that IE1 contains separable domains for transactivation and oligomerization-dependent DNA binding. Furthermore, they support a model wherein hr-mediated transactivation by IE1 involves sequence-specific DNA binding that contributes to transcriptional stimulation by interaction with components of the basal transcription complex.

Hypoxia-selective antitumor agents. 15. Modification of rate of nitroreduction and extent of lysosomal uptake by polysubstitution of 4-(alkylamino)-5-nitroquinoline bioreductive drugs.

Studies have shown that 4-(alkylamino)-5-nitroquinolines possess high selectivity (20-60-fold) for hypoxic tumor cells in vitro, but are not active as hypoxia-selective cytotoxins (HSCs) in vivo. The compounds show inadequate rates of extravascular diffusion, likely due both to sequestration of the bisbasic compounds into lysosomes and rapid nitroreduction. A further series of analogues, designed to counteract these limitations, has been synthesized and evaluated. Analogues bearing one to three electron-donating substituents on the quinoline have one-electron reduction potentials up to 100 mV lower than that of the unsubstituted compound (5), but do not have improved biological activity. The relationship between hypoxic selectivity and rates of metabolic reduction suggests at least two mechanisms of cytotoxicity for this series of 5-nitroquinolines. Compounds with high rates of reduction are toxic via oxygen-sensitive net bioreduction, while compounds which are poor substrates for nitroreduction are toxic through an oxygen-insensitive non-bioreductive mechanism. As rates of metabolic reduction are lowered, the non-bioreductive mechanism of toxicity becomes dominant and hypoxic selectivity is lost. A small series of analogues bearing hydrophilic but neutral side chains were also prepared. Compounds with a dihydroxypropyl side chain retained cytotoxic potency and hypoxic cell selectivity in cell culture assays, and had lowered uptake into lysosomes, but none of three analogues evaluated against KHT tumors in mice showed activity as an HSC in vivo.

Synthesis and evaluation of 4-substituted analogues of 5-[N,N-bis (2-chloroethyl)amino]-2-nitrobenzamide as bioreductively activated prodrugs using an Escherichia coli nitroreductase.

2,4-Dinitrobenzamide mustards, exemplified by the parent compound SN 23862 (2) are activated under aerobic conditions by an Escherichia coli nitroreductase enzyme (NR2) via selective reduction of the 2-nitro group, and are thus of interest as prodrugs for antibody-directed enzyme-prodrug therapy (ADEPT). A series of related compounds 12a-12d, where the 4-nitro group of 2 was replaced by other substituents of varying electronic properties, were prepared and evaluated as potential ADEPT prodrugs. One-electron reduction potentials of the compounds correlated well with the substituent sigma m values, with the exception of the unsubstituted (4-H) analogue 13, which had a much lower value than expected on electronic grounds, due to a coplanar conformation of the mustard. The cytotoxicities of the compounds towards aerobic UV4 cells correlated positively with the electron-donating ability of the 4-substituent (measured by sigma p values), indicating that the cytotoxicities of the compounds in the absence of the NR2 enzyme are due substantially to the parent (unreduced) compounds. A positive, although less strong, correlation was seen between the electronic properties of the 4-substituent and their cytotoxicities in the presence of the NR2 enzyme, suggesting that, in this closely related series, the degree of activation by the enzyme is significantly dependent on the reduction potential of the 2-nitro group. While the 4-SO2Me derivative 12d was the next most preferred substrate after the parent 2, it was considerably less so (degree of activation as measured by IC50 ratio of 26 compared with 145), despite the similar electronic properties of the two 4-substituents.

Tertiary amine N-oxides as bioreductive drugs: DACA N-oxide, nitracrine N-oxide and AQ4N.

Tertiary amine N-oxides of DNA intercalators with alkylamino sidechains are a new class of bioreductive drugs. N-oxidation masks the cationic charge of the amines, forming prodrugs with low DNA binding affinity and low toxicity which can be activated selectively by metabolic reduction under hypoxic conditions. This study compares three intercalator N-oxides (NC-NO, DACA-NO and AQ4N), which, respectively, give nitracrine (NC), DACA and AQ4 on reduction. In aerobic cell culture all three N-oxide were much less toxic than the corresponding amines, and showed large increases in cytotoxicity under hypoxia. The topoisomerase poisons DACA and AQ4 (and their N-oxides) were less active against non-cycling than cycling cells. However, only AQ4N was active against the mouse mammary tumour MDAH-MCa-4. This dialkylaminoanthraquinone-di-N-oxide has activity at least as great as the reference bioreductive drug RB 6145 against this tumour, both with and without radiation and when combined with the tumour blood flow inhibitor 5,6-dimethylxanthenone-4-acetic acid (DMXAA). It is suggested that the high in vivo activity of AQ4N relative to the other topoisomerase-targeted N-oxide, DACA-NO, may be in part due to release in hypoxic cells of an intracalator with sufficiently high DNA binding affinity that it is retained long enough to kill non-cycling cells when they eventually re-enter the cell cycle.

Toxicity and histopathology of the growth regulator pyriproxyfen to adults and eggs of the cat flea (Siphonaptera:Pulicidae).

Adult cat fleas, Ctenocephalides felis (Bouché), exposed continuously to pyriproxyfen died within 8-10 d. Microscopic examination of 7-d-old adults indicated death was caused by histopathological damage to fat body, Malpighian tubules, midgut epithelia, salivary gland cells, and other internal tissues. Fleas were killed by pyriproxyfen regardless of whether they were held as unfed adults on treated filter paper or as feeding adults on treated dog hair. In addition to these toxic effects on adults, pyriproxyfen also induced formation of large autophagic vacuoles in maturing oocytes leading to partial reabsorption of yolk, degeneration of the nucleus, and lysis of the follicular epithelium. Consequently, most of the eggs laid by treated fleas lacked a nucleus, had a poorly formed chorion, and were ruptured during ovulation. These laboratory results suggest that continuous exposure of fleas to pyriproxyfen on a host animal could prevent deposition of viable eggs and eventually kill adults, thereby controlling all stages of flea development.

Hypoxia-selective antitumor agents. 13. Effects of acridine substitution on the hypoxia-selective cytotoxicity and metabolic reduction of the bis-bioreductive agent nitracrine N-oxide.

A series of nuclear-substituted derivatives of nitracrine N-oxide (2; a bis-bioreductive hypoxia-selective cytotoxin) were prepared and evaluated, seeking analogues of lower nitroacridine reduction potential. Disubstitution with Me or OMe groups at the 4- and 5-positions did not provide analogues with one-electron reduction potentials significantly lower than those of the corresponding monosubstituted derivatives (E(1) ca. -350 mV for both the 4-OMe and 4,5-diOMe compounds). This appears not to be due to a concomitant raising of the acridine pKa but to a lack of direct electronic effect of substituents in the ring not bearing the nitro group. Conversely, placing two OMe groups in the nitro-bearing ring does result in a substantial further lowering of reduction potential (the 2,4-diOMe analogue has an E(1) of -401 mV). The mono- and disubstituted N-oxides have substantially lower cytotoxicities than the parent nitracrine N-oxide 2 but generally retain very high hypoxic selectivity. The OMe-substituted N-oxides all showed greater metabolic stability than 2 in hypoxic AA8 cell cultures, and the 4-OMe compound 6 had improved activity in EMT6 multicellular spheroids suggesting that this metabolic stabilization may allow more efficient diffusion in tumor tissue. The parent compound 2 was selectively toxic to hypoxic cells in KHT tumors in vivo and clearly superior to nitracrine itself (although only at doses which would eventually be lethal to the host). The analogues of lower E(1), including 6, were not superior to 2 in vivo, indicating that metabolic stabilization of the nitro group is not alone sufficient to improve therapeutic utility.