Ten-fold Lower Dose Research Articles

Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

Simple Summary2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is a cyclic oligosaccharide widely used as an excipient in pharmaceutical preparations, in addition to also being used as a cholesterol regulator. HP-β-CyD was used in clinical trials for patients with Niemann-Pick Type C disease to remove accumulated intracellular lipid. HP-β-CyD has anti-leukemia activity by inducing apoptosis and cell-cycle arrest; however, the antitumor activity of HP-β-CyD lacks tumor cell-selectivity. Taking advantage of the fact that folate receptors are highly expressed in many cancer cells, we synthesized folate-appended HP-β-CyD (FA-HP-β-CyD) to confer tumor cell-selectivity to HP-β-CyD. FA-HP-β-CyD inhibited the proliferation of chronic myeloid leukemia (CML) cells and the mechanism underlying the effect of FA-HP-β-CyD in inducing cell death may involve autophagy. The combination of FA-HP-β-CyD and ABL tyrosine kinase inhibitors (imatinib and ponatinib) had a synergistic inhibitory effect on CML cells. In a mouse model of BCR-ABL-induced leukemia, FA-HP-β-CyD had a stronger inhibitory effect on leukemia progression than HP-β-CyD or imatinib.2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is widely used as an enabling excipient in pharmaceutical formulations. We previously demonstrated that HP-β-CyD disrupted cholesterol homeostasis, and inhibited the proliferation of leukemia cells by inducing apoptosis and cell-cycle arrest. Recently developed drug delivery systems using folic acid (FA) and folic acid receptors (FR) are currently being used in cancer treatment. To confer tumor cell-selectivity to HP-β-CyD, we synthesized folate-appended HP-β-CyD (FA-HP-β-CyD) and evaluated the potential of FA-HP-β-CyD as an anticancer agent using chronic myeloid leukemia (CML) cells in vitro and in vivo. FA-HP-β-CyD inhibited the growth of FR-expressing cells but not that of FR-negative cells. FA-HP-β-CyD had stronger anti-leukemia and cell-binding activities than HP-β-CyD in CML cells. Unlike HP-β-CyD, FA-HP-β-CyD entered CML cells through endocytosis and induced both apoptosis and autophagy via mitophagy. FA-HP-β-CyD increased the inhibitory effects of the ABL tyrosine kinase inhibitors imatinib mesylate and ponatinib, which are commonly used in CML. In vivo experiments in a BCR-ABL leukemia mouse model showed that FA-HP-β-CyD was more effective than HP-β-CyD at a ten-fold lower dose. These results indicate that FA-HP-β-CyD may be a novel tumor-targeting agent for the treatment of leukemia.

Influence of herbicides, insecticides and fungicides on food consumption and body weight of Rossiulus kessleri (Diplopoda, Julidae)

Pesticides kill organisms harmful for the human organism, sometimes also harming beneficial ones. After treatment, pesticides remain on the soil surface in agrocenoses and adjacent plots for decades. For the laboratory experiment, we selected Rossiulus kessleri (Lochmander, 1927) – a species which lives 5–6 years on the soil surface and can dig in to soil to a depth of 30–40 cm. During a 20-day experiment we used herbicides (Roundup, Urahan Forte), insecticides (Omite, BI 58, Biotlin, Actellic, Nurelle D) and fungicides (Ridomil Gold, Thiovit Jet, Penncozeb 80 WP, Falcon, Tilt, Horus) which are often used in agrocenosises of Ukraine. Under the impact of Roundup and Urahan, body weight of R. kessleri reliably did not change, but food consumption and production of excrement increased. Pesticide-treated litter did not digest in the intestine of millipedes, but they survived to the end of the experiment (20 days). In the conditions of treating litter with insecticides Omite, BI 58, Biotlin, Actellic and Nurelle D, the amount of consumed food and body weight reliably did not change; most of these insecticides slowed the formation of feces in the millipedes. The highest studied concentrations of Actellic and Nurelle D preparations caused death to R. kessleri. Depending on the concentration in the litter, the studied fungicides Ridomil Gold, Thiovit Jet, Penncozeb, Falcon, Tilt, Horus had a varying effect on food consumption, body weight and the amount of excrement of R. kessleri. Thus, in agrocenoses and forest ecosystems adjacent to them (windbreaks, ravine and flood plain forests), R. kessleri can be significantly affected by the manufacturer-recommended doses of pesticides, as well as more than ten-fold lower doses.

Antigen dose affects disease kinetics in a murine model of demyelinating disease

Abstract Multiple sclerosis is an autoimmune disorder caused by immune cell damage to the myelin sheath lining nerve cells, leading to neuronal damage and dysfunction. The murine model of the disease - experimental autoimmune encephalomyelitis (EAE) - is induced by inoculation with MOG (myelin oligodendrocyte glycoprotein) peptide and an adjuvant, leading to recapitulation of disease that is mediated primarily by CD4 T cells. In this study, we look at the effect of MOG dosage on demyelinating disease kinetics in WT and MOG-deficient animals, as MOG-deficient mice have different numbers of MOG specific CD4 Teff and Tregs. The T cells from MOG deficient animals have a higher affinity for MOG, expand faster, and cause exacerbated disease in an adoptive transfer model compared to CD4 T cells from wildtype mice. Our results indicate that the dose of MOG peptide used to induce disease can also modulate disease course. Using tenfold lower doses changes T cell and disease kinetics resulting in disease that resolves compared to controls. Taken together, we can use varying doses of MOG as a tool to probe CD8 and CD4 T cell expansion and monitor migration of immune cells to the CNS. Further understanding of the cellular tides during disease course enables us to gain insight on how cell ratios in different sites contribute to disease pathogenesis. Using this information, we are developing mathematical modeling tools that could be used to analyze and predict disease progression.

Abstract 1619: The next-generation ALK inhibitor PF-06463922 overcomes primary resistance to crizotinib in neuroblastoma

Abstract Neuroblastomas (NBs) harboring activating point mutations in the kinase domain of anaplastic lymphoma kinase (ALK) are differentially sensitive to ALK inhibition by crizotinib (Bresler et al. 2011). The F1174L and F1245C amino acid substitutions, which comprise 38.5% (mycancergenome.org) of ALK aberrations in NB, also confer intrinsic resistance to crizotinib and present a significant obstacle in the clinic. Therefore, our goal has been to identify a next-generation ALK inhibitor with improved selectivity and potency to target these resistant mutants effectively. We demonstrate here that PF-06463922 (Pfizer), an ATP-competitive, macrocyclic ALK/ROS1 inhibitor, overcomes crizotinib resistance of these ALK variants in NB, and exerts unprecedented activity as a single agent against the F1174L and F1245C ALK-mutated tumors. At a tenfold lower dose (10 mg/kg) than typically used for crizotinib (100 mg/kg), PF-06463922 induces complete tumor regression in patient-derived (COG-N-426x, F1245C ALK-mutated) and cell line-derived (SY5Y, F1174L ALK-mutated) xenograft mouse models. Whereas crizotinib-treated mice in both models experience rapid tumor growth on therapy, PF-06463922-treated mice showed sustained complete responses for several weeks even after treatment was discontinued. Cell-based and biochemical analyses indicate that the superior activity of PF-06463922 is a function of both higher potency and a broader spectrum of effectiveness across mutants. ATP levels were measured in ten NB cell lines upon treatment with PF-06463922 and crizotinib; at 120 hours post-treatment, PF-06463922 is 26-38 times more effective at inducing growth inhibition in cells with primary resistance than crizotinib. These in vitro and in vivo results are corroborated by our biochemical experiments. In comparative kinase inhibition studies of several next-generation ALK inhibitors, PF-06463922 was most effective against frequently observed ALK variants accounting for approximately 75% of neuroblastoma samples, including those with F1174L mutations. In vitro, PF-06463922 showed at least 3-4 fold higher potency than crizotinib in peptide phosphorylation assays, and its superior inhibition was consistently observed in cellular analysis of transforming ability. Further investigation is necessary to elucidate PF-06463922's mechanism of action and reveal how it induces apoptotic signaling networks without abrogating phospho-ALK. Taken together, these results support the argument that a modified ALK inhibitor may improve outcomes for patients with primary resistance to crizotinib and provide the preclinical basis from which to design a clinical trial for PF-06463922 for the treatment of ALK-mutated NBs. Citation Format: Nicole Infarinato, Jin Park, Renata Sano, Kateryna Krytska, Hannah Ryles, Helen Zou, Nathan Lee, Tod Smeal, Mark A. Lemmon, Yael P. Mosse. The next-generation ALK inhibitor PF-06463922 overcomes primary resistance to crizotinib in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1619. doi:10.1158/1538-7445.AM2015-1619

Low-dose PTH increases osteoblast activity via decreased Mef2c/Sost in senescent osteopenic mice.

Intermittent administration of parathyroid hormone (PTH) 1-34 at a standard dose has been shown to induce anabolic effects in bone. However, whether low-dose PTH promotes bone formation during senescence is unknown. To address this issue, we determined the effects of low-dose PTH and analysed the underlying mechanisms in prematurely senescent mice that display osteopenia. Treatment of 9-week-old Samp6 mice for 6 weeks with PTH at a standard dose (100 μg/kg per day) increased vertebral and femoral bone mass and improved bone microarchitecture as a result of increased bone-forming surfaces and mineral apposition rate (MAR). At a tenfold lower dose (10 μg/kg per day), PTH increased axial bone volume and trabecular thickness, as detected by bone histomorphometry but not by micro-computed tomography analysis. This anabolic effect resulted from increased osteoblast activity, as reflected by increased serum N-terminal propeptide of type 1 procollagen (P1NP) levels and MAR, with unchanged bone-forming surface or osteoblast surface. Mechanistically, low-dose PTH increased the expression of osteoblast markers in bone marrow stromal cells and mature osteoblasts, which was associated with increased expression of the Wnt effector Wisp1. Moreover, low-dose PTH decreased the expression of the Mef2c transcription factor, resulting in decreased Sost expression in osteoblasts/osteocytes. These results indicate that PTH at a low dose is effective at promoting bone formation and increased bone volume in senescent osteopenic mice through increased osteoblast activity and modulation of specific Wnt effectors, which raises the potential therapeutic use of intermittent PTH at low dose to increase bone forming activity and bone mass in skeletal senescence.

Treatment with atacicept enhances neuronal cell death in a rat model of optic neuritis

We investigated the effect of atacicept, a recombinant fusion protein blocking BLyS and APRIL and acting on B cells, on degeneration of retinal ganglion cells (RGCs) during experimental autoimmune encephalomyelitis (EAE). We used myelin oligodendrocyte glycoprotein in Brown Norway rats to induce a variant of EAE which involves B cells and leads to severe optic neuritis. Intraperitoneal treatment with atacicept at some of the studied dose levels (100 or 200μg) resulted in increased apoptosis of retinal ganglion cells whereas at a tenfold lower dose or in vehicle-treated animals no such effect became apparent. Also the extent of inflammation, demyelination, and axonal loss of the optic nerve was more pronounced in rats treated with the higher atacicept dose level. The present study describes observational evidence for adverse effects of atacicept on neuronal survival during EAE.

Antidiuretic effect of desmopressin chimera agglomerates by nasal administration in rats

In the present study, a nasal powder of the antidiuretic peptide desmopressin (DDAVP) formulated as chimera agglomerates was studied to improve drug bioavailability and provide a flexible drug product. Firstly, DDAVP was spray-dried along with mannitol and lecithin to produce primary microparticles capable of instantaneous dissolution in water. The chimera agglomerates were spontaneously formed by mechanically vibrating the microparticles on two stacked sieves. Agglomerate formation and strength were favored by the presence of lecithin. Drug content and dissolution rate remained unmodified after agglomeration. However, owing to the agglomerate larger size, powder flowability was greatly improved in comparison with the original microparticles, allowing accurate powder dosing into the nasal delivery device. DDAVP in vitro permeation across excised rabbit nasal mucosa from the agglomerates was significantly higher than that obtained from a commercial liquid nasal spray. In rats, intranasal DDAVP agglomerates allowed for efficient administration with almost 80% of the loaded powder emitted from the device into the animal nose. The administration of DDAVP agglomerates induced a significant reduction in urine production. Moreover, the antidiuretic effect of agglomerates did not significantly differ from the one induced by an intravenous injection of DDAVP at a ten-fold lower dose.

Characterization of a model of lethal dengue virus 2 infection in C57BL/6 mice deficient in the alpha/beta interferon receptor.

Dengue virus (DENV) causes dengue fever and dengue haemorrhagic fever/dengue shock syndrome, both considered major public-health problems worldwide. We generated a lethal DENV-2 strain (D220) by 10 additional cycles of subcutaneous inoculation of mice with supernatant from mosquito cells infected with the previously characterized strain D2S10, followed by harvesting of serum. D220 induces mortality at ten-fold lower doses than D2S10 in mice lacking only the alpha/beta interferon (IFN-α/β) receptor in C57BL/6 or 129 backgrounds under both non-enhanced and antibody-enhanced conditions. Sequence analysis of the complete viral genome revealed five amino acid changes between D220 and D2S10, of which two (K122I in envelope and V115A in NS4B) appear to account for the observed phenotypic differences between the viruses. By causing mortality at lower doses in C57BL/6 mice lacking only the IFN-α/β receptor, D220 constitutes an improved tool for study of DENV-induced pathogenesis, as well as for testing potential vaccines and antiviral drugs against DENV.

Development of Peru-15 (CholeraGarde®), a live-attenuated oral cholera vaccine: 1991–2009

Epidemics of severe dehydrating cholera are on the increase in resource-limited settings around the world. Adults, children and young infants are all at risk of these infections. Considerable efforts have been made for the development of safe and efficacious oral cholera vaccines over the last three decades. Whole-cell-inactivated as well as live oral cholera vaccines have been developed and tested in different field settings to determine the efficacy and/or effectiveness of such vaccines for reducing life-threatening disease. This review follows the trail of the development of CholeraGarde®, a live-attenuated Vibrio cholerae O1 vaccine candidate of the El Tor biotype and Inaba serotype. CholeraGarde, also well known as Peru-15, was derived from V. cholerae O1 strain C6709, a clinical isolate from Peru. The vaccine has now been tested in over 500 individuals, adults and children and shows a good safety and immunogenicity profile. At a dose of around 108 CFU, it is immunogenic in adults in the USA, as well as in adults, children and infants in Bangladesh. The vaccine has been tested in infants of 9 months of age where a single 108 CFU dose was safe and immunogenic while a tenfold lower dose was not. Excretion of the strain was higher in adults in the USA and low in Bangladeshi participants in all age groups. Phase II studies of CholeraGarde are ongoing in cholera-endemic countries to concomitantly administer it to infants with the parenteral measles vaccine. Studies on HIV-positive individuals are also ongoing to determine safety, immunogenicity and contraindications, if any. Phase III studies are being targeted to determine the protective efficacy of CholeraGarde and for further development of a single-dose vaccine that will protect infants and also other age groups from endemic and epidemic cholera.

C-20 Cyclopropyl Vitamin D3 Analogs

The formal C-20 methylation of 1,25-dihydroxy vitamin D3 (calcitriol) and bridging of two methyl groups produces spiro[cyclopropane-1, 20'-calcitriol], colloquially referred to as C-20 cyclopropylcalcitriol, which is much more active in MLR for suppression of interferon-gamma release than calcitriol, and hypercalcemia in mice is elicited at a ten-fold lower dose when compared to calcitriol. Introduction of the Delta16,17-double bond, modification of the side chain by 23-unsaturation and replacement of the methyl groups at C-26 and C-27 with trifluoromethyl moieties create a highly active series of vitamin D analogs. As previously observed in the calcitriol series, the presence of the C-16 double bond in the cyclopropyl analogs also arrests metabolic side-chain oxidation in the at the C-24 oxo level in UMR 106 cells. The enhanced biological activity is ascribed, at least in part, to the improved resistance toward metabolic degradation.

Dose Sparing of CpG Oligodeoxynucleotide Vaccine Adjuvants by Nanoparticle Delivery

The main objective of these studies was to investigate whether the nanoparticle delivery has any immunopotentiation effect at modest doses of a few micro- or nanograms of CpG oligodeoxynucleotide (CpG ODN) and what would be the influence on T cell responses at such low doses. Various doses (5 to 0.05 microg) of a model CpG ODN adjuvant (#1826) along with 2 Lf tetanus toxoid (TT) were formulated in either nanoparticles using poly(D,L-lactic-co-glycolic acid) (PLGA) 50:50 co-polymer, or saline. Strong antigen specific ex vivo T cell proliferation was observed for the Balb/c mice receiving immunogens in nanoparticles. At 5 microg dose of CpG ODN, the T cell stimulation index (SI) was 241 as compared with 74 for the same dose when given in saline. Comparable SI value of 78 was observed at 100-fold lower dose (0.05 microg) using nanoparticles. Similarly, significantly higher (P<0.01) cytokine secretion was observed for nanoparticles groups. A ten-fold lower dose (0.5 microg instead of 5 microg) of CpG ODN in nanoparticles was adequate to obtain levels of IFN-gamma, TNF-alpha, and IL-2 comparable to those observed following immunisations in saline. The immunopotentiation effect of the particulate delivery on antibody response (total IgG and subtypes) was not so marked. These studies emphasise that antigen delivery in biodegradable nanoparticles can facilitate induction of strong T cell responses, particularly of the Th1 type, at extremely lower doses of CpG ODN. Such reduction in the effective dose would be advantageous for minimising the potential side effects of these novel adjuvants.

Differential toxicity of camptothecin, topotecan and 9-aminocamptothecin to human, canine, and murine myeloid progenitors (CFU-GM) in vitro

20(S)-Camptothecin (CAM), topotecan (TPT, active ingredient in Hycamtin) and 9-amino-20(S)-camptothecin (9AC) are topoisomerase I inhibitors that cause similar dose-limiting toxicities to rapidly renewing tissues, such as hematopoietic tissues, in humans, mice, and dogs. However, dose-limiting toxicity occurs at tenfold lower doses in humans than in mice. The purpose of the current study was to determine whether hematopoietic progenitors of the myeloid lineage from humans, mice, and dogs exhibit the differential sensitivity to these compounds that is evident in vivo. Drug-induced inhibition of in vitro colony formation by a myeloid progenitor in human, murine, and canine marrow colony-forming unit-granulocyte/macrophage (CFU-GM) provided the basis for interspecies comparisons at concentrations which inhibited colony formation by 50% (IC50) and 90% (IC90). Murine IC90 values were 2.6-, 2.3-, 10-, 21-, 5.9-, and 11-fold higher than human values for CAM lactone (NSC-94600) and sodium salt (NSC-100880), TPT (NSC-609699), and racemic (NSC-629971), semisynthetic and synthetic preparations (NSC-603071) of 9AC, respectively. In contrast, canine IC90 values were the same as, or lower than, the human IC90 values for all six compounds. The greater susceptibility of humans and dogs to the myelotoxicity of camptothecins, compared to mice, was evident in vitro at the cellular level. Differential sensitivity between murine and human myeloid progenitors explains why the curative doses of TPT and 9AC in mice with human tumor xenografts are not achievable in patients. Realizing the curative potential of these compounds in humans will require the development of therapies to increase drug tolerance of human CFU-GM at least to a level equal to that of murine CFU-GM. Because these interspecies differences are complicated by species-specific effects of plasma proteins on drug stability, not all in vitro assay conditions will yield results which can contribute to the development of such therapies.

Temperature, food intake, and locomotor activity effects of a 5-HT3 receptor agonist and two 5-HT3 receptor antagonists in rats.

This study investigated three physiologic functions known to be modulated by serotonin-temperature, food intake and locomotor activity - using the 5-HT3 receptor agonist, m-chlorophenylbiguanide (m-CPBG), and two 5-HT3 antagonists, MDL-72222 and ondansetron. m-CPBG produced dose-dependent elevations in rectal temperature. MDL-72222, which had no effects on temperature when given alone, significantly attenuated m-CPBG-induced hyperthermia. Food intake in food-deprived rats was reduced during the first hour by the highest dose of m-CPBG. Food intake was also dose-dependently reduced by MDL-72222; m-CPBG plus MDL-72222 led to greater reductions in food intake. Food intake in freely fed rats was unaffected by m-CPBG or MDL-72222. Locomotor activity was unaffected by m-CPBG, but was dose-dependently reduced by MDL-72222, an effect which may have contributed to its hypophagic effects. Ondansetron, used in ten-fold lower doses than MDL-72222, was inactive in all of these paradigms. These data: (1) provide some evidence for 5-HT3 receptor-mediated changes in temperature; (2) are in agreement with two prior studies which reported locomotor activity reductions following 5-HT3 antagonists; but (3) do not support an important role for 5-HT3 receptors in the regulation of food intake in rats.

Involvement of dopamine in the central effect of neurotensin on intestinal motility in rats

The effects of intracerebroventricular (ICV) administration of neurotensin (NT) before a meal on intestinal postprandial motility were examined in conscious rats chronically fitted with intraparietal Nichrome electrodes in the duodeno-jejunum. The effects were compared with those of two analogues, [D-Tyr 11]NT and [D-Trp 11]NT, resistant to degradation by brain peptidases. NT (10 μg ICV) delayed the occurrence of postprandial disruption of duodenal motility and blocked it on the jejunum. [D-Tyr 11]NT and [D-Trp 11]NT (1 μg ICV) elicited the same effects but at a ten-fold lower dose. NT administered peripherally just before a meal significantly lengthened the duration of the postprandial motor pattern. The central effect of NT on the fed pattern involved dopaminergic neurons as it was mimicked by dopamine, blocked by haloperidol and partly antagonized by either sulpiride or (+) SCH 23390. It is concluded that: 1) both D 1 and D 2 receptors are involved in the blocking effect of the postprandial disruption induced by central NT; 2) that [D-Tyr 11]NT and [D-Trp 11]NT are potent agonists at NT receptors in the brain.