Efficacy and safety of Wilms' tumor 1 helper peptide OCV-501 in elderly patients with acute myeloid leukemia: a multicenter, randomized, double-blind, placebo-controlled phase 2 trial.

PURPOSE: Complete remission (CR) of acute myeloid leukemia (AML) in elderly patients has a short duration, and there is no suitable post-remission therapy. We explored the role of the Wilms' tumor 1 helper peptide OCV-501 to prevent recurrence after remission. METHODS: This placebo-controlled phase 2 study was designed to evaluate accurately the efficacy and immunogenicity of OCV-501 in elderly AML patients. Elderly AML patients who achieved first CR were randomly allocated to receive either OCV-501 (N = 69) or placebo (N = 65) once a week for eight weeks and then every two weeks until week 104. The primary endpoint was disease-free survival (DFS). RESULTS: Nineteen (27.5%) patients in the OCV-501 group and 23 (35.4%) patients in the placebo group completed the study without relapse. The median DFS in the OCV-501 and placebo groups was 12.1 and 8.4 months, respectively (p = 0.7671, hazard ratio [95% confidence interval]: 0.933 [0.590, 1.477]). The major drug adverse reactions were injection-site reactions. Although treatment with OCV-501 did not prolong DFS for elderly AML patients, post hoc analysis found that immune responders to OCV-501 whose specific IgG was > 10,000 ng/mL (N = 16) and whose WT1-specific interferon-γ response was > 10 pg/mL (N = 26) had significantly longer overall survival compared with placebo. CONCLUSIONS: The placebo-controlled design of this study and quantitative immunological monitoring provides new insight into the relationship between peptide-induced immune responses and survival, suggesting future perspectives for cancer immunotherapy.

Pharmacokinetic Properties of DNA Aptamers with Base Modifications.

The addition of novel side chains at the 5-position of uracil is an effective means to increase chemical diversity of aptamers and hence the success rate for discovery of high-affinity ligands to protein targets. Such modifications also increase nuclease resistance, which is useful in a range of applications, especially for therapeutics. In this study, we assess the impact of these side chains on plasma pharmacokinetics of modified aptamers conjugated to a 40 kDa polyethylene glycol. We show that clearance from plasma depends on relative hydrophobicity: side chains with a negative cLogP (more hydrophilic) result in slower plasma clearance compared with side chains with a positive cLogP (more hydrophobic). We show that clearance increases with the number of side chains in sequences of ≥28 synthons, but this effect is dramatically diminished in shorter sequences. These results serve as a guide for the design of new therapeutic aptamers with diversity-enhancing side chains.

Choroid plexus-cerebrospinal fluid route for monocyte-derived macrophages after stroke.

BACKGROUND: Choroid plexus (CP) supports the entry of monocyte-derived macrophages (MDMs) to the central nervous system in animal models of traumatic brain injury, spinal cord injury, and Alzheimer's disease. Whether the CP is involved in the recruitment of MDMs to the injured brain after ischemic stroke is unknown. METHODS: Adult male C57BL/6 mice were subjected to focal cortical ischemia by permanent occlusion of the distal branch of the right middle cerebral artery. Choroid plexus tissues were collected and analyzed for Vcam1, Madcam1, Cx3cl1, Ccl2, Nt5e, and Ifnγ expression at different timepoints after stroke using qPCR. Changes of MDMs in CP and cerebrospinal fluid (CSF) at 1 day and 3 days after stroke were analyzed using flow cytometry. Infiltration of MDMs into CP and CSF were validated using ß-actin-GFP chimeric mice and Fgd5-CreERT2 x Lox-stop-lox-Tomato mice. CD115+ monocytes were isolated using a magnetic cell separation system from bone marrow of Cx3cr1-GFP or wild-type C57BL/6 donor mice. The freshly isolated monocytes or M2-like MDMs primed in vitro with IL4 and IL13 were stereotaxically injected into the lateral ventricle of stroke-affected mice to trace for their migration into ischemic hemisphere or to assess their effect on post-stroke recovery using open field, corridor, and active avoidance behavioral tests. RESULTS: We found that CP responded to cortical stroke by upregulation of gene expression for several possible mediators of MDM trafficking and, concomitantly, MDMs increased in CP and cerebrospinal fluid (CSF). We then confirmed that MDMs infiltrated from blood into CP and CSF after the insult using ß-actin-GFP chimeric mice and Fgd5-CreERT2 x Lox-stop-lox-Tomato mice. When MDMs were directly administered into CSF following stroke, they homed to the ischemic hemisphere. If they had been primed in vitro prior to their administration to become M2-like macrophages, they promoted post-stroke recovery of motor and cognitive function without influencing infarct volume. CONCLUSIONS: Our findings suggest the possibility that autologous transplantation of M2-like MDMs into CSF might be developed into a new strategy for promoting recovery also in patients with stroke.

Chemically Modified Interleukin-6 Aptamer Inhibits Development of Collagen-Induced Arthritis in Cynomolgus Monkeys.

Interleukin-6 (IL-6) is a potent mediator of inflammatory and immune responses, and a validated target for therapeutic intervention of inflammatory diseases. Previous studies have shown that SL1026, a slow off-rate modified aptamer (SOMAmer) antagonist of IL-6, neutralizes IL-6 signaling in vitro. In the present study, we show that SL1026 delays the onset and reduces the severity of rheumatoid symptoms in a collagen-induced arthritis model in cynomolgus monkeys. SL1026 (1 and 10 mg/kg), administered q.i.d., delayed the progression of arthritis and the concomitant increase in serum IL-6 levels compared to the untreated control group. Furthermore, SL1026 inhibited IL-6-induced STAT3 phosphorylation ex vivo in T lymphocytes from human blood and IL-6-induced C-reactive protein and serum amyloid A production in human primary hepatocytes. Importantly, SOMAmer treatment did not elicit an immune response, as evidenced by the absence of anti-SOMAmer antibodies in plasma of treated monkeys. These results demonstrate that SOMAmer antagonists of IL-6 may be attractive agents for the treatment of IL-6-mediated diseases, including rheumatoid arthritis.

Chemically modified DNA aptamers bind interleukin-6 with high affinity and inhibit signaling by blocking its interaction with interleukin-6 receptor.

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.

Crystal structure of interleukin-6 in complex with a modified nucleic acid ligand.

IL-6 is a secreted cytokine that functions through binding two cell surface receptors, IL-6Rα and gp130. Because of its involvement in the progression of several chronic inflammatory diseases, IL-6 is a target of pharmacologic interest. We have recently identified a novel class of ligands called SOMAmers (S low Off-rate Modified Aptamers) that bind IL-6 and inhibit its biologic activity. SOMAmers exploit the chemical diversity of protein-like side chains assembled on flexible nucleic acid scaffolds, resulting in an expanded repertoire of intra- and intermolecular interactions not achievable with conventional aptamers. Here, we report the co-crystal structure of a high affinity SOMAmer (Kd = 0.20 nm) modified at the 5-position of deoxyuridine in a complex with IL-6. The SOMAmer, comprised of a G-quartet domain and a stem-loop domain, engages IL-6 in a clamp-like manner over an extended surface exhibiting close shape complementarity with the protein. The interface is characterized by substantial hydrophobic interactions overlapping the binding surfaces of the IL-6Rα and gp130 receptors. The G-quartet domain retains considerable binding activity as a disconnected autonomous fragment (Kd = 270 nm). A single substitution from our diversely modified nucleotide library leads to a 37-fold enhancement in binding affinity of the G-quartet fragment (Kd = 7.4 nm). The ability to probe ligand surfaces in this manner is a powerful tool in the development of new therapeutic reagents with improved pharmacologic properties. The SOMAmer·IL-6 structure also expands our understanding of the diverse structural motifs achievable with modified nucleic acid libraries and elucidates the nature with which these unique ligands interact with their protein targets.

Novel oral formulation safely improving intestinal absorption of poorly absorbable drugs: utilization of polyamines and bile acids.

In order to develop a novel oral formulation that can safely improve the intestinal absorption of poorly absorbable drugs, polyamines such as spermine (SPM) and spermidine (SPD) was examined as an absorption enhancing adjuvant in rats. The absorption of rebamipide, classified into BCS Class IV, from colon was significantly improved by SPM or SPD, and the enhancing ability of SPM was larger than that of SPD. As a possible mixing and/or interaction of polyamines with bile acids were expected, the combinatorial use of sodium taurocholate (STC) with polyamines was also examined. The absorption of rebamipide was drastically improved by the combinatorial use of SPM or SPD with STC. As STC itself did not enhance the absorption of rebamipide so much, it was considered that polyamines and STC had a synergistic enhancing effect. In-vivo oral absorption study was also performed to investigate the effectiveness and safety of polyamines and their combinatorial use with STC in rats. Although the enhancing effect slightly attenuated comparing with the in-situ loop study, the absorption of rebamipide was significantly improved and the combinatorial use of 10 mM SPM with 25 mM STC showed the largest enhancing effect. Histopathological studies clearly showed that any significant change in stomach and duodenum was not caused by SPM (10 mM), SPD (10 mM) or their combinatorial use with STC (25 mM) at 1.5 or 8.0 h after oral administration. Taken all together, polyamines, especially SPM, and its combinatorial use with STC could improve the absorption of poorly absorbable drugs without any significant changes in gastrointestinal tract after oral administration in rats.

p38 MAPK associated with stereoselective priming by grepafloxacin on O2- production in neutrophils.

Grepafloxacin is an asymmetric fluoroquinolone derivative which possesses high tissue penetrability as well as strong, broad-spectrum antimicrobial activities. We recently found that grepafloxacin induced a priming effect on neutrophil respiratory burst induced by N-formylmethionylleucylphenylalanine. In this report, we elucidate the precise mechanism of the priming by grepafloxacin. The R(+) enantiomer of grepafloxacin induced a more potent priming effect than did S(-)-grepafloxacin. R(+)-Grepafloxacin also produced a more potent translocation of both p47- and p67-phox proteins to membrane fractions of neutrophils. Grepafloxacin-induced primed superoxide generation was significantly inhibited by pretreatment with PD169316 and SB203580, p38 mitogen-activated protein kinase (MAPK) inhibitors, but not with PD98059, a specific inhibitor of the upstream kinase that activates p44/42 MAPK, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Grepafloxacin strongly phosphorylated p38 MAP kinase but not p44/42 MAPK or JNK. R(+)-Grepafloxacin showed more potent phosphorylation of p38 MAPK than did S(-)-grepafloxacin, in a time- and concentration-dependent manner. PD169316 significantly inhibited R(+)-grepafloxacin-induced translocation of p47-phox protein to the membrane fraction. Interestingly, grepafloxacin stereospecifically bound to the membrane fractions of neutrophils. These results strongly suggest that grepafloxacin stereospecifically primes neutrophil respiratory burst, and p38 MAPK activation is closely related to the grepafloxacin priming.

Regulation of fluoroquinolone uptake by human neutrophils: involvement of mitogen-activated protein kinase.

Although human neutrophils actively internalize fluoroquinolones, the precise uptake mechanism is not fully understood. In this study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in fluoroquinolone uptake in neutrophils. Spontaneous grepafloxacin uptake was significantly enhanced by SB203580, a p38 MAPK inhibitor, in a dose-dependent manner, but not by PD98059, a specific inhibitor of the upstream kinase that activates p44/42 MAPK. Neither inhibitor affected spontaneous ciprofloxacin or ofloxacin uptake. Phorbol myristate acetate (PMA) treatment enhanced ciprofloxacin uptake, whereas it reduced grepafloxacin uptake. These effects by PMA were significantly inhibited by the pretreatment of neutrophils with GF109203X, a specific inhibitor of PKC. PMA had no effect on ofloxacin uptake. The PMA-induced enhancement of ciprofloxacin uptake was inhibited by PD98059, but not by SB203580. On the other hand, the PMA-induced reduction of grepafloxacin uptake was not inhibited by either MAPK inhibitor. Grepafloxacin, but not ciprofloxacin or ofloxacin, strongly phosphorylated p38 MAPK. This phosphorylation of p38 MAPK was not inhibited by GF109203X pretreatment. None of these three fluoroquinolones phosphorylated p44/42 MAPK. PMA phosphorylated both p38 and p44/42 MAPK. These findings indicate that grepafloxacin negatively regulates its uptake in neutrophils, and p38 MAPK activation is involved in this down-regulation of grepafloxacin uptake. Ciprofloxacin uptake is positively regulated by the activation of PKC, and p44/42 MAPK activation is involved in this up-regulation. Neither PKC, p38 nor p44/42 MAPK is involved in the regulation of ofloxacin uptake.

Lung tissue distribution after intravenous administration of grepafloxacin: comparative study with levofloxacin.

The aim of the present study is to study the pharmacokinetics in plasma, lung lymph and bronchial washing fluid after intravenous infusion of grepafloxacin (GPFX), in comparison with those of levofloxacin (LVFX). Four conscious sheep with chronically instrumented lung lymph fistulas and tracheotomy were prepared. GPFX and LVFX concentrations in plasma and lung lymph after intravenous infusion of the drugs (10 mg/kg) for over 10 min were measured. In addition serial bronchial washing with 50 mL normal saline was performed to obtain epithelial lining fluid (ELF) at 2, 4, 6, 8, 12, 24 h after the intravenous administration. The time courses of lung lymph concentration were almost identical to those of the concomitant levels of both GPFX and LVFX in plasma, suggesting that both GPFX and LVFX could be easily moved from plasma to pulmonary interstitium and/or lung lymph circulation. However, GPFX concentrations of ELF were significantly higher than LVFX concentrations over time after the administration. In addition, intracellular concentrations in ELF of GPFX were also extremely high compared with those of LVFX. These results demonstrated that penetration of GPFX in bronchial wall, bronchial epithelium and/or phagocytic cells was superior to that of LVFX. These observations suggest that the pharmacokinetic characteristics of GPFX in the lung may provide a new insight into the strategy for clinical treatment of various pulmonary infections, especially cytotropic bacterial infections.