A splice-switching oligonucleotide treatment ameliorates glycogen storage disease type 1a in mice with G6PC c.648G>T.

Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-α (G6Pase-α, encoded by G6PC), which is primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves life expectancy, patients still experience intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a but potentially require complicated glycemic management throughout life. Here, we present an oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. We generated a mouse strain with homozygous knockin of this variant that well reflected the pathophysiology of patients with GSD1a. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b had long-lasting efficacy of more than 12 weeks in mice that received a single dose and had favorable pharmacokinetics and tolerability in mice and monkeys. These findings together indicate that this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T.

Renadirsen, a Novel 2'OMeRNA/ENA® Chimera Antisense Oligonucleotide, Induces Robust Exon 45 Skipping for Dystrophin In Vivo.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients' DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2'-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2'OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

Efficacy of a single intravenous administration of laninamivir (an active metabolite of laninamivir octanoate) in an influenza virus infection mouse model.

Laninamivir, a potent neuraminidase (NA) inhibitor, is an active metabolite of laninamivir octanoate (code name: CS-8958) which is a long acting NA inhibitor and is commercially available under the brand name Inavir in Japan to complete the treatment of influenza by a single inhalation. It is supposed that the long acting character is provided by the long retention of laninamivir in the respiratory tract after intranasal administration of laninamivir octanoate in mice and with stable binding of laninamivir to NA of various influenza viruses such as N1, N2 subtypes and NA of B virus. Peramivir, another NA inhibitor, is also approved in Japan as a single intravenous infusion. In spite of the quick disappearance of peramivir from the blood after injection, the reason treatment can be completed by a single administration is thought to be that peramivir showed stable binding to NA with N9 subtype. Therefore, the stable binder, laninamivir is possibly effective by a single intravenous administration in the mouse model infected with influenza viruses. A single intravenous administration of laninamivir and peramivir at 30mg/kg significantly prolonged mice survival at a comparable level in the mouse lethal model infected with the A/PR/8/34 (H1N1) virus. Also, a single intravenous administration of laninamivir and peramivir significantly suppressed virus proliferation in the lungs of mice infected with influenza B virus. Thus, laninamivir may be effective by a single intravenous infusion in treating influenza, the same as peramivir.

Absorption, elimination, and metabolism of CS-1036, a novel α-amylase inhibitor in rats and monkeys, and the relationship between gastrointestinal distribution and suppression of glucose absorption.

The absorption, metabolism, and excretion of (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-ß-d-glucopyranosyl)-α-d-glucopyranoside (CS-1036), a novel and potent pancreatic and salivary α-amylase inhibitor, were evaluated in F344/DuCrlCrlj rats and cynomolgus monkeys. The total body clearance and volume of distribution of CS-1036 were low (2.67-3.44 ml/min/kg and 0.218-0.237 l/kg for rats and 2.25-2.84 ml/min/kg and 0.217-0.271 l/kg for monkeys). After intravenous administration of [(14)C]CS-1036 to rats and monkeys, radioactivity was mainly excreted into urine (77.2% for rats and 81.1% for monkeys). After oral administration, most of the radioactivity was recovered from feces (80.28% for rats and 88.13% for monkeys) with a low oral bioavailability (1.73-2.44% for rats and 0.983-1.20% for monkeys). In rats, intestinal secretion is suggested to be involved in the fecal excretion as a minor component because fecal excretion after intravenous administration was observed (15.66%) and biliary excretion was almost negligible. Although intestinal flora was involved in CS-1036 metabolism, CS-1036 was the main component in feces (70.3% for rats and 48.7% for monkeys) and in the intestinal contents (33-68% for rats up to 2 hours after the dose) after oral administration. In Zucker diabetic fatty rats, CS-1036 showed a suppressive effect on plasma glucose elevation after starch loading with a 50% effective dose at 0.015 mg/kg. In summary, CS-1036 showed optimal pharmacokinetic profiles: low oral absorption and favorable stability in gastrointestinal lumen, resulting in suppression of postprandial hyperglycemia by α-amylase inhibition.

High and continuous exposure of laninamivir, an anti-influenza drug, may work suppressively to generate low-susceptibility mutants in animals.

Laninamivir octanoate (Inavir(®); Daiichi Sankyo, Tokyo, Japan) is an anti-influenza drug that provides complete treatment by a single inhalation. It works as a long-acting neuraminidase (NA) inhibitor by means of high and continuous exposure of laninamivir, its active metabolite, in the lungs of mice after intranasal administration. Even after 6 days after intranasal administration of 236 µg/kg laninamivir octanoate, the concentration of laninamivir in the lungs was maintained more than 2-3 orders higher than 50% inhibitory concentrations of laninamivir to N1 NAs, about 2 orders higher than N2 NA of seasonal influenza A viruses, and more than about 50 times higher than influenza B virus NA. From A/H1N1 influenza virus-infected and laninamivir octanoate-treated mice, no low-susceptibility mutants to laninamivir were obtained. In contrast, four different mutants to oseltamivir were obtained from mice administered oseltamivir phosphate, which required repeated administration for treatment under the experimental condition, showing similar virus load reduction between both compounds. This finding suggested the unique characteristics of laninamivir octanoate in mice may work suppressively to generate low-susceptibility mutants.

In vivo pharmacodynamic activity of tomopenem (formerly CS-023) against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus in a murine thigh infection model.

Tomopenem (formerly CS-023) is a novel carbapenem with broad-spectrum activities against diverse hospital pathogens, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). We examined the in vivo pharmacodynamic characteristics of tomopenem against P. aeruginosa and MRSA by using a neutropenic murine thigh infection model with P. aeruginosa 12467 (MIC, 1 µg/ml) and MRSA 12372 (MIC, 2 µg/ml). The mice had 10(6) to 10(7) CFU/thigh of each strain 2 h after inoculation and were treated for 24 h with a fractionated administration of tomopenem given at intervals of 3, 6, 12, and 24 h. The serum protein binding of tomopenem was 17.4%. The efficacy of tomopenem in both infection models was enhanced by frequent dosing, which indicates that the efficacy is driven by the time above MIC (T(MIC)). In a sigmoid model, the cumulative percentages of the 24-h period that the concentrations of free, unbound fractions of the drug exceeded the MIC under steady-state pharmacokinetic conditions (f%T(MIC)s) were best correlated with efficacy when R(2) was 0.79 and 0.86 against P. aeruginosa and MRSA, respectively. Other pharmacokinetic and pharmacodynamic (PK-PD) indexes for the free, unbound fractions, the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC) and the maximum concentration of the drug in serum divided by the MIC (C(max)/MIC), showed poor correlation with efficacy when R(2) was ≤0.42. The f%T(MIC) values required for a static effect, 1-log kill, and 2-log kill against P. aeruginosa were 29, 39, and 51, respectively, which were similar to those for meropenem, for which the values were 24, 33, and 45, respectively. Against MRSA, the values for tomopenem were 27, 35, and 47. In conclusion, the pharmacodynamic characteristics of tomopenem were similar to those of meropenem against P. aeruginosa, and there was no difference between the target values for P. aeruginosa and MRSA required for efficacy in this study.

In vitro postantibiotic effects of tomopenem (CS-023) against Staphylococcus aureus and Pseudomonas aeruginosa.

The postantibiotic effect (PAE) of tomopenem was determined after a 2 h exposure of two strains of meticillin-susceptible and meticillin-resistant Staphylococcus aureus (MSSA and MRSA), and imipenem-susceptible and imipenem-resistant Pseudomonas aeruginosa, to tenfold the respective MIC. The PAEs on MSSA and P. aeruginosa were approximately 1 h and they were comparable to those of meropenem. The PAE on MRSA was 1.5 to 3 h, equal to or longer than those of vancomycin. The PAEs of tomopenem not only were found for MRSA, but also were present in the imipenem-resistant P. aeruginosa tested.

Laninamivir prodrug CS-8958, a long-acting neuraminidase inhibitor, shows superior anti-influenza virus activity after a single administration.

Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for use for the treatment and prophylaxis of influenza. We have reported on laninamivir (code name, R-125489), a novel neuraminidase inhibitor, and have discovered that the laninamivir prodrug CS-8958 worked as a long-acting neuraminidase inhibitor in a mouse influenza virus infection model when it is intranasally administered. In this study, CS-8958 was administered just once 7 days before infection and showed significant efficacy in vivo. The efficacy of a single administration of CS-8958 after viral infection was then compared with that of repeated administrations of oseltamivir phosphate or zanamivir in mice and ferrets. CS-8958 showed efficacy superior or similar to the efficacies of the two licensed NA inhibitors. CS-8958 also significantly reduced the titers of an oseltamivir-resistant H1N1 virus with a neuraminidase H274Y substitution in a mouse infection model. These results suggest that since CS-8958 is characteristically long lasting in the lungs, it may be ideal for the prophylaxis and treatment of influenza.

[In vitro and in vivo effects of a long-acting anti-influenza agent CS-8958 (laninamivir octanoate, Inavir) against pandemic (H1N1) 2009 influenza viruses].

Laninamivir is a novel neuraminidase inhibitor of influenza viruses and it has been reported that its prodrug, CS-8958 shows a long-lasting characteristics. Using viruses isolated in Nagasaki of pandemic (H1N1) 2009 influenza virus which cause pandemic in 2009, it was shown that laninamivir has a strong inhibitory activities against their neuraminidases and virus replication in cultured cells, and strong binding stability to the virus NA. Furthermore, a single intranasal administration of CS-8958 showed a superior reduction of virus load in lungs in mouse infection model. These suggest that CS-8958 will work as a long-acting neuraminidase inhibitor to an infection with pandemic (H1N1) 2009 influenza viruses as well.

CS-8958, a prodrug of the new neuraminidase inhibitor R-125489, shows long-acting anti-influenza virus activity.

Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for the treatment of and prophylaxis against influenza. In this paper, the new potent NA inhibitor R-125489 is reported for the first time. R-125489 inhibited the NA activities of various type A and B influenza viruses, including subtypes N1 to N9 and oseltamivir-resistant viruses. The survival effect of R-125489 was shown to be similar to that of zanamivir when administered intranasally in a mouse influenza virus A/Puerto Rico/8/34 infection model. Moreover, we found that the esterified form of R-125489 showed improved efficacy compared to R-125489 and zanamivir, depending on the acyl chain length, and that 3-(O)-octanoyl R-125489 (CS-8958) was the best compound in terms of its life-prolonging effect (P < 0.0001, compared to zanamivir) in the same infection model. A prolonged survival effect was observed after a single administration of CS-8958, even if it was given 7 days before infection. It is suggested that intranasally administered CS-8958 works as a long-acting NA inhibitor and shows in vivo efficacy as a result of a single intranasal administration.

Affinity of Tomopenem (CS-023) for penicillin-binding proteins in Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.

Tomopenem (formerly CS-023), a novel 1beta-methylcarbapenem, exhibited high affinity for penicillin-binding protein (PBP) 2 in Staphylococcus aureus, PBP 2 in Escherichia coli, and PBPs 2 and 3 in Pseudomonas aeruginosa, which are considered major lethal targets. Morphologically, tomopenem induced spherical forms in E. coli and short filamentation with bulges in P. aeruginosa, which correlated with the drug's PBP profiles. The potential of resistance of these bacteria to tomopenem was comparable to that to imipenem.

[In-vitro activity of panipenem against clinical isolates in 2006].

The antimicrobial activity of various antibiotics against clinical bacterial isolates recovered from patients with infectious diseases at the medical facilities in the Kanto region between March and September 2006 was evaluated. A total of 1030 clinical isolates were available for susceptibility tests: 420 aerobic Gram-positive organisms, 520 aerobic Gram-negative organisms, 30 anaerobic Gram-positive organisms and 60 anaerobic Gram-negative pathogens. Antimicrobial susceptibility data for Streptococcus pneumoniae and Haemophilus influenzae isolates from pediatric and adult patients were analyzed separately. Panipenem (PAPM), imipenem (IPM), meropenem (MEPM), biapenem (BIPM), doripenem (DRPM), cefozopran (CZOP), cefepime (CFPM), and sulbactam/cefoperazone (SBT/CPZ) were used as test antibiotics. PAPM, IPM and DRPM exhibited excellent in vitro antibacterial activities against methicillin-susceptible Staphylococcus, with all isolates exhibiting a MIC of < or =0.06 microg/mL. Against Streptococcus including penicillin-resistant S. pneumoniae, PAPM demonstrated the strongest antibacterial activity among the carbapenems with a MIC range of < or =0.06 to 0.12 microg/mL. Against Enterobacteriaceae, MEPM showed the strongest antibacterial activity, and PAPM had comparable activity to IPM. Against the extended-spectrum beta-lactamase producing Escherichia coli, Klebsiella species and Proteus species, the MICs for the cephems were high, however, those for the carbepenems were low. Against H. influenzae, PAPM had comparable activity to IPM. With respect to anaerobes, each of the carbapenems tested demonstrated almost the same strong antibacterial activity. In conclusion, 13 years has passed since PAPM was launched in 1993, PAPM still maintains potent antibacterial activity and is considered an effective antimicrobial agent for various types of infectious diseases.

Potent in vitro activity of tomopenem (CS-023) against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Tomopenem (formerly CS-023) is a novel 1beta-methylcarbapenem with broad-spectrum coverage of gram-positive and gram-negative pathogens. Its antibacterial activity against European clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was compared with those of imipenem and meropenem. The MICs of tomopenem against MRSA and P. aeruginosa at which 90% of the isolates tested were inhibited were 8 and 4 microg/ml, respectively, and were equal to or more than fourfold lower than those of imipenem and meropenem. The antibacterial activity of tomopenem against MRSA was correlated with a higher affinity for the penicillin-binding protein (PBP) 2a. Its activity against laboratory mutants of P. aeruginosa with (i) overproduction of chromosomally coded AmpC beta-lactamase; (ii) overproduction of the multidrug efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN; (iii) deficiency in OprD; and (iv) various combinations of AmpC overproduction, MexAB-OprM overproduction, and OprD deficiency were tested. The increases in the MIC of tomopenem against each single mutant compared with that against its parent strain were within a fourfold range. Tomopenem exhibited antibacterial activity against all mutants, with an observed MIC range of 0.5 to 8 microg/ml. These results suggest that the antibacterial activity of tomopenem against the clinical isolates of MRSA and P. aeruginosa should be ascribed to its high affinity for PBP 2a and its activity against the mutants of P. aeruginosa, respectively.

In vitro and in vivo antibacterial activities of CS-023 (RO4908463), a novel parenteral carbapenem.

CS-023 (RO4908463, formerly R-115685) is a novel 1beta-methylcarbapenem with 5-substituted pyrrolidin-3-ylthio groups, including an amidine moiety at the C-2 position. Its antibacterial activity was tested against 1,214 clinical isolates of 32 species and was compared with those of imipenem, meropenem, ceftazidime, ceftriaxone, ampicillin, amikacin, and levofloxacin. CS-023 exhibited a broad spectrum of activity against gram-positive and -negative aerobes and anaerobes, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, penicillin-resistant Streptococcus pneumoniae (PRSP), beta-lactamase-negative ampicillin-resistant Haemophilus influenzae, and Pseudomonas aeruginosa. CS-023 showed the most potent activity among the compounds tested against P. aeruginosa and MRSA, with MICs at which 90% of isolates tested were inhibited of 4 microg/ml and 8 microg/ml, respectively. CS-023 was stable against hydrolysis by the beta-lactamases from Enterobacter cloacae and Proteus vulgaris. CS-023 also showed potent activity against extended-spectrum beta-lactamase-producing Escherichia coli. The in vivo efficacy of CS-023 was evaluated with a murine systemic infection model induced by 13 strains of gram-positive and -negative pathogens and a lung infection model induced by 2 strains of PRSP (serotypes 6 and 19). Against the systemic infections with PRSP, MRSA, and P. aeruginosa and the lung infections, the efficacy of CS-023 was comparable to those of imipenem/cilastatin and vancomycin (tested against lung infections only) and superior to those of meropenem, ceftriaxone, and ceftazidime (tested against P. aeruginosa infections only). These results suggest that CS-023 has potential for the treatment of nosocomial bacterial infections by gram-positive and -negative pathogens, including MRSA and P. aeruginosa.

Antifungal activities of R-135853, a sordarin derivative, in experimental candidiasis in mice.

The activities of R-135853, a novel sordarin derivative that possesses a 1,4-oxazepane ring moiety, were evaluated in vitro and in vivo. R-135853 exhibited potent in vitro activities against Candida albicans (fluconazole-susceptible strains), Candida glabrata, Candida tropicalis, and Cryptococcus neoformans, with MICs at which 90% of isolates were inhibited of 0.03, 1, 0.5, and 0.5 microg/ml, respectively. R-135853 also exhibited potent activities against fluconazole-susceptible dose-dependent and fluconazole-resistant strains of C. albicans, with MICs ranging from 0.03 to 0.06 mug/ml. However, R-135853 exhibited weak or no activity against Candida parapsilosis, Candida krusei, and Aspergillus spp. R-135853 exhibited dose-dependent efficacy against experimental murine hematogenous candidiasis induced by C. albicans when it was administered by both the subcutaneous and the oral routes and reduced viable cell counts in the kidneys significantly when it was administered at 50 mg/kg of body weight/dose (administration three times a day). In this model, R-135853 also exhibited dose-dependent efficacy by single oral administration. Subcutaneous administration of R-135853 exhibited dose-dependent efficacy against experimental murine esophageal candidiasis induced by fluconazole-resistant C. albicans, against which fluconazole at 50 mg/kg/dose was ineffective, and reduced viable cell counts in the esophagus significantly when it was administered at 10 and 50 mg/kg/dose. R-135853 eradicated C. albicans from the esophagi of one and four of five mice when it was administered at 10 and 50 mg/kg/dose, respectively. These results suggest that R-135853 is promising for the treatment of disseminated or mucosal candidiasis, including fluconazole-refractory infections.

Activity of capuramycin analogues against Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare in vitro and in vivo.

OBJECTIVES: The antimycobacterial activities of RS-112997, RS-124922 and RS-118641, three capuramycin analogues that inhibit phospho-N-acetylmuramyl-pentapeptide translocase, were tested against clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare. METHODS AND RESULTS: MICs were determined by the broth microdilution method using a modified Middlebrook 7H9 broth. RS-118641 was the most potent compound overall. The MIC50/90 (mg/L) results for RS-118641 were: M. tuberculosis, 1/2; multidrug-resistant (MDR) M. tuberculosis, 0.5/2; M. avium, 4/8; and M. intracellulare, 0.06/0.5. No statistically significant differences in MIC distributions were observed between non-MDR and MDR M. tuberculosis for any of the capuramycin analogues tested. In order to evaluate the therapeutic efficacy of RS-112997 and RS-124922 in a murine lung model of tuberculosis, both compounds were administered intranasally at 0.1 or 1 mg/mouse/day for 12 days. The mycobacterial load in the lungs was significantly lower in all treatment groups than in the untreated controls. Additional experiments were performed to evaluate the therapeutic efficacy of the three compounds against the M. intracellulare infection in mice. All compounds were administered intranasally at 0.1 mg/mouse/day for 21 days. The mycobacterial load in the lungs was significantly lower in all treatment groups than in the untreated controls. CONCLUSIONS: These results suggest that capuramycin analogues exhibit strong antimycobacterial potential and should be considered for further evaluation in the treatment of M. tuberculosis and M. avium-M. intracellulare complex infections in humans.

Synthesis and antimycobacterial activity of capuramycin analogues. Part 1: substitution of the azepan-2-one moiety of capuramycin.

Capuramycin analogues with a variety of substituents in place of the azepan-2-one moiety were synthesized from A-500359E and were tested for their translocase I inhibitory activity and in vitro antimycobacterial activity. Phenyl-type moieties were found to be effective substituents for capuramycin analogues.

Synthesis and antimycobacterial activity of capuramycin analogues. Part 2: acylated derivatives of capuramycin-related compounds.

Acylated derivatives of capuramycin and A-500359A were synthesized and tested for antimycobacterial activity. Compound 20 having a decanoyl group showed very potent activity.

[Rapid bactericidal activities of carbapenems against Pseudomonas aeruginosa in simulating human plasma concentrations].

The rapid bactericidal activities of panipenem (PAPM), imipenem (IPM), and meropenem (MEPM) against Pseudomonas aeruginosa were investigated by using in vitro pharmacodynamic model simulating the human plasma concentrations after intravenous drip infusion at 500 mg for 0.5 hours. Against P. aeruginosa PAO1, PAPM and IPM showed rapider reduction in viable cell counts than MEPM at 0.5 hours after exposure. All drugs showed more than 3 log10 reduction in viable cell counts at 2 hours after exposure and bacterial regrowth was not observed throughout 6 hours. The initial bactericidal activities of the drugs against 4 clinical isolates within 1 hour after exposure were also investigated by the same method. Against P. aeruginosa strain 12,475, the 3 drugs showed similar initial bactericidal activity but PAPM and IPM showed stronger initial bactericidal activity than MEPM against the other strains as did against P. aeruginosa PAO1. The morphological change of a strain 12,489, for which the initial bactericidal activities were different largely, after 0.5 hours exposure to simulated drug-concentrations was investigated by scanning electron microscope. PAPM and IPM induced morphological changes in most of the cells and cell lysis and bulge formation. On the other hand, MEPM induced changes of the surface structure of cells and slightly elongated cells, but not cell lysis.