Anticancer drug development from traditional cytotoxic to targeted therapies: evidence of shorter drug research and development time, and shorter drug lag in Japan.

WHAT IS KNOWN AND OBJECTIVE: Concern about the drug lag, the delay in marketing approval between one country and another, for anticancer drugs has increased in Japan. Although a number of studies have investigated the drug lag, none has investigated it in relation to the transition of anticancer therapy from traditional cytotoxic drugs to molecularly targeted agents. Our aim was to investigate current trend in oncology drug lag between the US and Japan and identify oncology drugs approved in only one of the two countries. METHODS: Publicly and commercially available data sources were used to identify drugs approved in the US and Japan as of 31 December 2010 and the data used to calculate the drug lag for individual drugs. RESULTS AND DISCUSSION: Fifty-one drugs were approved in both the US and Japan, whereas 34 and 19 drugs were approved only in the US or Japan, respectively. Of the 19 drugs approved only in Japan, 12 had not been subject to development for a cancer indication in the US, and all were approved before 1996 in Japan. Of the 34 drugs approved only in the US, 20 had not been subject to development in Japan, and none was in the top 25 by annual US anticancer drug-class sales. For drugs approved in both countries, the mean approval lag of the molecularly targeted drugs (MTDs) was significantly shorter than that of the non-molecularly targeted drugs (non-MTDs) (3·3 vs. 5·4 years). Further, mean R&D time of the MTDs was significantly shorter than that of non-MTDs (10·0 vs. 13·7 years). The price of MTDs had increased on average by 6·6% annually in the US, whereas it had decreased on average by 4·3% biyearly in Japan. WHAT IS NEW AND CONCLUSION: The emergence of new molecularly targeted agents has contributed to reducing the approval lag, most likely due to improvements in R&D strategy.

Role of kidney-specific organic anion transporters in the urinary excretion of methotrexate.

BACKGROUND: High-dose folinic acid is used to accelerate methotrexate elimination to avoid renal toxicity of the drug. The present study was carried out to examine the role of the renal organic anion transporters OAT-K1 and OAT-K2 in the urinary excretion of methotrexate, especially in the methotrexate-folinic acid rescue therapy. METHODS: Madin-Darby canine kidney cells stably expressing OAT-K1 and OAT-K2 were used for the in vitro transport study; 5/6 nephrectomized rats were used to detect changes in mRNA expression levels of OAT-K1 and OAT-K2 and to evaluate methotrexate pharmacokinetics under conditions of renal insufficiency. RESULTS: Methotrexate efflux mediated by these transporters in stable transfectants was stimulated in the presence of extracellular folic acid and folinic acid, suggesting that they could serve as anion exchangers to enhance the apical efflux of methotrexate. The mRNA expression levels of OAT-K1 and OAT-K2 were markedly diminished after 5/6 nephrectomy, but those of multidrug resistance associated protein 2, which could transport methotrexate, were maintained. Renal clearance of methotrexate was markedly decreased in 5/6 nephrectomized rats compared with that in sham-operated rats. Additional folinic acid treatment resulted in a significant increase in methotrexate renal clearance in sham-operated rats but not in 5/6 nephrectomized rats. CONCLUSIONS: The decreased expressions of OAT-K1 and OAT-K2 may be attributable to the longer exposure to methotrexate and ineffective folinic acid rescue. In terms of contributing to patient safety, renal clearance of methotrexate, especially folinic acid-stimulated tubular secretion of the drug via these transporters, would be a key factor in methotrexate therapy.

Effects of multinutrient supplementation on antioxidant defense systems in healthy human beings.

Oxidative damage involved in the pathogenesis of many diseases, such as cardiovascular disease, cancer and diabetics. The antioxidant defense system plays an important role in protecting body from oxidative damage. Numerous studies have been shown that a single vitamin or mineral supplementation has the beneficial effect on the antioxidant defense system. However, the overall combined effect of multinutrient supplementation on antioxidant defense system remains to be clarified. In the present double blind, placebo-controlled study, the antioxidative defense system was measured in 34 healthy subjects before and after multinutrient supplementation. Plasma vitamin C, E and beta-carotene, erythrocyte vitamin E, as well as whole blood selenium all showed increase at 5 weeks of supplementation. The activities of catalase (CAT) and glutathione peroxidase (GPX), but not superoxide dismutase (SOD), as well as GSH level were significantly increased at 16 weeks of supplementation. Moreover, the resistance of erythrocytes to 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidation was elevated at 5 weeks after supplementation. These results clearly demonstrated that short-term supplementation (16 weeks) with multinutrient could markedly improve antioxidative vitamin status and enzymatic activities. These improvements also led to the reduction of RBC susceptibility to free radial peroxidation.

cDNA cloning and characterization of vascular apoptosis-inducing protein 1.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.

[Japanese cedar pollinosis in infants in the allergy clinic].

Recently, the number of patients with Japanese cedar (Cryptomeria japonica) pollinosis has increased, especially in children. However, little is known about the incidence in infants. We studied on the rate of sensitization and the onset of pollinosis in children under 6 years old. The percentage of positive CAP-RAST to Japanese cedar pollen was 27.6%, in 76 infants (51 male and 25 female, 2 months-5 years old) who visited National Mie Hospital pediatric allergy clinic due to bronchial asthma and/or atopic dermatitis. The youngest child who has been sensitized to pollen was 1 year 8 month old boy. The percentage of positive rate of CAP-RAST to house dust mite was 61.8%. Twenty-seven infants (20 male and 7 female, 2-5 years of age) were diagnosed as Japanese cedar pollinosis in National Mie Hospital Otorhinolaryngology clinic in 1999 and 2000. The youngest child with pollinosis was 2 year 5 month old boy. Most of the 27 infants complained of rhinorrhea and/or eye symptoms and some of them complained cough, snoring, or epistaxis. About 40% were sensitized to Japanese cedar and/or cupressaceae pollen alone, 60% were also sensitized to house dust mite. In conclusion, it is possible that the sensitization to Japanese cedar pollen occurs after 2 season of pollen exposure and pollinosis occurs in 2 years old. Japanese cedar pollen has been an important allergen not only in school children, but also in infants.

Possible involvement of activator protein-1 DNA binding in mechanisms underlying ischemic tolerance in the CA1 subfield of gerbil hippocampus.

Transcription factors are nuclear proteins with an ability to recognize particular nucleotide sequences on double stranded genomic DNAs and thereby modulate the activity of RNA polymerase II which is responsible for the formation of messenger RNAs in cell nuclei. Gel retardation electrophoresis revealed that transient forebrain ischemia for 5 min led to drastic potentiation of binding of a radiolabelled double-stranded oligonucleotide probe for the transcription factor activator protein-1, in the thalamus as well as the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils previously given ischemia for 2 min two days before, which is known to induce tolerance to subsequent severe ischemia in the CA1 subfield. By contrast, ischemia for 5 min resulted in prolonged potentiation of activator protein-1 binding in the vulnerable CA1 subfield of the gerbils with prior ischemia for 5 min 14 days before, which is shown to induce delayed death of the pyramidal neurons exclusively in this subfield. Similar prolongation was seen with activator protein-1 binding in the vulnerable thalamus but not in the resistant CA3 subfield and dentate gyrus of the gerbils with such repeated ischemia for 5 min. Limited proteolysis by Staphylococcus aureus V8 protease as well as supershift assays using antibodies against c-Fos and c-Jun proteins demonstrated the possible difference in constructive partner proteins of activator protein-1 among nuclear extracts of the CA1 subfield obtained from gerbils with single, tolerated and repeated ischemia. These results suggest that de novo protein synthesis may underlie molecular mechanisms associated with acquisition of the ischemic tolerance through modulation at the level of gene transcription by activator protein-1 composed of different constructive partner proteins in the CA1 subfield. Possible participation of glial cells in the modulation is also suggested in particular situations.

Erythropoietin prevents place navigation disability and cortical infarction in rats with permanent occlusion of the middle cerebral artery.

Erythropoietin (EPO) prevents the ischemia-induced delayed neuronal death in the hippocampal CA1 field in gerbils. EPO receptor (EPOR) is also expressed in the cerebral cortex but its function is not known. To examine whether EPO has a neuroprotective action in the cortex, EPO was infused into the cerebroventricles of stroke-prone spontaneously hypertensive rats with permanent occlusion of the left middle cerebral artery. Morris water maze test indicated that EPO infusion alleviated the ischemia-induced place navigation disability. The left (ischemic)-to-right (contralateral nonischemic) (L/R) ratio of cerebrocortical area in the EPO-infused ischemic group was larger than that in the vehicle-infused ischemic group. The occlusion caused secondary thalamic degeneration but infusion of EPO prevented the decrease in the L/R ratio of thalamic area and supported neuron survival in the ventroposterior thalamic nucleus. In situ hybridization indicated that EPOR mRNA was upregulated in the periphery (ischemic penumbra) of a cerebrocortical infarct after occlusion of the middle cerebral artery, suggesting that an increased number of EPOR in neurons facilitates the EPO signal transmission, thereby preventing the damaged area from enlarging.

[Japanese cedar pollinosis in children in our allergy clinic].

Recently, it is pointed out that the incidence of Japanese cedar pollinosis has increased in children. We studied on the rate of sensitization and the onset of the disease in children under sixteen who visited allergy clinic in the Department of Otorhinolaryngology in Mie University Hospital. The percentage of positive rate of skin test and IgE antibodies of house dust mite was about 80 to 90% in 1981, 1991, and 1996. However, the percentage of positive rate of skin test and IgE antibodies of Japanese cedar pollen was 43% and 26%, respectively in 1981, but both increased to 58% in 1996. Most of Japanese cedar pollinosis patients also had house dust mite allergy. Among 115 pediatric patients who visited our allergy clinics in the past seven years, 32.2% were allergic to house dust mite alone, 8.6% were allergic to Japanese cedar pollen alone, 40.9% were allergic to both, and 6.1% were allergic to house dust mite, Japanese cedar pollen, and orchard grass pollen. 68% of the total 115 patients were boys, but significantly more girls had the pollinosis. 17.4% of those who are sensitized to the pollen were asymptomatic during the pollen season. Thus, it was confirmed that the rate of children sensitized to the pollen has apparently increased for the past twenty years. We should take care of those children who are sensitized to the pollen but asymptomatic during the pollen season.

[A case of far-advanced gastric cancer treated with neoadjuvant combination chemotherapy of UFT, low-dose CDDP and leucovorin, followed by subtotal gastrectomy with curative intent].

We herein present a case of a 70-year-old man with the tentative diagnosis of far-advanced gastric cancer supposed to be beyond surgical intervention. Neoadjuvant chemotherapy enabled us to perform subtotal gastrectomy with curative intent. The man was admitted to our hospital with the chief complaint of poor appetite. Because preoperative examinations revealed a mass adjacent to the portal vein and common bile duct, which was suspected to be lymphnode metastasis or gastric cancer directly invading those vital structures, 4 weeks of neoadjuvant combination chemotherapy (NACC) (CDDP 10 mg/body, day 1 through day 5/week, UFT 600 mg/body, every day, Leucovorin 15 mg/body, every day) was given with resultant curative resection of the tumor one month after completion of NACC.

Simplified method for the determination of 25-hydroxy and 1alpha,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma. Application to nutritional studies.

A simplified method for the determination of 25-hydroxy and 1alpha,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C18OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1alpha,25-dihydroxyvitamin D [1,25(OH)2D] fractions. The 25-OH-D fraction was purified by a Bond Elut C18 cartridge and 25-OH-D2 and 25-OH-D3 were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)2D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)2D2 and 1,25(OH)2D3 fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies.

[The pollen count of Cryptomeria japonica and nasal symptom of Cryptomeria japonica pollinosis patients in recent three years in Mie Prefecture].

Airborne scattering of Cryptomeria japonica pollen depends on climate. Total pollen count and scattering pattern varies from one year to another. We counted Cryptomeria japonica pollen in February and March from 1993 to 1995 at several points in Mie Prefecture. One-third to two-thirds of all the patients who visited our clinic for the first time in the pollen period were occupied by allergic patients. The pollen count in 1995 amounted to 17943/cm2 at Tsu and 99512/cm2 in the whole prefecture, which is the largest in the past 12 years. The number of days in which patients severely suffered was correlated to the pollen count for the past three years. It was shown that nasal symptoms developed after certain amount of pollen had scattered, but the severity of symptoms thereafter largely depended on the total pollen count. At Tsu in 1995, we scarcely had pollen scattering in February but had tremendous amount of pollen count in March. Percentage of development of nasal symptoms among thirty patients was well correlated to the logarithm of accumulated pollen count. When accumulated pollen count reached to 100/cm2, half the patients developed symptoms. Most of the patients developed symptoms when it reached to 1000/cm2. Since there is a great interindivisual difference in when and how much patients have nasal symptoms, the choice of therapy should be based not only on the pollen count but also on the symptom and condition of each patient.

Radioprotective effects of antioxidative plant flavonoids in mice.

Radioprotective effects of tea infusions and plant flavonoids were investigated by using the micronucleus test for anticlastogenic activity and the thiobarbituric acid assay for antioxidative activity. A single gastric intubation of rooibos tea (Aspalathus linearis) infusion at 1 ml per mouse 2 h prior to gama-ray irradiation (1.5 Gy) reduced the frequency of micronucleated reticulocytes (MNRETs). After the fractionation of rooibos tea infusion, the flavonoid fraction was found to be most anticlastogenic and antioxidative. From this fraction, luteolin was isolated as an effective component. Then, anticlastogenic effects of 12 flavonoids containing luteolin and their antioxidative activities against lipid peroxidation by Fenton's reagent were examined. A good correlation (r=0.717) was observed between both activities. Luteolin showed the most effective potency. A gastric intubation of luteolin (10 micromoles/kg) 2 h prior to gamma-ray irradiation (6 Gy) suppressed lipid peroxidation in mouse bone marrow and spleen and a trend of protective effect of luteolin against the decrease of endogenous ascorbic acid in mouse bone marrow after gamma-ray irradiation (3 Gy) was observed. These results suggest that plant flavonoids, which show antioxidative potency in vitro, work as antioxidants in vivo and their radioprotective effects may be attributed to their scavenging potency towards free radicals such as hydroxyl radicals. Therefore, the flavonoids contained in tea, vegetables and fruits seem to be important as antioxidants in the human diet.

[The evaluation of hyposensitization with sugi pollen extracts in patients with nasal allergy to Japanese sugi pollen].

We evaluated the clinical and pathophysiological efficacy of hyposensitization with Japanese sugi pollen extracts to sugi pollinosis. Twenty patients with Japanese cedar pollinosis received hyposensitization with Japanese cedar pollen extracts for one to 15 years with a mean of 9.5 years were compared with 18 patients without hyposensitization. The improvement of nasal symptoms was not found in patients with hyposensitization of sugi pollen extracts compared with patients with non-hyposensitization, but the number of mast cells in nasal scraping, nasal hypersensitivity to histamine, and specific sugi IgG4 antibody in serum was improved significantly compared with non-hyposensitization group. These findings suggest that the continuation of hyposensitization with sugi pollen extracts is recommendable as a basic treatment against seasonal nasal allergy.

Rice protein 16KD--a major allergen in rice grain extract.

The allergenic activity of Rice protein 16 KD (RP16KD) isolated from water soluble rice proteins was examined by radioallergosorbent test (RAST), RAST inhibition and histamine release assay. All of the 31 sera which showed positive RAST values for rice grain extract were positive for RP16KD RAST. Furthermore, there was a significant correlation (r = 0.56, p less than 0.01) between these RAST values. PR16KD effectively inhibited IgE binding to the rice grain extract disc in RAST inhibition assays using 4 sera with positive RAST values for both antigens. In 17 subjects with positive RAST values for rice grain extract, a significant positive correlation (r = 0.53, p less than 0.05) was found between the maximum percent histamine releases from their leukocytes by rice grain extract and RP16KD. These data strongly suggest that RP16KD is one of the major allergens of rice grain.

[Allergic individuals to Japanese cedar or orchard grass consist of two subgroups based on the sensitization to Dermatophagoides pteronyssinus].

One hundred and twenty-six allergic subjects were divided into three groups based on the RAST results for Dermatophagoides pteronyssinus (D.p) and Japanese cedar (J.C.). We compared sex, age of examination for RAST, serum IgE, family history of allergic diseases, prevalence rate of bronchial asthma (BA) and allergic rhinitis (AR), and onset of AR among three groups. No significant difference in the other variables except serum IgE and prevalence rate of BA was found between the group with D.p-positive and J.C.-positive RAST and the group with D.p-positive and J.C.-negative RAST. The group with positive RAST for D.p and J.C. showed lower age of the examination, higher serum IgE, higher incidence of allergic family history, higher prevalence rate of BA, and lower onset age of AR than the group with negative RAST for D.p and positive RAST for J.C. We suggested that in J.C. RAST-positive group there might be two different subgroups distinguished by RAST result for D.p Similar results were obtained from the assessment of RAST for Orchard grass instead of J.C.

Bone marrow and serum concentrations of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1 alpha,25-dihydroxyvitamin D in patients with leukemia and normal subjects.

Concentrations of 25-hydroxyvitamin D (25-OH-D), 24,25-dihydroxyvitamin D [24,25(OH)2D], and 1 alpha,25-dihydroxyvitamin D [1,25(OH)2D] in bone marrow and serum of patients with leukemia and normal subjects were assayed. There were highly significant correlations between the bone marrow and serum concentrations of the respective vitamin D metabolites. Especially, the concentrations of 25-OH-D and 1,25(OH)2D in the bone marrow gave very similar values to those in serum. This is a big advantage in controlling the bone marrow levels of vitamin D metabolites in patients with leukemia, because doctors can calculate the bone marrow levels from the serum levels of the respective vitamin D metabolites without bone marrow aspiration. When 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3) was administered orally to eight patients with leukemia, clinical conditions were improved in seven patients: four complete remissions (CR), one partial response (PR), and two minor responses (MR) without severe hypercalcemia. The results suggest that the therapy with 1 alpha-OH-D3 is fairly effective for curing human leukemia although it is not dramatic.

Changes in the concentrations of vitamin D and its metabolites in the plasma of healthy subjects orally given physiological doses of vitamin D2 by multivitamin or vitamin D preparations.

Changes in the concentrations of vitamin D and its metabolites in plasma of healthy subjects orally given physiological doses of vitamin D2 by multivitamin or vitamin D liquid preparations were determined and the bioavailability of vitamin D was studied. Separative assay on the D2 and D3 compounds of vitamin D, 25-hydroxyvitamin D (25-OH-D), 24R,25-dihydroxyvitamin D [24,25(OH)2D], and 1 alpha,25-dihydroxyvitamin D [1,25(OH)2D] was performed in plasma of eight healthy male volunteers. When the concentrations of vitamin D and its metabolites in plasma of volunteers were assayed after daily oral administration of 400 IU of vitamin D2 in a form of multivitamin tablet for 1 week, the variations of vitamin D3 and its metabolites in plasma levels were very small. In contrast, the concentrations of 25-OH-D2 and 1,25(OH)2D2 slightly increased after the administration, while neither vitamin D2 nor 24,25(OH)2D2 was detected. A single dose of 4,000 IU of vitamin D2 was orally given to the volunteers in a form of a vitamin D liquid preparation and the hourly variations were observed during 24 h. These concentrations of vitamin D2, 25-OH-D2, and 1,25(OH)2D2 were slightly higher than those of the repeated doses. The result suggests that even the high dose of 4,000 IU has little effect on the plasma levels of vitamin D2 and its metabolites by a single dose, indicating a low risk for hypervitaminosis D.