[Current Topics on Vitamin D. Protein interaction and biological action of active vitamin D compounds].

To express its biological action, active vitamin D3 (1α,25-dihydroxyvitamin D3) interacts with three proteins : vitamin D binding protein (DBP), vitamin D receptor (VDR), and CYP24A1. This article explains how eldecalcitol interacts with these three proteins to achieve its mode of action. The main feature of eldecalcitol's molecular structure is the 3-hydroxypropyloxy group (3-HP group) at the 2ß-position. When interacting with DBP, eldecalcitol tightly binds to DBP via the 3-HP group and enhances systemic stability of eldecalcitol. By binding to VDR, the 3-HP group stabilizes the eldecalcitol-VDR complex, which leads to high efficacy. The 3-HP group also interferes with the binding to CYP24A1, which induces residence to CYP24A1 and liver metabolism. These protein interactions constitute the mode of action of eldecalcitol.

Effects of fluorines on nonsecosteroidal vitamin D receptor agonists.

From our research of nonsecosteroidal vitamin D(3) derivatives with gamma hydroxy carboxylic acid, we identified compound 6, with two CF(3) groups in the side chain, as a most potent vitamin D receptor (VDR) agonist that shows superagonistic activity in VDRE reporter gene assay, MG-63 osteocalcin production assay and HL-60 cell differentiation assay. Compound 6 demonstrated that fluorination is as effective in the case of our nonsecosteroidal scaffold as in the case of secosteroidal VD(3) analogs. X-ray analysis of the VDR with compound 6 revealed all of the six fluorine atoms of the hexafluoropropanol (HFP) moiety in the side chain effectively interacting with the VDR by both steric (van der Waals) and electrostatic (hydrogen bond, NH-F and CH-F) interactions. The HFP moiety of 6 effectively interacts with helix 12 (H12) of the VDR and stabilizes the position and the orientation of H12, which could result in stabilizing the coactivator and enhancing the VDR agonistic activity.

A series of nonsecosteroidal vitamin D receptor agonists for osteoporosis therapy.

In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonsecosteroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)2D3 had been replaced by aryl acetic acid. These analogs showed very potent activity in vitro compared with 1,25(OH)2D3. An X-ray analysis of 8d showed that the inserted phenyl ring well mimicked the folded methylene linker of the gamma hydroxy carboxylic acid moiety but the carboxylic acid of 8d interacted with VDR in a different manner from gamma hydroxy carboxylic acids. Through our in vivo screening in an osteoporosis rat model using immature rats, we identified a potent active vitamin D3 analog, compound 7e. In mature rats of the same model, compound 7e also showed good PK profiling and excellent ability to prevent bone mineral density loss without severe hypercalcemia. Our nonsecosteroidal VDR agonist 7e (CH5036249) could be a possible new drug candidate for treating osteoporosis in human.

The Effects of Topiroxostat, a Selective Xanthine Oxidoreductase Inhibitor, on Arterial Stiffness in Hyperuricemic Patients with Liver Dysfunction: A Sub-Analysis of the BEYOND-UA Study.

BACKGROUND: The effects of uric acid (UA)-lowering therapy with xanthine oxidoreductase (XOR) inhibitors on the development of cardiovascular diseases remain controversial. Based on recent findings that plasma XOR activity increased in liver disease conditions, we conducted a sub-analysis of the BEYOND-UA study to examine the differential effects of topiroxostat on arterial stiffness based on liver function in hyperuricemic individuals with hypertension. METHODS: Sixty-three subjects treated with topiroxostat were grouped according to baseline alanine aminotransferase (ALT) levels (above or below cut-off values of 22, 30, or 40 U/L). The primary endpoint was changes in the cardio-ankle vascular index (CAVI) from baseline to 24 weeks. RESULTS: Significant reductions in CAVI during topiroxostat therapy occurred in subjects with baseline ALT ≥30 U/L or ≥40 U/L, and significant between-group differences were detected. Brachial-ankle pulse wave velocity significantly decreased in the ALT-high groups at all cut-off values. Reductions in morning home blood pressure and serum UA were similar regardless of the baseline ALT level. For eleven subjects with available data, ALT-high groups showed high plasma XOR activity, which was significantly suppressed by topiroxostat. CONCLUSIONS: Topiroxostat improved arterial stiffness parameters in hyperuricemic patients with liver dysfunction, which might be related to its inhibitory effect on plasma XOR.

Systematic SAR study of the side chain of nonsecosteroidal vitamin D3 analogs.

A series of nonsecosteroidal vitamin D(3) analogs with carboxylic acid were explored. Through our systematic SAR studies on the side chain moiety, compound 6b was identified as the optimal compound showing excellent vitamin D receptor (VDR) agonistic activity. Compound 6b had the diethyl group in the terminal which was bound by (E)-olefin linker to the bisphenyl core. Calculating the volume of the side chain showed that the diethyl group in 6b filled the hydrophobic region of VDR with the ideal packing coefficient based on the 55% rule, and that this resulted in the most potent in vitro activity.

Discovery of novel tetracyclic compounds as anaplastic lymphoma kinase inhibitors.

Anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is considered a promising therapeutic target for human cancers. We identified novel tetracyclic derivatives as potent ALK inhibitors. Among them, compound 27 showed strong cytotoxicity against KARPAS-299 with an IC(50) value of 21 nM and significant antitumor efficacy in ALK fusion-positive blood and solid cancer xenograft models in mice without body weight loss.

Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis.

Novel vitamin D(3) analogs with carboxylic acid were explored, focusing on a nonsecosteroidal analog, LG190178, with a bisphenyl skeleton. From X-ray analysis of these analogs with vitamin D receptor (VDR), the carboxyl groups had very unique hydrogen bonding interactions in VDR and mimicked 1α-hydroxy group and/or 3ß-hydroxy group of 1α,25-dihydroxyvitamin D(3). A highly potent analog, 6a, with good in vitro activity and pharmacokinetic profiles was identified from an SAR study. Compound 6a showed significant prevention of bone loss in a rat osteoporosis model by oral administration.

Design and synthesis of peptidomimetic factor VIIa inhibitors.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. In previous reports, we described a S3 subsite found in the X-ray crystal structure of compound 2 that bound to FVIIa/soluble tissue factor (sTF). Based on the X-ray crystal structure information and with the aim of improving the inhibition activity for FVIIa/TF and selectivity against other serine proteases, we synthesized derivatives by introducing substituents at position 5 of the indole ring of compound 2. Among them, compound 16 showed high selectivity against other serine proteases. Contrary to our expectations, compound 16 did not occupy the S3-subsite; X-ray structure analysis revealed that compound 16 improved selectivity by forming hydrogen bonds with Gln217, Thr99 and Asn100.

Successful pregnancy after intracytoplasmic sperm injection with testicular spermatozoa transported only under refrigeration.

PURPOSE: This case report describes two successful pregnancies after intracytoplasmic sperm injection (ICSI) with testicular spermatozoa that were transported under refrigeration. METHODS: Two first-time couples consulted our clinic concerned about their primary infertility. No sperm were present in the semen samples from either of the husbands and they were referred to the urology department (UD) of a neighbouring hospital. At the UD, seminiferous tubules were obtained by testicular sperm extraction. The tissue samples were put in a centrifuge tube with phosphate-buffered saline at 6°C and placed with refrigerant in a cushioned styrofoam box that was then transported to our clinic. Immediately upon arrival at our clinic, testicular spermatozoa were extracted. On the same day, ovum pickup was performed and mature oocytes were extracted that were then inseminated by conventional ICSI. Fertilized eggs were cultured for 2 days, and then cleaved embryos were cryopreserved. In one case after 4 months and in the other case after 2 months of cryopreservation, the frozen-thawed embryos were transferred. RESULT: Both patients became pregnant and normal, healthy babies were born. CONCLUSIONS: These results suggest that cases of obstructive azoospermia can be treated with ICSI by refrigerated transport of the seminiferous tubules, in cooperation with a UD, in a small single departmental obstetrics and gynecology clinic.

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.

Selective TRK Inhibitor CH7057288 against TRK Fusion-Driven Cancer.

Members of the tropomyosin receptor kinase (TRK) family are expressed in their constitutively activated forms as a result of a gene fusion that occurs across a wide variety of cancer types. We have identified CH7057288 as a potent and selective TRK inhibitor that belongs to a novel chemical class. CH7057288 showed selective inhibitory activity against TRKA, TRKB, and TRKC in cell-free kinase assays and suppressed proliferation of TRK fusion-positive cell lines, but not that of TRK-negative cell lines. Strong in vivo tumor growth inhibition was observed in subcutaneously implanted xenograft tumor models of TRK fusion-positive cells. Furthermore, in an intracranial implantation model mimicking brain metastasis, CH7057288 significantly induced tumor regression and improved event-free survival. Recently, resistant mutations in the kinase domain of TRK have been reported in patients who show disease progression after treatment with the TRK inhibitors now under clinical development. Our compound maintained similar levels of in vitro and in vivo activity against one of these resistant mutants as it did to wild-type TRK. An X-ray crystal structure of the TRKA and CH7057288 complex supported the activity against the mutant. In addition, gene expression analysis revealed that CH7057288 suppressed MAPK and E2F pathways as downstream signaling of TRK fusion. Therefore, CH7057288 could be a promising therapeutic agent for TRK fusion-positive cancer.

Synthesis of putative metabolites of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71).

1alpha,25-Dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71), an analog of active vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is under phase III clinical trials in Japan for the treatment of osteoporosis and bone fracture prevention. Since ED-71 has a substituent at the 2beta-position of the A-ring, it is recognized that the metabolic pathway of ED-71 might be more complicated than 1,25(OH)(2)D(3) because of metabolism at the 2beta-position substituent in addition to the inherent metabolism of the side chain. To clarify the metabolism of hydroxypropoxy substituent of the 2beta-positon and a combination of metabolism between side chain and 2beta-positon, four putative metabolites of ED-71 have been prepared as authentic samples. The metabolites at the 2beta-positon, the methyl ester derivative considered as an ester standard of the oxidized metabolite and the tetraol derivative as the truncated metabolite were synthesized from alpha-epoxide, a key intermediate of ED-71 synthesis. The combination metabolites between side chain and 2beta-positon, the 24(S)- and 24(R)-pentaols were synthesized using Trost's convergent method.

Percentage of motile spermatozoa at 22 hours after swim-up procedure: An indicator for intracytoplasmic sperm injection?

OBJECTIVE: The decision to use in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or split insemination (IVF-ICSI) in the first cycle is based on the number of motile sperm. Hence, total fertilization failure (TFF) often occurs during IVF cycles, despite normozoospermia. To investigate whether the cumulative motile swim-up spermatozoa percentage at 22 hours post-insemination (MSPPI) is an indicator for ICSI, we analyzed TFF, fertilization, blastocyst development, chemical pregnancy, clinical pregnancy, and live birth rates. METHODS: This prospective study was performed using data obtained from 260 IVF cycles. At 22 hours after insemination, the remaining swim-up spermatozoa were observed and divided into six groups according to MSPPI (<10%, 10% to <30%, 30% to <50%, 50% to <70%, 70% to <90%, and 90% to 100%). RESULTS: Regardless of the ejaculated motile sperm concentration (0.6-280×10(6)/mL motile spermatozoa), the incidence of TFF significantly increased when MSPPI was <10%, and the fertilization rate significantly decreased when MSPPI was <30%. We found that cumulative MSPPI correlated with the cumulative fertilization rate (Spearman correlation, 0.508, p<0.001). Regarding embryo development, we observed no significant differences in the rates of blastocyst development, chemical pregnancy, clinical pregnancy, or live birth among all groups. CONCLUSION: Our findings suggest that MSPPI is a viable indicator for split IVF-ICSI and ICSI. Taken together, by employing the MSPPI test in advance before IVF, ICSI, or split IVF-ICSI cycles, unnecessary split IVF-ICSI and ICSI may be avoided.

Structure of human factor VIIa/tissue factor in complex with a peptide-mimetic inhibitor: high selectivity against thrombin by introducing two charged groups in P2 and P4.

The crystal structure of human factor VIIa/soluble tissue factor (FVIIa/sTF) in complex with a highly selective peptide-mimetic FVIIa inhibitor which shows 1670-fold selectivity against thrombin inhibition has been solved at 2.6 A resolution. The inhibitor is bound to FVIIa/sTF at the S1, S2 and S3 sites and at the additional S1 subsite. Two charged groups, the amidino group in P2 and the carboxylate group in P4, form ionic interactions with Asp60 and Lys192 of FVIIa, respectively. Structural comparisons between factor VIIa and thrombin show that thrombin has oppositely charged residues, Lys60F and Glu192, in the S2 site and the S1 subsites, respectively. These data suggest that the utilization of the differences of charge distribution in the S2 site and the S1 subsites between FVIIa and thrombin is critical for achieving high selectivity against thrombin inhibition. These results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.

Sural nerve sorbitol in patients with diabetic sensorimotor polyneuropathy.

OBJECTIVE: Nerve sorbitol levels have been measured in sural nerve biopsy samples from patients with diabetic sensorimotor polyneuropathy in several studies using different methods and measurement units. In this study, we compared the results of sorbitol assays to determine the required sensitivity of analytical methods for nerve sorbitol measurements. RESEARCH DESIGN AND METHODS: We performed a literature search using PaperChase for reports of nerve sorbitol in diabetic patients and selected those with nerve conduction studies to delineate the severity of nerve damage. RESULTS: In patients who had undergone a nerve conduction study, the standardized nerve sorbitol levels were 0.034-0.300 nmol/mg wet nerve. CONCLUSIONS: Our results showed the level of sensitivity required in laboratory methodology to perform this assay in the target population and aid in the planning of clinical research trials of aldose reductase inhibitor agents.

Eldecalcitol reduces osteoporotic fractures by unique mechanisms.

Eldecalcitol shows higher binding affinity for vitamin D-binding protein (DBP), tighter binding to vitamin D receptor (VDR), and resistance to metabolic degradation via 24-hydroxylation. In silico analysis of the mode of binding demonstrated that the 3-hydroxypropyloxy (3-HP) group of eldecalcitol offers additional hydrogen bond and CH-π interaction for the binding to DBP and VDR. However, the 3-HP group interferes with the binding of eldecalcitol to CYP24A1, causing poor metabolic clearance of eldecalcitol by this enzyme. These characteristics may contribute to the stronger effect of eldecalcitol than calcitriol. The present post-hoc analysis also demonstrate that the incidence of hypercalcemia and hypercalciuria is slightly higher in eldecalcitol than in alfacalcidol group especially in patients with CKD stage 3B, that both serum and urinary calcium return to the baseline levels shortly after cessation of the treatment in both treatment groups, that the incidence of urolithiasis is higher in patients with higher eGFR and is similar between alfacalcidol and eldecalcitol groups, and that eGFR is transiently reduced by both alfacalcidol and eldecalcitol treatment especially among patients with higher eGFR but recovers after the end of both treatment. Eldecalcitol can be used for the treatment of osteoporosis without Ca supplementation to reduce the incidence of hypercalcemia and hypercalciuria, and enough hydration is recommended in order to avoid hypercalcemia, urolithiasis and deterioration of renal function.

Multifunctional and potent roles of the 3-hydroxypropoxy group provide eldecalcitol's benefit in osteoporosis treatment.

Eldecalcitol (1α,25-dihydroxy-2ß-(3-hydroxypropoxy)vitamin D3, [developing code: ED-71]), a new osteoporosis treatment drug that was recently approved in Japan, is a best-in-class drug in the class of calcitriol (1α,25-dihydroxyvitamin D3) and its prodrug alfacalcidol (1α-hydroxyvitamin D3), which have been used to treat osteoporosis for 30 years. In a comparative Phase III clinical study with alfacalcidol in osteoporosis patients, eldecalcitol demonstrated superior efficacy in the endpoints of increment of bone mineral density and reduction of bone fracture with equivalent safety to alfacalcidol. Eldecalcitol was discovered by searching synthetic analogs of calcitriol and alfacalcidol, and its main structural characteristic is having the 3-hydroxypropoxy group at the 2ß-position. This review discusses why introducing the group leads to excellent efficacy and safety in osteoporosis treatment and elucidates the functional roles of the 3-hydroxypropoxy group. Briefly, the functional roles of the group are, first, realizing the metabolism switching in which eldecalcitol shows resistance to CYP24A1 and is metabolized in the liver; second, increasing the affinity to the serum carrier protein and prolonging the half-life to 53h; and third, stabilizing the eldecalcitol-receptor complex. Taken together, these functional roles of the 3-hydroxypropoxy group are beneficial in osteoporosis treatment. This review attempts to give a detailed account of the mode of action of eldecalcitol by clarifying these multifunctional roles of the 3-hydroxypropoxy group from the medicinal chemist's perspective.

Synthesis and preliminary biological evaluation of 20-epi-eldecalcitol [20-epi-1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3: 20-epi-ED-71].

Eldecalcitol [1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3, developing code: ED-71] is an analog of active vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] that possesses a hydroxypropoxy substituent at the 2beta-position of 1,25(OH)2D3. Eldecalcitol has potent biological effects on bone and is now in preparation for approval as a promising medicine for the treatment of osteoporosis in Japan. To explore chemical structure-biological activity relationships between eldecalcitol and related analogs, we have already synthesized 1-epi-eldecalcitol, 3-epi-eldecalcitol, and 1,3-diepi-eldecalcitol with inherent biological interests of each targeted analog and evaluated their biological responses. It has been reported that 20-epi-1,25(OH)2D3, a diastereomer of 1,25(OH)2D3 that possesses an inverted methyl substituent at the 20-position of the side chain, shows remarkably enhanced biological activities compared to parental compound, 1,25(OH)2D3. As a continuation of our modification studies on eldecalcitol, we took great interest in 20-epi-eldecalcitol and its biological responses. In this paper, the synthesis of 20-epi-eldecalcitol by the Trost coupling reaction between the A-ring fragment and the C/D-ring fragment as well as in vitro preliminary biological evaluation of 20-epi-eldecalcitol are described. In the induction of human myeloid leukemia cell (HL-60) differentiation, inhibition of the human histiocytic lymphoma cell (U937) proliferation, and increase in osteocalcin concentration in the human osteosarcoma cell (MG-63), 20-epi-eldecalcitol showed significantly enhanced activity compared to eldecalcitol.

Improvement of motor nerve conduction velocity in diabetic rats requires normalization of the polyol pathway metabolites flux.

Using ranirestat, an aldose reductase (AR) inhibitor, we investigated the relationship between sorbitol and fructose levels in the sciatic nerve and motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-treated diabetic rats. Ranirestat inhibited rat and recombinant human AR with similar IC50 values and equipotently prevented sorbitol accumulation in rat erythrocytes and sciatic nerves in vitro. One week after STZ administration, sorbitol levels in rat erythrocytes and sciatic nerves significantly increased while MNCV decreased. Oral administration of ranirestat (0.03-1.0 mg/kg per day) for 3 weeks dose-dependently decreased the elevated sorbitol and fructose levels in the rat sciatic nerves without affecting blood glucose level. Particularly, at doses of 0.1 mg/kg per day or higher, ranirestat normalized both sorbitol and fructose levels in the sciatic nerves of STZ-treated rats. Ranirestat (0.1-1.0 mg/kg per day) also improved the STZ-induced decrease in MNCV in a dose-dependent manner. This improvement correlated with the decrease of sorbitol and fructose levels in the rat sciatic nerves. These findings indicate that ranirestat improves MNCV via normalization of sorbitol and fructose accumulation in the sciatic nerve.