Rho-associated coiled-coil-forming protein kinase (ROCK) is pivotally involved in invasion by tumor cells and their evolution to metastasis. We have developed a novel inhibitor of ROCK, Wf-536 ...(+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride. In the present study, we investigated its effect on in vitro invasion and in vivo pulmonary metastasis of B16 melanoma.
The following were evaluated: the anti-invasive effect of Wf-536 against the motility of mouse B16BL6 melanoma cells through a culture insert layered with reconstituted basement membrane (Matrigel); the cytotoxic effect of Wf-536 in the same cell line; the antimetastatic effect of Wf-536, administered by osmotic pump, on spontaneous pulmonary metastasis following subcutaneous injection of B16BL6 melanoma in mice; and the inhibitory effect of orally administered Wf-536, alone or in combination with the antineoplastic drug paclitaxel, on pulmonary metastasis of intravenously injected B16F10 melanoma in mice.
Wf-536 inhibited in vitro invasion by B16BL6 cells significantly and in a concentration-dependent manner and displayed an anti-invasive effect under conditions of both chemotaxis and chemokinesis. No cytotoxic effect was observed at any of the concentrations used. In vivo, Wf-536 administration suppressed tumor colony formation on the lung surface in a dose-dependent manner (0.3-3 mg/kg per day), with a metastasis inhibition rate of 95% at 3 mg/kg per day. In experimental metastasis of B16F10 melanoma, oral administration of Wf-536 significantly decreased tumor colony formation in the lung, with an inhibition rate of 41% at 3 mg/kg per day. The inhibition rate of paclitaxel (5 mg/kg per day) was 27%. The combination of Wf-536 and paclitaxel produced a synergistic effect on B16F10 metastasis and a 68% inhibition rate. Wf-536 administration at the doses used did not alter body weight, blood pressure or the health of treated animals as compared to vehicle-treated controls.
The results suggest that Wf-536 is a potentially valuable drug for preventing tumor metastasis both in monotherapy and in combination with an antineoplastic drug.
Abstract 4571
Severe combined immunodeficiency (SCID) is the most severe form of the primary immunodeficiencies (PID), the most frequent type of which is X-linked SCID (X-SCID; T-B+NK-SCID). ...Hematopoietic stem cell transplantation (SCT) is the only curative treatment for these high-risk patients. Recently, some PID cases treated with unrelated cord blood stem cell transplantation (CBT) have been reported. To avoid severe infection during SCT and late complications, such as mental and growth retardation, reduced-intensity conditioning (RIC) regimens have been used for unrelated SCT for PID patients. However, reports of X-SCID patients receiving CBT with reduced-intensity stem cell transplantation (RIST) are quite limited. We report successful allogenic RIST from unrelated cord blood (CB) for treatment of X-SCID in our single centre. Three patients with X-SCID who had no HLA-matched sibling received unrelated CBT. Mutations in the common gamma chain gene were detected in all patients. Pre-transplantation conditioning for all patients consisted of fludarabine (FLU) (30 mg/m2/day) from day -7 to day -2 (total dose 180 mg/m2) and busulfan (BU) 4 mg/kg/day from day -3 to day -2 (total dose 8 mg/kg). All CB units were serologically matched HLA-A, B, and DR loci. Though two of the patients suffered from fungal or bacterial pneumonia before transplantation, there were no additional infectious complications during transplantation. All patients achieved engraftment with an absolute neutrophil count > 500/μ l with a mean of 22 days (range, 19–27 days), and they showed 100% donor chimerism by fluorescence in situ hybridization of their peripheral mononuclear cells using × and Y chromosome probes at one-year post transplantation. Full donor chimerism of both T and B cells was also confirmed. Only one patient (patient 2) developed grade III acute graft versus host disease (GVHD), which resolved with increased oral corticosteroid. None of the patients have developed chronic GVHD, received intravenous immunoglobulin replacements after the transplantations, or showed delayed psychomotor development during 21 to 77 months of follow-up. Only one patient (patient 3) had short stature (-2.4SD) at 21-month follow-up. The most commonly used RIC regimens worldwide have been the FLU/melphalan (LPAM) and the FLU/BU regimens. LPAM has broad stem cell toxicity to both primitive and committed stem cells, while BU may spare committed stem cells, resulting in early onset and a prolonged duration of neutropenia with the FLU/LPAM regimen. Moreover, it has been reported that the LPAM-containing conditioning regimen is a risk factor for hepatic veno-occlusive disease. Although conditioning regimens including standard-dose BU may be associated with a high rate of treatment-related complications due to organ toxicity, reduced-dose BU in combination with FLU is less myelosuppressive and less toxic than the FLU/LPAM regimen. Moreover, CBT has some advantages with regard to incidence and intensity of GVHD and availability compared with other alternative stem cell sources. Patients 1 and 2 had overcome pre-existing fungal and bacterial pneumonias, respectively, and recovered after transplantation. Thus, immediate SCT using an RIC regimen could be indicated for patients with X-SCID to reconstitute the immune system regardless of pre-existing infections. Since pre-existing infections are the principal risk factors for poor SCT outcomes, early diagnosis and transplantation before any infectious episodes is necessary for X-SCID patients. In conclusion, CBT is a suitable alternative to bone marrow transplantation for X-SCID patients requiring urgent SCT with no sibling donors. RIC consisting of FLU and BU is an effective and safe treatment for such cases.
No relevant conflicts of interest to declare.
We describe an 8-year-old girl with chronic active Epstein-Barr virus (EBV) infection (CAEBV) who was treated successfully by reduced-intensity stem cell transplantation (RIST) from unrelated cord ...blood (CB). She had been suffering from fever, abdominal pain, and interstitial lymphadenopathy, and CAEBV was diagnosed. After chemotherapy that included etoposide, the amount of EBV decreased transiently below the detection level. However, the disease due to CAEBV worsened despite the chemotherapy, and she finally needed chemotherapy every week. Therefore, instead of conventional myeloablative transplantation, we performed CB transplantation with reduced-intensity conditioning regimens consisting of low-dose total body irradiation, fludarabine, and etoposide. CB, for which human leukocyte antigen (HLA) was 2-loci mismatched on the DR loci from an unrelated donor, was infused after conditioning. Although grade III acute graft-versus-host disease (GVHD) in the gut and chronic GVHD in the lung developed, the symptoms of GVHD disappeared with immunosuppressive therapy. After 15 months, the patient remained a complete chimera, with undetectable levels of EBV in peripheral blood and bone marrow. We conclude that RIST from unrelated CB can be indicated for some cases of CAEBV who are refractory to chemotherapy and have no HLA-matched related and unrelated donors as the source of bone marrow or peripheral blood stem cells.
Summary
1. Rho‐associated coiled‐coil forming protein serine/threonine kinase (ROCK) is involved in the development of tumour metastasis. Wf‐536, (+)‐(R)‐4‐(1‐Aminoethyl)‐N‐(4‐pyridyl) benzamide ...monohydrochloride, a novel inhibitor of ROCK, inhibits tumour metastasis in some animal models. To metastasise, tumour cells have to disturb the tight intercellular junctions and the basement membrane matrix of the host tissue, which, respectively, create an intercellular barrier and the extracellular membrane. To clarify the mechanism of Wf‐536 in inhibition of tumour metastasis, we analysed the effect of Wf‐536 on the transition of tumour cells through the host cell layer and the basement membrane in in vitro systems.
2. In a coculture system of human fibrosarcoma HT1080 cells plated on a monolayer of human ECV304 cells, Wf‐536 (0.3–3 µmol/L) inhibited the paracellular infiltration of tumour cells.
3. Wf‐536 (3–30 µmol/L) inhibited the invasion of tumour cells through the reconstituted basement membrane (Matrigel) layer.
4. Wf‐536 (10–30 µmol/L) inhibited the migration of tumour cells. At 0.3–3 µmol/L, Wf‐536 also restrained hepatocyte growth factor/scatter factor (HGF)‐induced increases in paracellular permeability of the ECV304 cell layer.
5. These results suggest that Wf‐536 suppresses tumour metastasis by both enhancing the barrier function of host cell layers and inhibiting tumour cell motility at the stage of host tissue penetration by metastatic tumour cells.
Potent human chymase inhibitors with high enzymatic selectivity and satisfactory metabolic stability were obtained by replacing the Val-Pro (P3−P2) dipeptide portion of the previously described ...inhibitor 1 with a nonpeptidic pyrimidinone skeleton. The potency of the novel compounds was further enhanced by the introduction of carbamoyl-substituted difluoromethylene ketone moieties. The most potent chymase inhibitor of the newly created series was 2u (Y-40018), which had a K i of 2.62 nM. Compound 2u possessed high selectivity for human chymase since it lacked significant activity toward other representative human proteolytic enzymes. Moreover its strict specificity for human chymase suggested that 2u strongly inhibited human and canine chymases but not rat and mouse ones. Pharmacokinetic studies in rats and dogs indicated that 2u was absorbed rapidly after oral administration and had satisfactory bioavailability in these experimental animal species (rat, 17%; dog, 32%). In conclusion, 2u is a novel, potent, and orally active chymase inhibitor which would prove very useful in revealing the precise roles of the latter in various pathophysiological processes.
We examined the result of cord blood transplantation (CBT) for acute lymphoblastic leukemia (ALL) in children. Fifty ALL patients underwent stem cell transplantation in our hospital. Among these, 23 ...patients received related bone marrow transplantation and peripheral blood stem cell transplantation (R-BMT/PBSCT), 17 patients received unrelated bone marrow transplantation (U-BMT), and 10 patients received unrelated cord blood transplantation (U-CBT). The 5-year overall survival rates after R-BMT/PBSCT, U-BMT and U-CBT were 64.6%, 32.3%, and 85.7%, respectively. Event-free survivals after 5 years were 59.6%, 14.7%, and 70.0%, respectively. The relapse rate in the U-CBT group was equal to that in the R-BMT/PBSCT group, and the transplant-related mortality of U-CBT was 0%. Our data show that U-CBT should be the first choice for patients with refractory or relapsed ALL who have no related HLA-matched donor.
We designed nonpeptidic chymase inhibitors based on the structure of a peptidic compound (1) and demonstrated that the combination of a pyrimidinone skeleton as a P3−P2 scaffold and heterocycles as ...P1 carbonyl-activating groups can function as a nonpeptidic chymase inhibitor. In particular, introduction of heterobicycles such as benzoxazole resulted in more potent chymase-inhibitory activity. Detailed structure−activity relationship studies on the benzoxazole moiety and substituents at the 2-position of the pyrimidinone ring revealed that 2r (Y-40079) had the most potent chymase-inhibitory activity (K i = 4.85 nM). This compound was also effective toward chymases of nonhuman origin and showed good selectivity for chymases over other proteases. Pharmacokinetic studies in rats indicated that 2r was absorbed slowly after oral administration and showed satisfactory bioavailability (BA) (T max = 6.0 ± 2.3 h, BA = 19.3 ± 6.6%, t 1/2 = 35.7 ± 13.3 h). In conclusion, 2r is a novel, potent, and orally active chymase inhibitor which would be a useful tool in elucidating the pathophysiological roles of chymase.
Organ transplant recipients are generally considered to be at greater risk for developing malignant disorders because of prolonged immunosuppression for organ grafting, but acute leukemia is a rare ...complication after organ transplantation (0.2 -2.5%). We encountered two girls with acute promyelocytic leukemia (APL) after living donor partial orthotopic liver transplantation. In one patient, APL developed 21 months after liver transplantation for ornithine transcarbamylase deficiency. She had been administered tacrolimus for prophylaxis of graft-versus-host reaction. In the other patient, APL was diagnosed 46 months after liver transplantation for congenital biliary atresia. Both patients were successfully treated by chemotherapy including all-trans retinoic acid (ATRA), and after reaching complete remission, they have subsequently been in continuous remission. Although leukemia after liver transplantation is generally thought of as a rare complication, increases in survival rate following liver transplantation is likely to lead to more such cases, and documentation of these cases is therefore of importance.
It has been suggested that progressive pathophysiologic modifications of endothelium are associated with aging. Aging has been shown to influence some specific functions at the cellular level. In the ...present study, the effects of aging on levels of prostacyclin (PGI2) production were examined in cultured rat aortic endothelial cells from young (six-week-old) and old (100-week-old) Wistar rats. The level of PGI2 production from rat aortic endothelial cells decreased significantly with increasing age, suggesting decreased function of the endothelial cells. The production of PGI2 stimulated by thrombin was decreased in old rat aortic endothelial cells compared to young rat aortic endothelial cells, whereas there was no difference in the rate of intracellular calcium mobilization caused by thrombin. These data indicate that aging nonuniformly affects both basal and agonist-induced levels of PGI2 production in rat aortic endothelial cells, and that this diminution in PGI2 production may be related to the age-related potentiation of various thrombotic events.
