Pharmacokinetics and pharmacodynamics of a novel protein kinase inhibitor, UCN-01

Purpose: 7-Hydroxystaurosporine (UCN-01) is a potent protein kinase inhibitor and is being developed as a novel anticancer agent. We describe here its pharmacokinetics and pharmacodynamics in experimental animals. Methods: The pharmacokinetics of UCN-01 were studied following intravenous (i.v.) administration to mice, rats and dogs at doses of 1–9, 0.35–3.5 and 0.5 mg/kg, respectively. We also studied the pharmacodynamics of UCN-01 (9 mg/kg per day) during and after five consecutive i.v. administrations to nude mice bearing xenografted human pancreatic tumor cells (PSN-1). The concentrations of UCN-01 in plasma and tumor were measured by HPLC using a fluorescence detector. Results: UCN-01 in plasma after i.v. administration was eliminated biphasically in mice and rats, and triphasically in dogs. The elimination half-lives in mice, rats and dogs were 3.00–3.98, 4.02–4.46 and 11.6 h, respectively. The total clearance (Cl_total) values in mice, rats and dogs were high (1.93–2.64, 2.82–3.86 and 0.616 l/h per kg, respectively). The hepatic clearance (Cl_hepatic) in rats represented 54.0–81.3% of Cl_total. The volumes of distribution at steady-state in mice, rats and dogs were large (7.89–8.42, 13.0–16.9 and 6.09 l/kg, respectively). These pharmacokinetic parameters were dose-independent in mice and rats. UCN-01 produced significant inhibition of tumor growth during five consecutive i.v. administrations in mice bearing the xenografted PSN-1 cells, and the inhibitory effect continued for 3 days after the final administration. UCN-01 concentrations in tumor tissue were much higher than those in the plasma, and the ratio of tumor to plasma concentrations was about 500 at 24 h after five consecutive doses. Conclusions: The pharmacokinetic studies showed that UCN-01 has a high clearance and large distribution volume in various experimental animals, and its disposition is linear over the range of doses tested. The pharmacodynamic study showed that UCN-01 is distributed at much higher concentrations in tumor than those in plasma and that it significantly inhibits tumor growth. The high distribution of UCN-01 into tumor cells may contribute to the potent inhibition of tumor growth in vivo. Received: 29 April 1998 / Accepted: 28 October 1998     

Raf激酶抑制蛋白与恶性肿瘤

Raf激酶抑制蛋白(RKIP)是磷脂酰乙醇胺结合蛋白家族的成员。RKIP在Raf、核转录因子-κB(NF-κB)及G蛋白偶联受体(GPCR)等蛋白激酶信号传导通路中起重要调节作用。RKIP参与神经发育、心脏功能、精子发生、细胞迁移及巨噬细胞分化,同时还具有丝氨酸蛋白酶活性。近年来众多资料表明,RKIP在多种恶性肿瘤中表达减弱或丢失,其在恶性肿瘤中作用机制已经成为研究的热点,该类研究将为今后肿瘤治疗提供新的靶点。     

Raf激酶抑制蛋白与恶性肿瘤

Raf激酶抑制蛋白（RKIP）是磷脂酰乙醇胺结合蛋白家族的成员。RKIP在Raf、核转录因子-κB（NF-κB）及G蛋白偶联受体（GPCR）等蛋白激酶信号传导通路中起重要调节作用。RKIP参与神经发育、心脏功能、精子发生、细胞迁移及巨噬细胞分化，同时还具有丝氨酸蛋白酶活性。近年来众多资料表明，RKIP在多种恶性肿瘤中表达减弱或丢失，其在恶性肿瘤中作用机制已经成为研究的热点，该类研究将为今后肿瘤治疗提供新的靶点。     

甘油二酯激酶α、蛋白激酶C在肝癌组织中的表达及临床意义

 目的 研究肝癌组织中甘油二酯激酶α (Diacylglycerol Kinase α, DGKα)和蛋白激酶C（PKC）的表达及临床意义。方法 应用免疫组化检测DGKα在60例肝癌、癌周组织及10例正常肝组织中的表达,统计分析DGKα的表达与临床病理因素的关系。 结果 DGKα和PKC蛋白在正常肝组织、癌周组织和肝癌组织的阳性表达率逐渐降低,但在肝细胞的表达部位却逐渐从胞浆移至胞膜。DGKα阳性表达率的差异与原发性肝癌的分化类型、是否有门静脉癌栓和TNM分期有统计学意义(P <0.05)。结论 在越晚期、分化越差的肝癌中DGKα的活性越强,DGKα促进了原发性肝癌的病理进程。     

The protein kinase C inhibitor CGP 41251, a staurosporine derivative with antitumor activity,reverses multidrug resistance

Multidrug resistance (MDR) is frequently associated with overexpression of a 170-kDa P-glycoprotein (Pgp). Data suggest altered protein kinase C (PKC) activity in cells expressing the multidrug-resistant phenotype. The staurosporine derivative CCP A1251, an experimental anticancer drug, has been shown to exert selectivity for inhibition of protein kinase C activity and to exhibit antitumor activity in Wtro and in vivo. Here we show that CGP A1251 is also able to reverse MDR. After treatment of the mult id rug-resistant human lymphoblastoid cell line CCRF-VCR1000 with 500 nM Adriamydn, cell proliferation was reduced to 81% of untreated controls. A combination of 500 nM Adriamydn with a non-toxic concentration of 150 nM CGP 41251 (IC₅0 for inhibition of cell proliferation 420 nM CGP 41251) inhibits cell proliferation of CCRF-VCR1000 cells to 29% of untreated controls. In sensitive CCRF-CEM cells no enhancement of Adriamycin-induced cytotoxicity was observed upon addition of 150 nM CGP 41251. Strong synergism of the inhibition of cell proliferation was also observed after concomitant treatment of KB-851 I cells with CGP 41251 and Vinblas-tine or Adriamydn. Drug-sensitive KB-31 cells could not be further sensitized to Adriamycin or Vinblastine with CGP 41251 doses above 100 nM. Pretreatment with 50-1000 nM CGP 41251 for 30 min led to a dose-dependent increase in the intracellular accumulation of rhodamine 123, a substrate of P-glycoprotein. Treatment of multidrug-resistant CCRF-VCR1000 cells with CGP 41251 for 10 min was sufficient to inhibit the efflux of rhodamine 123. Preincubation with CGP 41251 for 12 or 24 hr did not alter multidrug resistance gene (mdrl)-mRNA levels. CGP 41251, a drug with antitumor efficacy in experimental systems, might offer an attractive combination partner for the treatment of tumors expressing the MDR phenotype. © Wiley-Liss, Inc.     

Synergistic anti-tumor efficacy of lovastatin and protein kinase C-beta inhibitor in hepatocellular carcinoma

Purpose  Hepatocellular carcinoma (HCC) is characterized by hypervascularity and chemoresistance. Protein kinase C (PKC) participates in cancer progression by enhancing anti-apoptotic signals, angiogenesis, and chemoresistance. Statins have a selective anti-cancer effect due to over-expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR) in cancer cells, but statins may also activate PKC. Thus, we hypothesized that simultaneous treatment with statin and PKC inhibitor might synergistically enhance their anti-tumor efficacies against HCCs. Methods  Hepatocellular carcinoma cell growth was assessed using MTS assays, apoptotic cell death by DAPI staining, and apoptotic signaling cascades were explored by immunoblotting. An in vivo model of HCC was established in C3H mice intradermally implanted with MH134 cells. Lovastatin and/or a PKC inhibitor (enzastaurin) was subsequently administered. Anti-tumor efficacies were evaluated by measuring tumor volumes and quantifying apoptotic cells and microvessel densities (MVD). Results  Co-treatment with lovastatin and enzastaurin was found to synergistically suppress HCC cell growth in vitro. Lovastatin induced HCC cellular apoptosis by activating mitochondrial apoptotic signals, and although enzastaurin alone did not induce apoptosis, its addition significantly enhanced lovastatin-induced apoptosis. This enhanced apoptosis was attributed to increased caspase-9 activation in these cells. Moreover, tumor growth was significantly suppressed in mice co-treated with lovastatin and enzastaurin, and percentages of TUNEL-positive cells were significantly increased and MVDs were significantly decreased in those mice. Conclusion  Combinatorial treatment with statin and PKC inhibitor was found to enhance anti-tumor efficacy in vivo and in vitro. Further studies are warranted to prove anti-tumor efficacy of this potential therapy in human HCCs.     

蛋白激酶C抑制剂逆转肾癌多药耐药机制的探讨

目的探讨蛋白激酶C（PKC）抑制剂对肾癌细胞多药耐药（MDR）逆转作用的机制。方法应用荧光显色法、RT-PCR和Western blot方法,检测PKCα cDNA转染肾癌786-0细胞前后,细胞中多药耐药基因1（MDR1）、多药耐药相关蛋白1（MRP1）和肺耐药蛋白（LRP）表达的变化。采用MTT方法,分别测定阿霉素（ADM）与PKC激动剂以及与PKC抑制剂协同作用后,786-0细胞和肾癌转染细胞系PKCα／786-0耐药性的变化。结果RT—PCR结果显示,肾癌转染细胞系PKCα／786-0的MDR1表达水平高于肾癌786-0细胞。ADM与PKC抑制剂协同作用的PKCα／786-0细胞系耐药性明显降低。786-0细胞对ADM的IC50为7．8015e^-7（5．7046e^-7～1．0669e^-6）;PKCα／786-0对ADM的IC50为1．6588e^-6（1．1621e^-6～2．3677e^-6）。PKC激动剂佛波醇酯（PMA）联合ADM处理PKCα／786-0的IC50为2．6794e^-6（2．0521e^-6-3．4983e^-6）;PKC抑制剂Calphostin C联合ADM处理PKCα／786-0的IC50为9．2506e^-8（5．9337e^-8～1．4422e^-7）。结论PKC抑制剂可以逆转入肾癌细胞的多药耐药性,其途径可能与改变MDR1的表达有关。     

The bisindolylmaleimide protein kinase C inhibitor, Ro 32-2241, reverses multidrug resistance in KB tumour cells

Ro 32-2241 is a bisindolylmaleimide that selectively inhibits protein kinase C (PKC) as compared with other protein kinases. Experiments were carried out to examine its potential as a multidrug resistance-reversing agent. Ro 32-2241 inhibited efflux, and increased accumulation, of [³H]-daunomycin in multidrug-resistant (MDR) KB-8-5 and KB-8-5-11 cells and had no effect on drug-sensitive KB-3-1 cells. Ro 32-2241 completely reversed the doxorubicin resistance of KB-8-5 and KB-8-5-11 cells, showing no effect on the sensitivity of drug-sensitive KB-3-1 cells. The potency of Ro 32-2241 was comparable with that of cyclosporin A and better than that of verapamil, known modulators of multidrug resistance. Ro 32-2241 also completely reversed the taxol resistance of KB-8-5 cells and partially reversed the resistance of KB-8-5-11 cells. Vinblastine resistance was also partially reversed. Mechanistic experiments were carried out to determine whether Ro 32-2241 interacted with P-glycoprotein (Pgp) directly. Increased efflux of [¹⁴C]-Ro 32-2241 was seen with the more resistant KB-8-5-11 cells (although the percentage effluxed was very low as compared with [³H]-daunomycin), suggesting that Ro 32-2241 can act as a substrate for Pgp. Direct interaction of Ro 32-2241 with Pgp was confirmed by demonstration that it inhibited binding of [³H]-azidopine to Pgp in KB-8-5-11 membranes. In conclusion, Ro 32-2241, acting directly on Pgp (rather than, or in addition to, an effect on PKC), is effective in reducing or reversing resistance to doxorubicin, taxol and vinblastine in human tumour cells with a clinically relevant degree of MDR. However, results of in vivo experiments conducted to investigate the effects of Ro 32-2241 on resistance to doxorubicin suggest that it may not be possible to achieve sufficiently high levels of Ro 32-2241 in vivo to modulate MDR. Received: 23 February 1997 / Accepted: 22 October 1998     

Phase I study of bryostatin 1, a protein kinase C modulator, preceding cisplatin in patients with refractory non-hematologic tumors

Purpose  Preclinical data suggested that bryostatin-1 (bryo) could potentiate the cytotoxicity of cisplatin when given prior to this drug. We designed a phase I study to achieve tolerable doses and schedules of bryo and cisplatin in combination and in this sequence. Methods  Patients with non-hematologic malignancies received bryo followed by cisplatin in several schedules. Bryo was given as an 1 and a 24 h continuous infusion, while cisplatin was always given over 1 h at 50  and 75 mg/m²; the combined regimen was repeated on an every 3-week and later on an every 2-week schedule. Bryo doses were escalated until recommended phase II doses were defined for each schedule. Patients were evaluated with computerized tomography every 2 cycles. Results  Fifty-three patients were entered. In an every 2-week schedule, the 1-h infusion of bryo became limited by myalgia that was clearly cumulative. With cisplatin 50 mg/m² its recommended phase II dose was 30 μg/m². In the 3-week schedule, dose-limiting toxicities were mostly related to cisplatin effects while myalgias were tolerable. Pharmacokinetics unfortunately proved to be unreliable due to bryo’s erratic extraction. Consistent inhibition of PKC isoform eta (η) in peripheral blood mononuclear cells was observed following bryo. Conclusions  Bryo can be safely administered with cisplatin with minimal toxicity; however, only four patients achieved an objective response. Modulation of cisplatin cytotoxicity by bryo awaits further insight into the molecular pathways involved. The work was supported by U01 CA76642, P30 CA 16087 and GCRC MO1 RR00096.     

Factors influencing a second myeloid malignancy in patients with Philadelphia-negative -7 or del(7q) clones during tyrosine kinase inhibitor therapy for chronic myeloid leukemia

The detection of Philadelphia-negative (Ph(neg)) cells with non-random karyotypic abnormalities after tyrosine kinase inhibitor (TKI) therapy of chronic myeloid leukaemia (CML) can be associated with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). To our knowledge, however, there have been no studies on variables influencing the risk of MDS/AML in patients with specific Ph(neg) karyotypes. We systematically examined studies reporting -7 or del(7q) within Ph(neg) cells in TKI-treated CML patients, and abstracted clinical and cytogenetic data from individual reports into a standardized format for further analysis. Of 53 patients, 43 had Ph(neg) -7 clones [as the sole abnormality (-7(sole)) in 29, or with other clones (-7(dual)) in 14], and del(7q) was present in 10. A total of 16/51 evaluable patients, all with -7, transformed to MDS/AML. Transformation was more frequent (15/16 patients) within 6 months of Ph(neg) -7 detection rather than subsequently (P < 0.0001). At first detection after TKI therapy, Ph(neg) abnormal clones comprised ≥50% of Ph(neg) cells in a greater proportion of patients with -7 than del(7q) (P = 0.035). Upon comparing -7(sole) and -7(dual), the latter was likely to be transient (P = 0.004), and AML was frequently observed with persistent -7 clones (P = 0.03). By logistic regression analysis (n = 36), clone size (P = 0.017), time-to-detection longer than 15 months (P = 0.02), and CML response (P = 0.085) were associated with MDS/AML. Validation of these novel associations in registry-based studies will help develop predictive criteria that define the MDS/AML risk in individual patients.     

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol,a valosin‐containing protein inhibitor

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin‐containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.     

Antitumor activity of erbstatin, a tyrosine protein kinase inhibitor

A tyrosine protein kinase inhibitor, erbstatin, showed no antineoplastic effect on L-1210 mouse leukemia when it was injected alone. Erbstatin was found to be inactivated by incubation in serum, but not in dialyzed serum. It was also inactivated in reconstituted serum containing dialyzed serum components and ferric or ferrous ion. Because erbstatin was considered to be inactivated by the ferric or ferrous ion in serum, foroxymithine, which is a potent chelator for the ferric ion, was given to the mice together with erbstatin. Administration of both erbstatin and foroxymithine showed antineoplastic activity against L-1210 leukemia.     

Antiangiogenic and antitumor activity of a selective PDGFR tyrosine kinase inhibitor, CP-673,451

CP-673,451 is a potent inhibitor of platelet-derived growth factor beta-receptor (PDGFR-beta) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-beta in cells (IC(50) = 1 nmol/L) being more than 450-fold selective for PDGFR-beta versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-beta inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-beta phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-beta phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC(50) of 120 ng/mL in plasma at C(max). In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. x 5, p.o., corresponding to 5.5 ng/mL at C(max)). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. x 10 days dosing routinely inhibited tumor growth (ED(50) < or = 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-beta phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.     

ABC294640, a sphingosine kinase 2 inhibitor,enhances the antitumor effects of TRAIL in non-small cell lung cancer

Evidences suggest that tumor microenvironment may play an important role in cancer drug resistance. Sphingosine kinase 2 (SphK2) is proposed to be the key regulator of sphingolipid signaling. This study is aimed to investigate whether the combination of molecular targeting therapy using a specific inhibitor of SphK2 (ABC294640), with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can enhance the apoptosis of non-small cell lung cancer (NSCLC) cells. Our results revealed that NSCLC cells'' sensitivity to TRAIL is correlated with the level of SphK2. Compared with TRAIL alone, the combination therapy enhanced the apoptosis induced by TRAIL, and knockdown of SphK2 by siRNA presented a similar effect. Combination therapy with ABC294640 increased the activity of caspase-3/8 and up-regulated the expression of death receptors (DR). Additional investigations revealed that translocation of DR4/5 to the cell membrane surface was promoted by adding ABC294640. However, expression of anti-apoptosis proteins such as Bcl^-2 and IAPs was not significantly modified by this SphK2 inhibitor. Overall, this work demonstrates that SphK2 may contribute to the apoptosis resistance in NSCLC, thus indicating a new therapeutic target for resistant NSCLC cells.     

SH-7, a new synthesized shikonin derivative, exerting its potent antitumor activities as a topoisomerase inhibitor

1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enylfuran-2-caroxylate (SH-7), a new naphthoquinone compound, derived from shikonin, exhibited obvious inhibitory actions on topoisomerase II (Topo II) and topoisomerase I (Topo I), which were stronger than its mother compound shikonin. Notably, the SH-7's inhibitory potency on Topo II was much stronger than that on Topo I. In addition, SH-7 significantly stabilized Topo II-DNA cleavable complex and elevated the expression of phosphorylated-H2AX. The in vitro cell-based investigation demonstrated that SH-7 displayed wide cytotoxicity in diversified cancer cell lines with the mean IC(50) value of 7.75 microM. One important finding is SH-7 displayed significant cytotoxicity in the 3 MDR cell lines, with an average IC(50) value nearly equivalent to that of the corresponding parental cell lines. The average resistance factor (RF) of SH-7 was 1.74, which was much lower than those of reference drugs VP-16 (RF 145.92), ADR (RF 105.97) and VCR (RF 197.39). Further studies illustrated that SH-7 had the marked apoptosis-inducing function on leukemia HL-60 cells, which was validated to be of mitochondria-dependence. The in vivo experiments showed that SH-7 had inhibitory effects on S-180 sarcoma implanted to mice, SMMC-7721, BEL-7402 human hepatocellular carcinoma and PC-3 human prostate cancer implanted to nude mice. Taken together, these results suggest that SH-7 induces DSBs as a Topo II inhibitor, which was crucial to activate the apoptotic process, and subsequently accounts for its both in vitro and in vivo antitumor activities. The well-defined Topo II inhibitory activity, antitumor effects particularly with its obvious anti-MDR action, better solubility and less toxicity make SH-7 as a potential antitumor drug candidate for further research and development.