A Phase I Study of Pelabresib (CPI-0610), a Small-Molecule Inhibitor of BET Proteins, in Patients with Relapsed or Refractory Lymphoma.

Purpose: NF-κB, a transcription factor essential for inflammatory responses, is constitutively activated in many lymphomas. In preclinical studies, pelabresib (CPI-0610), an investigational (BET) bromodomain inhibitor, downregulated NF-κB signaling and demonstrated antitumor activity in vitro. Here we report the safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity from the first-in-human phase I study of pelabresib in patients with relapsed/refractory lymphomas (NCT01949883). Experimental Design: Sixty-four patients with relapsed/refractory lymphoma (median of 4 prior lines of therapy) were treated with either capsule (6, 12, 24, 48, 80, 120, 170, 230, 300 mg) or tablet (125, 225 mg) doses of pelabresib orally once daily on a 14 days on, 7 days off schedule. Results: The MTD was determined as the 225 mg tablet daily. The most frequent adverse events were fatigue, nausea, and decreased appetite. Thrombocytopenia, a class effect for all BET inhibitors, was dose-dependent, reversible, and noncumulative. Pelabresib exhibited dose-proportional increases in systemic exposure, rapid absorption, and a half-life of approximately 15 hours (supporting once daily dosing). The bioavailability of the tablet formulation was 60% greater than the capsules. Pelabresib suppressed IL8 and CCR1 mRNA at doses above 120 and 170 mg, respectively. Four patients (6.2%) had an objective response (2 complete response and 2 partial response) and 5 patients had prolonged stable disease. Conclusions/Discussion: Pelabresib is capable of BET target gene suppression in an exposure-dependent manner with an acceptable safety profile leading to the recommended phase II dose of the 125 mg tablet once daily. Significance: BET proteins inhibition can potentially modify the pathogenic pathways which contribute to many diseases including malignancies. Pelabresib (CPI-0610), a potent and selective small molecule BET proteins inhibitor, has a MTD of 225 mg once daily for 14 days with a 7-day break, clear pharmacokinetic/pharmacodynamic relationship, and manageable clinical safety profile. These findings are part of the foundation for the ongoing pivotal study of pelabresib in patients with myelofibrosis.

Paediatric Strategy Forum for medicinal product development of epigenetic modifiers for children: ACCELERATE in collaboration with the European Medicines Agency with participation of the Food and Drug Administration.

The fifth multistakeholder Paediatric Strategy Forum focussed on epigenetic modifier therapies for children and adolescents with cancer. As most mutations in paediatric malignancies influence chromatin-associated proteins or transcription and paediatric cancers are driven by developmental gene expression programs, targeting epigenetic mechanisms is predicted to be a very important therapeutic approach in paediatric cancer. The Research to Accelerate Cures and Equity (RACE) for Children Act FDARA amendments to section 505B of the FD&C Act was implemented in August 2020, and as there are many epigenetic targets on the FDA Paediatric Molecular Targets List, clinical evaluation of epigenetic modifiers in paediatric cancers should be considered early in drug development. Companies are also required to submit to the EMA paediatric investigation plans aiming to ensure that the necessary data to support the authorisation of a medicine for children in EU are of high quality and ethically researched. The specific aims of the forum were i) to identify epigenetic targets or mechanisms of action associated with epigenetic modification relevant to paediatric cancers and ii) to define the landscape for paediatric drug development of epigenetic modifier therapies. DNA methyltransferase inhibitors/hypomethylating agents and histone deacetylase inhibitors were largely excluded from discussion as the aim was to discuss those targets for which therapeutic agents are currently in early paediatric and adult development. Epigenetics is an evolving field and could be highly relevant to many paediatric cancers; the biology is multifaceted and new targets are frequently emerging. Targeting epigenetic mechanisms in paediatric malignancy has in most circumstances yet to reach or extend beyond clinical proof of concept, as many targets do not yet have available investigational drugs developed. Eight classes of medicinal products were discussed and prioritised based on the existing level of science to support early evaluation in children: inhibitors of menin, DOT1L, EZH2, EED, BET, PRMT5 and LSD1 and a retinoic acid receptor alpha agonist. Menin inhibitors should be moved rapidly into paediatric development, in view of their biological rationale, strong preclinical activity and ability to fulfil an unmet clinical need. A combination approach is critical for successful utilisation of any epigenetic modifiers (e.g. EZH2 and EED) and exploration of the optimum combination(s) should be supported by preclinical research and, where possible, molecular biomarker validation in advance of clinical translation. A follow-up multistakeholder meeting focussing on BET inhibitors will be held to define how to prioritise the multiple compounds in clinical development that could be evaluated in children with cancer. As epigenetic modifiers are relatively early in development in paediatrics, there is a clear opportunity to shape the landscape of therapies targeting the epigenome in order that efficient and optimum plans for their evaluation in children and adolescents are developed in a timely manner.

A phase I and pharmacokinetic study of oral perifosine with different loading schedules in patients with refractory neoplasms.

PURPOSE: To determine the maximum tolerated dose (MTD) of perifosine (NSC 639966), an alkylphospholipid modulator of signal transduction, using different oral loading and maintenance regimens in an effort to avoid gastrointestinal toxicity while seeking maximal sustained plasma concentrations. METHODS: Thirty-one patients with advanced neoplasms were treated with monthly cycles of perifosine loading doses of 300, 600, 900, 1,200 and 1,500 mg (dose levels 1 through 5, respectively) on days 1-2 depending on the actual dose of the initial cycle. For subsequent cycles, perifosine loading doses were reduced to 100, 200, 300, 400 and 1,000 mg at the respective corresponding dose levels. Daily perifosine "maintenance" doses of 50, 100, 150, 200 and 250 mg for levels 1 through 5, respectively, commenced on days 2 or 3 and continued for a total of 21 days. No treatment was given for days 22-27. The pharmacokinetics of perifosine with these schedules was characterized. RESULTS: Dose-limiting diarrhea developed at or above dose level 4. The MTD and recommended phase II dose was dose level 3B, with a loading dose of 900 mg on day 1 divided into two doses of 450 mg administered 6 h apart and a maintenance dose of 150 mg on day 2 through 21. On subsequent cycles, the loading dose was reduced to 300 mg. Non-gastrointestinal toxicities included three episodes of gout or gout-like syndromes observed at doses above the MTD. The median peak plasma concentration of perifosine achieved at the MTD was approximately 8.3 µg/mL. Four patients had stable disease ranging from 167 to 735 days. CONCLUSIONS: Perifosine given according to a loading and maintenance schedule can safely sustain concentrations of drug, approaching concentrations achieved in preclinical models with evidence of anti-tumor effect.

Similarities and differences in the oncology drug approval process between FDA and European Union with emphasis on in vitro companion diagnostics.

Drug approval [U.S. Food and Drug Administration (FDA), or market authorization for the European Union's European Medicines Agency (EMA)] is the most significant regulatory milestone for any drug, as drugs can only be marketed after marketing approval by a health authority. This article focuses on the main regulatory aspects of the drug approval process in the European Union (EU) and the United States. Although the procedures, requirements, and timelines for drug approvals are different between the EU and the United States, several global harmonization efforts have been developed during the past few years to have more consistent regulatory procedures/outcomes in different parts of the world. One of the most different procedures/requirements among these regions is co-development, also known as in vitro companion diagnostic. In the United States, it is expected that for a drug that requires an in vitro diagnostic test to select the population to be treated, the companion diagnostic should be already/concomitantly approved by the FDA. In the EU, these requirements are not as stringent as in the United States. However, it is anticipated that in the very near future, legislation changes in the EU will lead to similar requirements for the companion diagnostics for EMA. In summary, although the principles, procedures, and requirements for drug approvals may differ between the United States and EMA, novel efforts to harmonize them are being considered and implemented, thereby leading to simpler global drug development. It is of outmost importance that drug developers understand and appreciate differences in regional regulations. Otherwise, lack of understanding may lead to rejection or delays in drug approvals for useful anticancer agents. See all articles in this CCR Focus section, "The Precision Medicine Conundrum: Approaches to Companion Diagnostic Co-development."

Phase I dose-escalation study of AZD7762, a checkpoint kinase inhibitor, in combination with gemcitabine in US patients with advanced solid tumors.

PURPOSE: AZD7762 is a Chk1 kinase inhibitor which increases sensitivity to DNA-damaging agents, including gemcitabine. We evaluated the safety of AZD7762 monotherapy and with gemcitabine in advanced solid tumor patients. EXPERIMENTAL DESIGN: In this Phase I study, patients received intravenous AZD7762 on days 1 and 8 of a 14-day run-in cycle (cycle 0; AZD7762 monotherapy), followed by AZD7762 plus gemcitabine 750-1,000 mg/m(2) on days 1 and 8, every 21 days, in ascending AZD7762 doses (cycle 1; combination therapy). RESULTS: Forty-two patients received AZD7762 6 mg (n = 9), 9 mg (n = 3), 14 mg (n = 6), 21 mg (n = 3), 30 mg (n = 7), 32 mg (n = 6), and 40 mg (n = 8), in combination with gemcitabine. Common adverse events (AEs) were fatigue [41 % (17/42) patients], neutropenia/leukopenia [36 % (15/42) patients], anemia/Hb decrease [29 % (12/42) patients] and nausea, pyrexia and alanine aminotransferase/aspartate aminotransferase increase [26 % (11/42) patients each]. Grade ≥3 AEs occurred in 19 and 52 % of patients in cycles 0 and 1, respectively. Cardiac dose-limiting toxicities occurred in two patients (both AZD7762 monotherapy): grade 3 troponin I increase (32 mg) and grade 3 myocardial ischemia with chest pain, electrocardiogram changes, decreased left ventricular ejection fraction, and increased troponin I (40 mg). AZD7762 exposure increased linearly. Gemcitabine did not affect AZD7762 pharmacokinetics. Two non-small-cell lung cancer patients achieved partial tumor responses (AZD7762 6 mg/gemcitabine 750 mg/m(2) and AZD7762 9 mg cohort). CONCLUSIONS: The maximum-tolerated dose of AZD7762 in combination with gemcitabine 1,000 mg/m(2) was 30 mg. Although development of AZD7762 is not going forward owing to unpredictable cardiac toxicity, Chk1 remains an important therapeutic target.

Information needed to conduct first-in-human oncology trials in the United States: a view from a former FDA medical reviewer.

Any drug product not previously authorized for marketing in the United States requires the submission of an Investigational New Drug application (IND). Although the IND submission is regulated by law (21CFR 312), there are several issues that are not covered in the law or U.S. Food and Drug Administration (FDA) guidances that are important for a successful IND submission. For oncology products, the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S9 guidance (still in draft) is the most relevant. The most difficult issues to solve in an IND are chemistry, manufacturing and control information, and pharmacology and toxicology. In the United States, pivotal toxicological studies are done in two species: one rodent (i.e., rats) and one nonrodent (i.e., dogs). The safe starting dose is based on toxicological findings observed in the most sensitive species. Most first-in-humans studies in oncology include patients with advanced and/or metastatic disease, as serious to severe side effects of anticancer therapies are often less threatening to advanced cancer patients than their disease, and acceptable levels of toxicity are higher. For other indications (adjuvant therapy, chemoprevention, or healthy volunteers), first-in-human studies need to follow ICH M3 guidelines as the risk to benefit ratio in those subjects and/or patients without evidence of tumor is different. The division welcomes submissions before the IND, also known as pre-INDs, particularly for products with "atypical issues."

Enhanced skin carcinogenesis and lack of thymus hyperplasia in transgenic mice expressing human cyclin D1b (CCND1b).

Cyclin D1b is an alternative transcript of the cyclin D1 gene (CCND1) expressed in human tumors. Its abundance is regulated by a single base pair polymorphism at the exon 4/intron 4 boundary (nucleotide 870). Epidemiological studies have shown a correlation between the presence of the G870A allele (that favors the splicing for cyclin D1b) with increased risk and less favorable outcome in several forms of cancer. More recently, it has been shown that, unlike cyclin D1a, the alternative transcript D1b by itself has the capacity to transform fibroblasts in vitro. In order to study the oncogenic potential of cyclin D1b, we developed transgenic mice expressing human cyclin D1b under the control of the bovine K5 promoter (K5D1b mice). Seven founders were obtained and none of them presented any significant phenotype or developed spontaneous tumors. Interestingly, K5D1b mice do not develop the fatal thymic hyperplasia, which is characteristic of the cyclin D1a transgenic mice (K5D1a). Susceptibility to skin carcinogenesis was tested in K5D1b mice using two-stage carcinogenesis protocols. In two independent experiments, K5D1b mice developed higher papilloma multiplicity as compared with wild-type littermates. However, when K5D1b mice were crossed with cyclin D1KO mice, the expression of cyclin D1b was unable to rescue the carcinogenesis-resistant phenotype of the cyclin D1 KO mice. To further explore the role of cyclin D1b in mouse models of carcinogenesis we carried out in silico analysis and in vitro experiments to evaluate the existence of a mouse homologous of the human cyclin D1b transcript. We were unable to find any evidence of an alternatively spliced transcript in mouse Ccnd1. These results show that human cyclin D1b has different biological functions than cyclin D1a and confirm its oncogenic properties.

CDK2 activation in mouse epidermis induces keratinocyte proliferation but does not affect skin tumor development.

It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21(Cip1) and p27(Kip1). Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4(D158N) mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4(D158N), but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21(Cip1) in K5Cdk2, but not in K5Cdk4(D158N), epidermis, suggesting that CDK2 overexpression elicits a p21(Cip1) response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis.

Expression of CDK4 or CDK2 in mouse oral cavity is retained in adult pituitary with distinct effects on tumorigenesis.

The keratin 5 (K5) promoter drives transgenic expression to the basal cell layer of stratified epithelia. Surprisingly, analysis of K5CDK4 and K5CDK2 transgenic mouse embryos showed CDK4 and CDK2 expression not only in the expected tissues, but also in the adenohypophysis. This organ is derived from an upwards growth of the primitive oropharynx, a K5-expressing tissue. We show that transgenic expression of CDKs in the embryonic oral ectoderm is specifically retained in undifferentiated cells from the pars intermedia of the adenohypophysis. Interestingly, we found that K5CDK4 mice show a decreased number of pituitary stem cells, even though CDK4 is not expressed in the stem cells but in transit-amplifying (TA)-like cells. Interestingly, CDK4-expressing cells, but not CDK2-expressing cells, strongly synergize with lack of p27(Kip1) to generate pituitary carcinomas that appear with shortened latency and are drastically more aggressive than those arising in p27(-/-) mice. Thus, we show that deregulation of CDK expression in the primitive oral epithelium plays a unique function, providing a selective advantage that gives rise to transgene-positive TA-like pituitary cells. Furthermore, retention of CDK4 in these TA-like pituitary cells synergizes with loss of p27(Kip1) to induce pituitary adenocarcinomas. This model suggests that forced expression of CDK4 sensitizes cells and synergizes with a second change resulting in tumor development.

S-Phase-specific activation of PKC alpha induces senescence in non-small cell lung cancer cells.

Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.

Erlotinib/gemcitabine for first-line treatment of locally advanced or metastatic adenocarcinoma of the pancreas.

Erlotinib (Tarceva) is a human epidermal growth factor receptor type 1/epidermal growth factor receptor (HER1/EGFR) tyrosine kinase inhibitor initially approved by the US Food and Drug Administration for the treatment of patients with locally advanced or metastatic non-small-cell lung cancer after failure of at least one prior chemotherapy regimen. In this report, we present the pivotal study that led to the approval of erlotinib in combination with gemcitabine (Gemzar) in patients with locally advanced/metastatic chemonaive pancreatic cancer patients. The combination demonstrated a statistically significant increase in overall survival accompanied by an increase in toxicity. Physicians and patients now have a new option for the treatment of locally advanced/metastatic adenocarcinoma of the pancreas.

Inhibitors of cyclin-dependent kinase modulators for cancer therapy.

Most human malignancies have an aberration in the Rb pathway due to 'cdk hyperactivation'. Several small-molecule cdk modulators are being discovered and tested in the clinic. The first ATP-competitive cdk inhibitors tested in clinical trials, flavopiridol and UCN-01, have shown promising results with evidence of antitumor activity and plasma concentrations sufficient to inhibit cdk-related functions. The best schedule to be administered, combination with standard chemotherapeutic agents, best tumor types to be targeted, and demonstration of cdk modulation from tumor samples from patients in these trials are important issues that need to be answered to advance these agents to the clinical arena.

Phenoxodiol, a novel isoflavone, induces G1 arrest by specific loss in cyclin-dependent kinase 2 activity by p53-independent induction of p21WAF1/CIP1.

Phenoxodiol, an isoflavone derivative of genistein with unknown mechanism of action, is currently being evaluated in early human cancer clinical trials. To determine the mechanism of antiproliferative effects of phenoxodiol, we examined its effects in a battery of human cell lines. Although we observed caspase-dependent apoptosis in HN12 cells as early as 24 hours after exposure, clonogenic death occurred only after 48-hour exposure despite caspase blockade by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (ZVAD)-fmk. Moreover, clear evidence of cell death as determined by nuclear morphology and plasmatic membrane damage occur despite ZVAD, suggesting that another mechanism besides caspase-dependent apoptosis is required for clonogenic death induced by phenoxodiol. In search for other potential antiproliferative effects, we assessed the effects of phenoxodiol in the cell cycle progression of human carcinoma cell lines. A significant G(1)-S arrest was observed by 12 hours of exposure in HN12 cell lines at concentrations > or =5 microg/mL. Cell cycle arrest occurred several hours (approximately 12 hours) before induction of apoptosis. Analysis of in vitro purified cyclin-dependent kinase (cdk) activity showed that phenoxodiol did not inhibit cdk activity. In contrast, cellular cdk2 activity obtained from HN12 cell lines exposed to phenoxodiol for 12 hours decreased by 60%, whereas cdk6 activity remained unaltered, suggesting that the loss of cdk2 activity was specific. Loss in cdk2 activity was preceded by the accumulation of the endogenous cdk inhibitor p21(WAF1). To assess the role of p21(WAF1) induction by phenoxodiol, we used HCT116 isogenic cell lines and showed that phenoxodiol induced G(1) arrest together with p21(WAF1) expression in wild-type clones. In contrast, p21(-/-) variants failed to show G(1) arrest. Finally, induction of p21 by phenoxodiol is p53 independent, as phenoxodiol induced p21 in HCT116 lacking p53. These data therefore indicate that phenoxodiol promotes G(1)-S arrest by the specific loss in cdk2 activity due to p53-independent p21(WAF1) induction. This novel feature of phenoxodiol may have clinical implications, as the majority of human malignancies have aberrations in cell cycle progression regulation.

RNA silencing of checkpoint regulators sensitizes p53-defective prostate cancer cells to chemotherapy while sparing normal cells.

p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G(2) checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.

Targeting cell cycle and apoptosis for the treatment of human malignancies.

Oncogenic transformation leads to cell cycle aberration and apoptosis dysregulation. Targeting cell cycle and apoptosis pathways has emerged as an attractive approach for the treatment of cancer. The activity of cdks can be modulated by targeting these kinases with small molecules that bind to the ATP binding pocket of cdks, or by altering the composition of the cdk/endogenous cdk inhibitor complexes by different mechanisms. Apoptosis can be modulated by targeting pro-apoptotic or pro-survival pathways. Several proteins relevant to oncogenic and proliferative processes, such as p53, bcl-2, AKT, ras and epidermal growth factor receptor, are also important in blocking apoptosis. Several small molecules that modulate cell cycle control and apoptosis have been approved recently and many will be approved in the near future. Several challenges remain, including finding ways of targeting these agents specifically to tumors (sparing normal cells), and the development of rationales for combining these new agents with standard therapies and for prioritizing the development of an overwhelming number of novel small molecules targeting cell cycle and apoptosis. Novel technologies such as genomics and proteomics will be instrumental in designing combinatorial regimens tailored to patients on the basis of the genetic makeup of tumors. Irrespective of all shortcomings, the future of modulation of apoptosis and cell cycle machinery for oncology therapy is quite exciting.

Effects of alpha1-acid glycoprotein on the clinical pharmacokinetics of 7-hydroxystaurosporine.

OBJECTIVE: UCN-01 (7-hydroxystaurosporine) is a small molecule cyclin-dependent kinase modulator currently under clinical development as an anticancer agent. In vitro studies have demonstrated that UCN-01 is strongly bound to the acute-phase reactant alpha (1)-acid glycoprotein (AAG). Here, we examined the role of protein binding as a determinant of the pharmacokinetic behavior of UCN-01 in patients. EXPERIMENTAL DESIGN: Pharmacokinetic data were obtained from a group of 41 patients with cancer receiving UCN-01 as a 72-hour i.v. infusion (dose, 3.6 to 53 mg/m(2)/day). RESULTS: Over the tested dose range, total drug clearance was distinctly nonlinear (P = 0.0076) and increased exponentially from 4.33 mL/hour (at 3.6 mg/m(2)/day) to 24.1 mL/hour (at 54 mg/m(2)/day). As individual values for AAG increased, values for clearance decreased in a linear fashion (R(2) = 0.264; P = 0.0008), although the relationship was shallow, and the data showed considerable scatter. Interestingly, no nonlinearity in the unbound concentration (P = 0.083) or fraction at the peak plasma concentration of UCN-01 was apparent (P = 0.744). CONCLUSION: The results suggest the following: (1) that extensive binding to AAG may explain, in part, the unique pharmacokinetic profile of UCN-01 described previously with a small volume of distribution and slow systemic clearance, and (2) that measurement of total UCN-01 concentrations in plasma is a poor surrogate for that of the pharmacologically active fraction unbound drug.

UCN-01-induced cell cycle arrest requires the transcriptional induction of p21(waf1/cip1) by activation of mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway.

The small molecule UCN-01 is a cyclin-dependent kinase (CDK) modulator shown to have antiproliferative effects against several in vitro and in vivo cancer models currently being tested in human clinical trials. Although UCN-01 may inhibit several serine-threonine kinases, the exact mechanism by which it promotes cell cycle arrest is still unclear. We have reported previously that UCN-01 promotes G(1)-S cell cycle arrest in a battery of head and neck squamous cancer cell lines. The arrest is accompanied by an increase in both p21(waf1/cip1) and p27(kip1) CDK inhibitors leading to loss in G(1) CDK activity. In this report, we explore the role and the mechanism for the induction of these endogenous CDK inhibitors. We observed that p21 was required for the cell cycle effects of UCN-01, as HCT116 lacking p21 (HCT116 p21(-/-)) was refractory to the cell cycle effects of UCN-01. Moreover, UCN-01 promoted the accumulation of p21 at the mRNA level in the p53-deficient HaCaT cells without increase in the p21 mRNA half-life, suggesting that UCN-01 induced p21 at the transcriptional level. To study UCN-01 transcriptional activation of p21, we used several p21(waf1/cip1) promoter-driven luciferase reporter plasmids and observed that UCN-01 activated the full-length p21(waf1/cip1) promoter and a construct lacking p53 binding sites. The minimal promoter region required for UCN-01 (from -110 bp to the transcription start site) was the same minimal p21(waf1/cip1) promoter region required for Ras enhancement of p21(waf1/cip1) transcription. Neither protein kinase C nor PDK1/AKT pathways were relevant for the induction of p21 by UCN-01. In contrast, the activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase mitogen-activated protein kinase pathways was required for p21 induction as UCN-01 activated this pathway, and genetic or chemical MEK inhibitors blunted p21 accumulation. These results demonstrated for the first time that p21 is required for UCN-01 cell cycle arrest. Moreover, we showed that the accumulation of p21 is transcriptional via activation of the MEK pathway. This novel mechanism, by which UCN-01 exerts its antiproliferative effect, represents a promising strategy to be exploited in future clinical trials.

Metabolism of UCN-01 in isolated perfused rat liver: role of Mrp2 in the biliary excretion of glucuronides.

UCN-01 is a promising, novel kinase inhibitor currently undergoing clinical development. Though UCN-01 shows pronounced antitumor activity, its metabolism and hepatic transport system is still unknown. To investigate the biotransformation and biliary excretion of UCN-01, livers of Wistar and Mrp2-deficient TR- rats were perfused with UCN-01 (0.2 microM) in a single pass system. In bile and perfusate, native UCN-01 and 5 novel metabolites (M1-M5) were quantified by HPLC and identified as glucuronides by enzymatic hydrolysis with beta-glucuronidase and mass spectroscopy. Cumulative efflux of UCN-01 and its metabolites M1-M5 into perfusate of Wistar rats was low (<0.14%) whereas total biliary excretion was up to 53-fold higher, representing 1.74, 0.54, 0.21, 1.17, 0.85 and 0.52% of infused UCN-01, respectively. After 60 min of perfusion, liver cells still contained approximately 95% of applied UCN-01. Biliary excretion greatly differs in TR- rats. While cumulative biliary excretion of UCN-01 and its metabolites M1-M5 was significantly reduced to 8.3, 5.3, 31.8, 10.4, 13.2 and 7.8%, efflux into perfusate was increased up to 2.2-fold. This indicates that in control rats, UCN-01 and its glucuronides are almost exclusively eliminated into bile by Mrp2. In summary, UCN-01 is extensively metabolized in the rat liver to 5 novel glucuronides mainly excreted into bile by Mrp2. Metabolism and biliary excretion of UCN-01 must be taken into consideration also during cancer therapy of patients.

Advances in molecular carcinogenesis: current and future use of mouse models to screen and validate molecularly targeted anticancer drugs.

Survival of patients with advanced solid tumors has not significantly improved over the past 30 years. Although molecularly targeted anticancer drugs offer promise, few drugs make it through the end of the Food and Drug Administration approval process. Animal models that more closely resemble human carcinogenesis may bridge the gap between preclinical success and benefits for patients. We discuss pros and cons of several mouse models, including genetically engineered mice that each represent different aspects of human cancer, and the screening of targeted drugs in these models.