A randomized phase II trial of CRLX101 in combination with bevacizumab versus standard of care in patients with advanced renal cell carcinoma.

BACKGROUND: Nanoparticle-drug conjugates enhance drug delivery to tumors. Gradual payload release inside cancer cells augments antitumor activity while reducing toxicity. CRLX101 is a novel nanoparticle-drug conjugate containing camptothecin, a potent inhibitor of topoisomerase I and the hypoxia-inducible factors 1α and 2α. In a phase Ib/2 trial, CRLX101 + bevacizumab was well tolerated with encouraging activity in metastatic renal cell carcinoma (mRCC). We conducted a randomized phase II trial comparing CRLX101 + bevacizumab versus standard of care (SOC) in refractory mRCC. PATIENTS AND METHODS: Patients with mRCC and 2-3 prior lines of therapy were randomized 1 : 1 to CRLX101 + bevacizumab versus SOC, defined as investigator's choice of any approved regimen not previously received. The primary end point was progression-free survival (PFS) by blinded independent radiological review in patients with clear cell mRCC. Secondary end points included overall survival, objective response rate and safety. RESULTS: In total, 111 patients were randomized and received ≥1 dose of drug (CRLX101 + bevacizumab, 55; SOC, 56). Within the SOC arm, patients received single-agent bevacizumab (19), axitinib (18), everolimus (7), pazopanib (4), sorafenib (4), sunitinib (2), or temsirolimus (2). In the clear cell population, the median PFS on the CRLX101 + bevacizumab and SOC arms was 3.7 months (95% confidence interval, 2.0-4.3) and 3.9 months (95% confidence interval 2.2-5.4), respectively (stratified log-rank P = 0.831). The objective response rate by IRR was 5% with CRLX101 + bevacizumab versus 14% with SOC (Mantel-Haenszel test, P = 0.836). Consistent with previous studies, the CRLX101 + bevacizumab combination was generally well tolerated, and no new safety signal was identified. CONCLUSIONS: Despite promising efficacy data on the earlier phase Ib/2 trial of mRCC, this randomized trial did not demonstrate improvement in PFS for the CRLX101 + bevacizumab combination when compared with approved agents in patients with heavily pretreated clear cell mRCC. Further development in this disease is not planned. CLINICAL TRIAL IDENTIFICATION: NCT02187302 (NIH).

Efficacy of the nanoparticle-drug conjugate CRLX101 in combination with bevacizumab in metastatic renal cell carcinoma: results of an investigator-initiated phase I-IIa clinical trial.

BACKGROUND: Anti-angiogenic therapies are effective in metastatic renal cell carcinoma (mRCC), but resistance is inevitable. A dual-inhibition strategy focused on hypoxia-inducible factor (HIF) is hypothesized to be active in this refractory setting. CRLX101 is an investigational camptothecin-containing nanoparticle-drug conjugate (NDC), which durably inhibits HIF1α and HIF2α in preclinical models and in gastric cancer patients. Synergy was observed in the preclinical setting when combining this NDC and anti-angiogenic agents, including bevacizumab. PATIENTS AND METHODS: Patients with refractory mRCC were treated every 2 weeks with bevacizumab (10 mg/kg) and escalating doses of CRLX101 (12, 15 mg/m(2)) in a 3 + 3 phase I design. An expansion cohort of 10 patients was treated at the recommended phase II dose (RP2D). Patients were treated until progressive disease or prohibitive toxicity. Adverse events (AEs) were assessed using CTCAE v4.0 and clinical outcome using RECIST v1.1. RESULTS: Twenty-two patients were response-evaluable in an investigator-initiated trial at two academic medical centers. RCC histologies included clear cell (n = 12), papillary (n = 5), chromophobe (n = 2), and unclassified (n = 3). Patients received a median of two prior therapies, with at least one prior vascular endothelial tyrosine kinase inhibitor therapy (VEGF-TKI). No dose-limiting toxicities were observed. Grade ≥3 AEs related to CRLX101 included non-infectious cystitis (5 events), fatigue (3 events), anemia (2 events), diarrhea (2 events), dizziness (2 events), and 7 other individual events. Five of 22 patients (23%) achieved partial responses, including 3 of 12 patients with clear cell histology and 2 of 10 patients (20%) with non-clear cell histology. Twelve of 22 patients (55%) achieved progression-free survival (PFS) of >4 months. CONCLUSIONS: CRLX101 combined with bevacizumab is safe in mRCC. This combination fulfilled the protocol's predefined threshold for further examination with responses and prolonged PFS in a heavily pretreated population. A randomized phase II clinical trial in mRCC of this combination is ongoing.

Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2).

Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients. Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea. To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR- rats were perfused with FLAP (30 microM) in a single pass system. Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR- rats. While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold. This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2. Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination. FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR- rats further supporting an efficient transport of FLAP glucuronides by Mrp2. FLAP (30 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates. In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin. This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.

Cyclin-dependent kinase modulators: a novel class of cell cycle regulators for cancer therapy.

With the understanding of the role of cdks in cell cycle regulation and the discovery that approx. 90% of all neoplasias are the result of 'cdk hyperactivation' leading to the abrogation of the Rb pathway, novel ATP competitive cdk inhibitors are being developed. The first two tested in clinical trials, flavopiridol and UCN-01, showed promising results with evidence of antitumor activity and plasma concentrations sufficient to inhibit cdk-related functions. Best schedule to be administered, combination with standard chemotherapeutic agents and demonstration of cdk modulation from tumor samples from patients in these trials are important issues that need to be answered in order to obtain the best possible results with these agents.

Characterization of autoantibodies against uridine-diphosphate glucuronosyltransferase in patients with inflammatory liver diseases.

Uridine diphosphate glucuronosyltransferase (UGT) was identified as an antigenic target in a subgroup of liver-kidney microsomal autoantibodies and was termed LKM-3. To evaluate the nature of LKM-3 antibodies, we screened sera from 80 untreated patients with autoimmune hepatitis (AIH) type 1 and 2, primary biliary cirrhosis (PBC), AIH/PBC, hepatitis C virus (HCV) infection, and 12 healthy individuals (controls) against 7 recombinant human UGT isoenzymes (UGT1A1, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, and UGT2B7). Autoantibodies reacting against various UGT isoenzymes were observed in sera from 3 of 18 AIH type 2 and 1 of 27 of the HCV patients. The anti-UGT-positive sera from AIH type 2 patients revealed the strongest immunoreactivity against UGT1A1, the main UGT-isoform involved in the bilirubin glucuronidation. Additionally, these sera were able to block UGT-mediated substrate glucuronidation in vitro. The prevalence for UGT1A1 was shown by 2 independent techniques: (1) UGT1A1 was identified as the main antigen by Western blotting. Preabsorption of sera with UGT1A1 prevented reaction against all tested UGT-isoforms. (2) In vitro immunoinhibition experiments showed that glucuronidation of the anticancer drug flavopiridol by UGT1A1 was more strongly inhibited than its UGT1A9-mediated biotransformation. In contrast, the serum from the HCV-patient reacted predominately with UGT1A6, and moreover, the immunoreactivity pattern was different from that of the AIH group. To summarize, we show the subtype preference of antibodies against UGT1A1 in a subgroup of AIH type 2 patients. These autoantibodies inhibit UGT-mediated glucuronidation in vitro, but it is unlikely that anti-UGT antibodies will have a marked effect on the patients capacity for drug biotransformation, as serum bilirubin levels in patients remained within the normal range.

Histone acetylation and the cell-cycle in cancer.

A number of distinct surveillance systems are found in mammalian cells that have the capacity to interrupt normal cell-cycle progression. These are referred to as cell cycle check points. Surveillance systems activated by DNA damage act at three stages, one at the G1/S phase boundary, one that monitors progression through S phase and one at the G2/M boundary. The initiation of DNA synthesis and irrevocable progression through G1 phase represents an additional checkpoint when the cell commits to DNA synthesis. Transition through the cell cycle is regulated by a family of protein kinase holoenzymes, the cyclin-dependent kinases (Cdks), and their heterodimeric cyclin partner. Orderly progression through the cell-cycle checkpoints involves coordinated activation of the Cdks that, in the presence of an associated Cdk-activating kinase (CAK), phosphorylate target substrates including members of the "pocket protein" family. One of these, the product of the retinoblastoma susceptibility gene (the pRB protein), is phosphorylated sequentially by both cyclin D/Cdk4 complexes and cyclin E/Cdk2 kinases. Recent studies have identified important cross talk between the cell-cycle regulatory apparatus and proteins regulating histone acetylation. pRB binds both E2F proteins and histone deacetylase (HDAC) complexes. HDAC plays an important role in pRB tumor suppression function and transcriptional repression. Histones are required for accurate assembly of chromatin and the induction of histone gene expression is tightly coordinated. Recent studies have identified an important alternate substrate of cyclin E/Cdk2, NPAT (nuclear protein mapped to the ATM locus) which plays a critical role in promoting cell-cycle progression in the absence of pRB, and contributes to cell-cycle regulated histone gene expression. The acetylation of histones by a number of histone acetyl transferases (HATs) also plays an important role in coordinating gene expression and cell-cycle progression. Components of the cell-cycle regulatory apparatus are both regulated by HATs and bind directly to HATs. Finally transcription factors have been identified as substrate for HATs. Mutations of these transcription factors at their sites of acetylation has been associated with constitutive activity and enhanced cellular proliferation, suggesting an important role for acetylation in transcriptional repression as well as activation. Together these studies provide a working model in which the cell-cycle regulatory kinases phosphorylate and inactivate HDACs, coordinate histone gene expression and bind to histone acetylases themselves. The recent evidence for cross-talk between the cyclin-dependent kinases and histone gene expression on the one hand and cyclin-dependent regulation of histone acetylases on the other, suggests chemotherapeutics targeting histone acetylation may have complex and possibly complementary effects with agents targeting Cdks.

Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms.

PURPOSE: To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV). PATIENTS AND METHODS: Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed. RESULTS: The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m(2)/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m(2)/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m(2)/d for 3 days included mean total plasma concentration of 36.4 microM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G(2) checkpoint. CONCLUSION: UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.

Possible mechanisms of diarrheal side effects associated with the use of a novel chemotherapeutic agent, flavopiridol.

The novel cyclin-dependent kinase inhibitor flavopiridol has recently completed Phase I trials for the treatment of refractory neoplasms. The dose-limiting toxicity observed with this agent was severe diarrhea. Because the compound otherwise showed promise, the present study sought to determine possible mechanisms underlying the diarrheal side effects. Flavopiridol was tested for its ability to modify chloride secretory responses of the human colonic epithelial cell line, T84. Studies were conducted in vitro in modified Ussing chambers. High concentrations of flavopiridol (10(-4) M), above those likely to be clinically relevant, had a direct stimulatory effect on chloride secretion, probably ascribable to an increase in cyclic AMP. Lower, clinically relevant concentrations of flavopiridol (10(-6) M) had no effect on chloride secretion by themselves but potentiated responses to the calcium-dependent secretagogue, carbachol. The drug also potentiated responses to thapsigargin and taurodeoxycholate and reversed the inhibitory effects of carbachol and epidermal growth factor on calcium-dependent chloride secretion. Pretreatment with the cyclic AMP-dependent secretagogue, forskolin, potentiated responses to flavopiridol, but not vice versa. Thus, diarrheal side effects induced by flavopiridol are likely multifactorial in origin and may involve interactions with endogenous secretagogues such as acetylcholine and bile acids. A better understanding of the diarrhea induced by flavopiridol should allow optimization of therapy with this otherwise promising drug and/or the development of related agents with improved toxicity profiles.

Development of cyclin-dependent kinase modulators as novel therapeutic approaches for hematological malignancies.

The majority of hematopoietic malignancies have aberrancies in the retinoblastoma (Rb) pathway. Loss in Rb function is, in most cases, a result of the phosphorylation and inactivation of Rb by the cyclin-dependent kinases (cdks), main regulators of cell cycle progression. Flavopiridol, the first cdk modulator tested in clinical trials, is a flavonoid that inhibits several cdks with evidence of cell cycle block. Other interesting preclinical features are the induction of apoptosis, promotion of differentiation, inhibition of angiogenic processes and modulation of transcriptional events. Initial clinical trials with infusional flavopiridol demonstrated activity in some patients with non-Hodgkin's lymphoma, renal, prostate, colon and gastric carcinomas. Main side-effects were secretory diarrhea and a pro-inflammatory syndrome associated with hypotension. Phase 2 trials with infusional flavopiridol in CLL and mantle cell lymphoma, other schedules and combination with standard chemotherapies are ongoing. The second cdk modulator tested in clinical trials, UCN-01, is a potent protein kinase C inhibitor that inhibits cdk activity in vitro as well. UCN-01 blocks cell cycle progression and promotes apoptosis in hematopoietic models. Moreover, UCN-01 is able to abrogate checkpoints induced by genotoxic stress due to modulation in chk1 kinase. The first clinical trial of UCN-01 demonstrated very prolonged half-life (approximately 600 h), 100 times longer than the half-life observed in preclinical models. This effect is due to high binding affinity of UCN-01 to the human plasma protein alpha-1-acid glycoprotein. Main side-effects in this trial were headaches, nausea/vomiting, hypoxemia and hyperglycemia. Clinical activity was observed in patients with melanoma, non-Hodgkin's lymphoma and leiomyosarcoma. Of interest, a patient with anaplastic large cell lymphoma refractory to high-dose chemotherapy showed no evidence of disease after 3 years of UCN-01 therapy. Trials of infusional UCN-01 in combination with Ara-C or gemcitabine in patients with acute leukemia and CLL, respectively, have commenced. In conclusion, flavopiridol and UCN-01 are cdk modulators that reach biologically active concentrations effective in modulating CDK in vitro, and show encouraging results in early clinical trials in patients with refractory hematopoietic malignancies. Although important questions remain to be answered, these positive experiences will hopefully increase the therapeutic modalities in hematological malignancies.

In vitro glucuronidation of the cyclin-dependent kinase inhibitor flavopiridol by rat and human liver microsomes: involvement of UDP-glucuronosyltransferases 1A1 and 1A9.

The metabolism of flavopiridol (FLAP), a novel anticancer drug currently undergoing clinical development, was investigated in rat and human liver microsomes. In the presence of uridine 5'-diphosphoglucuronic acid, two biotransformation products (M1 and M2) could be detected. Formation of metabolite M1 and M2 in terms of enzymatic efficacy (Vmax/K(M)) was about 50- and 5-fold higher in rat (1.58 +/- 2.23 and 7.22 +/- 1.17 microl/min/mg) as compared with human liver microsomes (0.032 +/- 0.016 and 1.52 +/- 0.93 microl/min/mg), indicating species-related differences in FLAP glucuronidation. Incubation in the presence of human recombinant UDP-glucuronosyltransferases (UGTs) demonstrated that M1 is almost exclusively catalyzed by UGT1A1, whereas M2 is formed by UGT1A9 and only to a minor extent by UGT1A1 and UGT1A10. Chemical inhibition experiments further prove the involvement of UGT1A1 and UGT1A9 in the formation of M1 and M2, as the UGT1A1 substrate bilirubin preferably inhibited M1 over M2 (K(i): 36 and 258 microM, respectively), whereas the UGT1A9 substrate propofol showed a more pronounced decrease in M2 but not in M1 formation (K(i): 47 and 142 microM, respectively). Both conjugates were purified from rat liver microsomes and analyzed by mass spectrometry, NMR, and UV experiments. On the basis of these results, M1 was identified as 5-O-beta-glucopyranuronosyl-flavopiridol and M2 as 7-O-beta-glucopyranuronosyl-flavopiridol. In conclusion, our results elucidate the enzymatic pathways of FLAP in rat and human liver, which must be considered during cancer therapy of patients.

Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells.

We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol.

Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25.

Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were recently described as potent, ATP-competitive, inhibitors of the cell cycle regulating cyclin-dependent kinases (CDKs). We here report that paullones also act as very potent inhibitors of glycogen synthase kinase-3beta (GSK-3beta) (IC50: 4-80 nM) and the neuronal CDK5/p25 (IC50: 20-200 nM). These two enzymes are responsible for most of the hyperphosphorylation of the microtubule-binding protein tau, a feature observed in the brains of patients with Alzheimer's disease and other neurodegenerative 'taupathies'. Alsterpaullone, the most active paullone, was demonstrated to act by competing with ATP for binding to GSK-3beta. Alsterpaullone inhibits the phosphorylation of tau in vivo at sites which are typically phosphorylated by GSK-3beta in Alzheimer's disease. Alsterpaullone also inhibits the CDK5/p25-dependent phosphorylation of DARPP-32 in mouse striatum slices in vitro. This dual specificity of paullones may turn these compounds into very useful tools for the study and possibly treatment of neurodegenerative and proliferative disorders.

Induction of growth inhibition and apoptosis in prostate cancer cells by flavopiridol.

Flavopiridol is an inhibitor of several cyclin-dependent kinases, and exhibits potent growth-inhibitory activity against a number of human tumor cell lines both in vitro, and when grown as xenografts in mice. It has shown promising antineoplastic activity and is currently undergoing clinical phase II testing. Prostate cancer (PCa) remains a leading cause of morbidity and mortality among males in the United States. There are no effective treatments for hormone and/or radiation refractory PCa, suggesting that novel and newer treatment strategy may be useful in the management of PCa. Our previous study showed that flavopiridol induces cell growth inhibition and apoptosis in breast cancer cells. Here, we investigated whether flavopiridol was effective against prostate cancer cells. Flavopiridol was found to inhibit growth of PC3 prostate cancer cells. Induction of apoptosis was also observed in PC3 cells treated with flavopiridol, as measured by DNA laddering and PARP cleavage. We also found a significant down-regulation of Bcl-2 in flavopiridol-treated cells. These findings suggest that down-regulation of Bcl-2 may be one of the molecular mechanisms through which flavopiridol induces apoptosis and inhibits cell growth, suggesting that flavopiridol may be an effective chemotherapeutic agent against prostate cancer.

P21-dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228.

Depsipeptide, FR901228, a novel cyclic peptide inhibitor of histone deacetylase with a unique cytotoxicity profile is currently in phase I clinical trials. Here we demonstrate that, in addition to G2/M arrest, FR901228 causes G1 arrest with Rb hypophosphorylation. In vitro kinase assays demonstrated no direct inhibition of CDK activity, however, an inhibition was observed in CDKs extracted from cells exposed to FR901228. Cyclin D1 protein disappeared between 6 and 12 hours after treatment with FR901228, whereas cyclin E was upregulated. While it did not induce wt p53, FR901228 did induce p21(WAF1/CIP1)in a p53-independent manner. Cell clones lacking p21 were not arrested in G1 phase, but continued DNA synthesis and were arrested in G2/M phase following FR901228 treatment. Finally, FR901228 blunted ERK-2/MAPK activation by EGF whereas early signal transduction events remained intact since overall cellular tyrosine phosphorylation after EGF stimulation was unaffected. Thus, FR901228, while not directly inhibiting kinase activity, causes cyclin D1 downregulation and a p53-independent p21 induction, leading to inhibition of CDK and dephosphorylation of Rb resulting in growth arrest in the early G1 phase. In contrast to the G1 arrest, the G2/M arrest is p21-independent, but is associated with significant cytotoxicity.

Flavopiridol inhibits P-TEFb and blocks HIV-1 replication.

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor that is in clinical trials as a cancer treatment because of its antiproliferative properties. We found that the flavonoid potently inhibited transcription by RNA polymerase II in vitro by blocking the transition into productive elongation, a step controlled by P-TEFb. The ability of P-TEFb to phosphorylate the carboxyl-terminal domain of the large subunit of RNA polymerase II was inhibited by flavopiridol with a K(i) of 3 nm. Interestingly, the drug was not competitive with ATP. P-TEFb composed of Cdk9 and cyclin T1 is a required cellular cofactor for the human immunodeficiency virus (HIV-1) transactivator, Tat. Consistent with its ability to inhibit P-TEFb, flavopiridol blocked Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocked HIV-1 replication in both single-round and viral spread assays with an IC(50) of less than 10 nm.

Inhibition of CDKs as a therapeutic modality.

Altered cell cycle control has emerged as a recurring theme in neoplasia. Strategies that would return toward normal the altered cell cycle control present in tumor cells have appeal as novel approaches to cancer treatment. Cyclin-dependent kinases (CDKs) control the progression through the cell cycle, operating at the transition from the G2 to M and G1 to S phases, and progression through S. CDKs are regulated by a complex set of mechanisms, including the presence of activating cyclins, regulatory phosphorylations, and endogenous CDK inhibitors at "checkpoints." This overview focuses on progress in defining compounds that can antagonize directly the action of CDKs. These have emerged as various types of ATP site-directed inhibitors, including flavopiridol, N-substituted adenine derivatives, the natural product butyrolactone, staurosporine derivatives, and, more recently, the synthetic paullones. Paullones appear to be of interest in that one of the most active members of the class, 9-nitropaullone (alsterpaullone), requires relatively brief periods of exposure to living cells in order to effect lasting effects on cellular and proliferative potential. Two of these compounds, flavopiridol and UCN-01 (7-hydroxy-staurosporine), have entered early clinical trials and achieved concentrations that might potentially modulate CDK function. In the case of UCN-01, unexpected human plasma protein binding might prevent direct inhibition of CDKs but allow drug concentrations to be achieved that indirectly affect CDKs by checkpoint abrogation. Further studies with CDK inhibitors should define the expected end point of CDK inhibition more clearly in preclinical models and clinical systems, including cytostasis, apoptosis, or differentiation.

A phase I study of combination therapy with immunotoxins IgG-HD37-deglycosylated ricin A chain (dgA) and IgG-RFB4-dgA (Combotox) in patients with refractory CD19(+), CD22(+) B cell lymphoma.

This study used an 8-day continuous infusion regimen of a 1:1 mixture of two immunotoxins (ITs) prepared from deglycosylated ricin A chain (dgA) conjugated to monoclonal antibodies directed against CD22 (RFB4-dgA) and CD19 (HD37-dgA; Combotox) in a Phase I trial involving 22 patients with refractory B cell lymphoma to determine the maximum tolerated dose, clinical pharmacology, and toxicity profile and to characterize any clinical responses. Adult patients received a continuous infusion of Combotox at 10, 20, or 30 mg/m2/192 h. No intrapatient dose escalation was permitted. Patients with > or =50 circulating tumor cells (CTCs)/mm3 in peripheral blood tolerated all doses without major toxicity. The maximum level of serum IT (Cmax) achieved in this group was 345 ng/ml of RFB4-dgA and 660 ng/ml of HD37-dgA (1005 ng/ml of Combotox). In contrast, patients without CTCs (<50/mm3) had unpredictable clinical courses that included two deaths probably related to the IT. Additionally, patients exhibited a significant potential for association between mortality and a history of either autologous bone marrow or peripheral blood stem cell transplants (P2 = 0.003) and between mortality and a history of radiation therapy (P2 = 0.036). In patients with CTCs, prior therapies appeared to have little impact on toxicity. Subsequent evaluation of the ITs revealed biochemical heterogeneity between two lots of HD37-dgA. In addition, HD37-dgA thawed at the study site tended to contain significant particulates, which were not apparent in matched controls stored at the originating site. This suggests that a tendency to aggregate may have resulted from shipping, storage, and handling of the IT that occurred prior to preparation for administration. It is not clear to what extent, if any, the aggregation of HD37-dgA IT was related to the encountered clinical toxicities; however, the potential to aggregate does suggest one possible basis for problems in our clinical experience with HD37-dgA and leads us to the conclusion that non-aggregate-forming formulations for these ITs should be pursued prior to future clinical trials.

Preclinical and clinical development of cyclin-dependent kinase modulators.

In the last decade, the discovery and cloning of the cyclin-dependent kinases (cdks), key regulators of cell cycle progression, have led to the identification of novel modulators of cdk activity. Initial experimental results demonstrated that these cdk modulators are able to block cell cycle progression, induce apoptotic cell death, promote differentiation, inhibit angiogenesis, and modulate transcription. Alteration of cdk activity may occur indirectly by affecting upstream pathways that regulate cdk activity or directly by targeting the cdk holoenzyme. Two direct cdk modulators, flavopiridol and UCN-01, are showing promising results in early clinical trials, in which the drugs reach plasma concentrations that can alter cdk activity in vitro. Although modulation of cdk activity is a well-grounded concept and new cdk modulators are being assessed for clinical testing, important scientific questions remain to be addressed. These questions include whether one or more cdks should be inhibited, how cdk inhibitors should be combined with other chemotherapy agents, and which cdk substrates should be used to assess the biologic effects of these drugs in patients. Thus, modulation of cdk activity is an attractive target for cancer chemotherapy, and several agents that modulate cdk activity are in or are approaching entry into clinical trials.