Early Phase Clinical Trial Designs - State of Play and Adapting for the Future.

The process of anti-cancer drug development is complex, with high attrition rates. Factors that may optimise this process include well-constructed and relevant pre-clinical testing and use of biomarkers for patient selection. However, the design of early phase clinical trials will probably play a vital role in both the robust clinical investigation of new targeted therapies and in streamlining drug development. In this overview, we assess current concepts in phase I clinical trials, highlighting issues and opportunities to improve their meaningfulness. The particular challenge of how to design combination trials is addressed, with focus on the potential of new adaptive and model-based designs.

Paclitaxel and CYC3, an aurora kinase A inhibitor, synergise in pancreatic cancer cells but not bone marrow precursor cells.

BACKGROUND: Amplification of aurora kinase A (AK-A) overrides the mitotic spindle assembly checkpoint, inducing resistance to taxanes. RNA interference targeting AK-A in human pancreatic cancer cell lines enhanced taxane chemosensitivity. In this study, a novel AK-A inhibitor, CYC3, was investigated in pancreatic cancer cell lines, in combination with paclitaxel. METHODS: Western blot, flow cytometry and immunostaining were used to investigate the specificity of CYC3. Sulforhodamine B staining, time-lapse microscopy and colony-formation assays were employed to evaluate the cytotoxic effect of CYC3 and paclitaxel. Human colony-forming unit of granulocyte and macrophage (CFU-GM) cells were used to compare the effect in tumour and normal tissue. RESULTS: CYC3 was shown to be a specific AK-A inhibitor. Three nanomolar paclitaxel (growth inhibition 50% (GI(50)) 3 nM in PANC-1, 5.1 nM in MIA PaCa-2) in combination with 1 µM CYC3 (GI(50) 1.1 µM in MIA PaCa2 and 2 µM in PANC-1) was synergistic in inhibiting pancreatic cell growth and causing mitotic arrest, achieving similar effects to 10-fold higher concentrations of paclitaxel (30 nM). In CFU-GM cells, the effect of the combination was simply additive, displaying significantly less myelotoxicity compared with high concentrations of paclitaxel (30 nM; 60-70% vs 100% inhibition). CONCLUSION: The combination of lower doses of paclitaxel and CYC3 merits further investigation with the potential for an improved therapeutic index in vivo.

Management of metastatic castration-resistant prostate cancer after first-line docetaxel.

Although chemotherapy, based on docetaxel, is now established in the management of metastatic castration-resistant prostate cancer (mCRPC), until recently, there has been no treatment licensed for use in the second line in men whose disease progresses during or after docetaxel therapy. This article reviews the classes of agents that have shown potential in this setting, notably chemotherapy drugs, hormonal therapies, immunotherapies, anti-angiogenic drugs, and clusterin-targeted therapy.

Small-bore hollow waveguides for delivery of 3-µm laser radiation.

Flexible hollow glass waveguides with bore diameters as small as 250 µm have been developed for 3-µm laser delivery. All the guides exhibit straight losses between 0.10 and 1.73 dB/m, and the loss increases to between 2.4 and 5.1 dB/m upon bending 1 m of the guides into 15-cm-diameter coils. This behavior is shown to depend strongly on the launch conditions and mode quality of the input beam. The waveguides are capable of efficiently delivering up to 8 W of Er:YAG laser power with proper input coupling, and they are suitable for use in both medical and industrial applications.

Power delivery of free electron laser light by hollow glass waveguides.

Hollow glass waveguides are used to deliver free electron laser (FEL) energy for applications in medicine and laser surgery. The hollow guides, optimized for the delivery of 6.45-µm FEL radiation, exhibited losses for the 1000-µm bore as low as 0.39 dB/m when the guide was straight and 1.75 dB/m when bent to a radius of 25 cm. Hollow glass guides are flexible, and their broadband capability provides an ideal fiber optic for the tunable FEL.

Enzymatic elimination of fluoride from alpha-fluoro-beta-alanine.

Rat liver homogenates catalyzed the elimination of fluoride from (R,S)-alpha-fluoro-beta-alanine. The substrate specificity and physical properties of the defluorinating enzyme were similar to those of mitochondrial L-alanine-glyoxylate aminotransferase II (EC 2.6.1.44, AlaAT-II). Furthermore, AlaAT-II activity, measured with L-alanine and glyoxylate as substrates, copurified with the alpha-fluoro-beta-alanine-defluorinating enzyme. The NH2-terminal sequence (18 residues) of the enzyme did not show significant sequence similarity with any of the proteins currently listed in GenBank. The purified enzyme catalyzed the transamination of L-alanine (Ala) and glyoxylate (glyx) at pH 8.5 by a ping-pong mechanism with kinetic parameters of kcat = 17 sec-1, KL-Ala = 3.2 mM, and Kglyx = 0.3 mM, respectively. The kinetic parameters for the defluorination of (R)-alpha-fluoro-beta-alanine and (S)-alpha-fluoro-beta-alanine were kcat = 6.2 and 2.6 min-1, respectively, and Km = 2.7 and 0.88 mM, respectively. L-Alanine potently inhibited the defluorination reaction with an apparent Ki of 0.024 mM. (R,S)-alpha-Fluoro-beta-alanine converted the optical spectrum of the enzyme-bound cofactor from the pyridoxal form to the pyridoxamino form, which indicated that this cofactor may participate in the defluorination reaction. The product of the enzymatic reaction, malonic semialdehyde, reacted nonenzymatically with (R,S)-alpha-fluoro-beta-alanine to form an adduct that was detected spectrally. AlaAT-II was not inactivated during dehalogenation of (R,S)-alpha-fluoro-beta-alanine but was inactivated completely during dehalogenation of beta-chloro-L-alanine.

Attenuation of the antitumor activity of 5-fluorouracil by (R)-5-fluoro-5,6-dihydrouracil.

5-Ethynyluracil (5-EU; 776C85) is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase that improves the antitumor activity of 5-fluorouracil (5-FU) to a greater extent than can be accounted for by the improved 5-FU pharmacokinetics that result from preventing the catabolism of 5-FU. We therefore tested the effects of (R)-5-fluoro-5,6-dihydrouracil (5-FUH2), the 5-FU catabolite extensively formed in the absence of 5-EU, on the antitumor activity and toxicity of 5-FU in 5-EU-treated rats bearing large s.c. tumors. Rats were dosed once weekly for 3 weeks with the following regimens: 100 mg/kg 5-FU (maximum tolerated dose), 10 mg/kg 5-FU 1 h after 1 mg/kg 5-EU, or 10 mg/kg 5-FU plus 90 mg/kg 5-FUH2 1 h after 1 mg/kg 5-EU. The latter regimen was designed to approximate the exposure produced from 5-FU in the absence of 5-EU, where > 80% of the dose is catabolized. 5-FU produced complete and sustained tumor regressions in 94% of the animals pretreated with 5-EU. In contrast, 5-FU in combination with 5-FUH2 produced complete regression in only 38% of the 5-EU-treated rats, which was similar to the antitumor activity of 5-FU in the absence of 5-EU. All treatments resulted in 7-11% transient weight loss. 5-FU produced no other notable toxicity in 5-EU-treated rats. However, 5-FUH2 added to this regimen caused transient diarrhea and stomatitis in 13% of the animals, which was similar to the toxicity produced by 5-FU in the absence of 5-EU. Thus, 5-FUH2, or other downstream catabolites of 5-FU, impaired the antitumor activity and slightly increased the toxicity of 5-FU. Accordingly, 5-EU approved to improve the efficacy of 5-FU by preventing the formation of 5-FU catabolites.

Solgel alumina coating for hollow waveguide delivery of CO(2) laser radiation.

Solgel alumina films were prepared by use of the Yoldas process and were characterized optically and microstructurally. From nitrogen adsorption and electron microscopy, we determined that the material was highly porous, with pores and crystallites of the order of tens of nanometers in size. The infrared transmission and reflectance of the films were measured, and dispersion curves were calculated as a function of firing temperature by extracting the film optical constants from the reflectance and thickness data. The use of this material in a hollow waveguide structure for the delivery of CO(2) laser radiation for surgical applications is discussed. Calculated waveguide losses indicate that solgel-based alumina is a good candidate material for this application.

Ab-interno neodymium:YAG versus erbium:YAG laser sclerostomies in a rabbit model.

This study was undertaken to determine whether thermally-induced tissue necrosis was a factor in ab-interno contact-laser sclerostomy failure. A rabbit model was used to compare the continuous-wave Neodymium (Nd):YAG with the pulsed Erbium (Er):YAG laser with respect to such failure. Laser energy was focused into a fused-silica fiber optic (400 microns) for the Nd:YAG laser (12 W; 3 to 5 seconds), and into a single-crystal, uncladded sapphire fiber optic (250 microns) for the Er:YAG laser (7 to 8 mJ; 250 microseconds; 6 to 8 pulses). The Nd:YAG and Er:YAG lasers required from 21 to 35 J and from 42 to 64 mJ, respectively, to create the sclerostomies. Filtering blebs and intraocular pressure reduction lasted longer (log-rank test; P less than .03) and surgical complications were fewer in the Er:YAG group than in the Nd:YAG group. By creating sclerostomies with minimal thermal damage, the Er:YAG laser may offer significant clinical advantages over lasers producing larger thermal effects.

Human ribonucleotide reductase. Activation and inhibition by analogs of ATP.

Sixteen ATP analogs were studied as activators of CDP reduction catalyzed by human ribonucleotide reductase. Activation constants were determined. Three analogs, 3-deazaATP, 5'-adenylimidodiphosphate, and 3'-dATP, activated approximately as efficiently as ATP. Four analogs were partial activators. These seven activators were also accessory activators of GDP reduction. Furthermore, two other analogs, adenosine-5'-O-(1-thiotriphosphate) and 8-bromoATP, selectively stimulated GDP reduction. Ten analogs, at equal molar concentrations with ATP, inhibited ATP-dependent activation of CDP reduction and/or accessory activation of GDP reduction by greater than 45%. No analog inhibited as potently as 2'-dATP, which had an IC50 of 30-50 microM versus the stimulation of CDP and GDP reduction by 2.0 mM ATP.

Attenuation of incoherent infrared radiation in hollow sapphire and silica waveguides.

The attenuation of incoherent infrared radiation for wavelengths from 6 to 20 microm was investigated for hollow sapphire and silica waveguides suitable for applications in spectroscopy and thermometry. A low-attenuation region was exhibited between 9.6 and 17.2 microm for hollow sapphire fibers and between 7.25 and 9.5 microm for hollow silica fibers as a result of the cladding index of refraction dipping below that of the air core (n approximately 1). Losses have been characterized as a function of fiber diameter, launch conditions, and waveguide bend radius for cladding regions of both n > 1 and n < 1. In addition, the remote infrared sensing capability of the hollow waveguides was demonstrated by the detection of CO(2) in N(2) by utilizing hollow sapphire fibers capped with ZnSe windows.

Herpes simplex virus type 1 ribonucleotide reductase: selective and synergistic inactivation by A1110U and its iron complex.

2-Acetylpyridine-5-[(dimethylamino)thiocarbonyl]thiocarbonohydr azone (A1110U) inactivated herpes simplex virus Type 1 ribonucleotide reductase (EC 1.17.4.1) by a first-order process (kinact) which had a maximum value (Mkinact) of 8 hr-1 and a Kd that was less than 1 microM. The stable complex between iron and A1110U, (A1110U)2Fe+i, inactivated this enzyme with a Mkinact of 7 hr-1 and a Kd of 7 microM. Free A1110U and its iron-complex synergized as inactivators of the enzyme. For example, the kinact for the combination of 2 microM A1110U and 1 microM (A1110U)2Fe+i as independent inactivators was calculated to be about 9 hr-1, while the observed value was 32 hr-1. The bimolecular rate constant for inactivation of the viral enzyme by (A1110U)2Fe+i in the presence of a saturating concentration of A1110U was 2.5 10(7) M-1 hr-1 at 30 degrees. Human ribonucleotide reductase was less sensitive to the inhibitory effects of A1110U and its iron-complex. This enzyme was neither inhibited nor inactivated by A1110U and was weakly inhibited by (A1110U)2Fe+i. Furthermore, inactivation required the combination of A1110U and (A1110U)2Fe+i. The bimolecular rate constant for inactivation of human ribonucleotide reductase by (A1110U)2Fe+i in the presence of a saturating concentration of A1110U was considerably smaller (3.8 10(6) M-1 hr-1 at 37 degrees) than the analogous constant for the viral enzyme. Several iron-chelating reagents with unrelated structures substituted for free A1110U in its various roles with both enzymes. However, the iron complexes of these alternative chelators did not substitute for (A1110U)2Fe+i. The rates of inactivation of both enzymes were independent of the oxidation state of iron in (A1110U)2Fe+i and of CDP concentration. The inactivated enzymes were reactivated rapidly by FeSO4, but were not reactivated by CoCl2, CuSO4, or NiCl2. MnCl2 inhibited reactivation of the viral enzyme by FeSO4.

Purine deoxynucleoside salvage in Giardia lamblia.

Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines, including deoxythymidine. Dependence on deoxynucleoside salvage is extremely unusual among eucaryotic cells (Moore, E. C., and Hurlbert, R. B. (1985) Pharmacol & Ther. 27, 167-196). The present study investigates the possibility that giardia lacks ribonucleotide reductase and depends entirely on deoxynucleoside salvage. A ribonucleotide reductase inhibitor, hydroxyurea, at concentrations up to 2 mM had no effect on the growth of giardia. This is 15-20 times the ED50 of hydroxyurea for the protozoans Trypanosoma cruzi, Trypanosoma gambiense, and Leishmania donovani. A lysate of giardia had no detectable ribonucleotide reductase. Although radiolabeled adenine, adenosine, guanine, and guanosine were readily incorporated into RNA by cultured cells, no adenine or adenosine and only trace amounts of guanine and guanosine were detectable in DNA. This is in contrast to deoxynucleosides, where 58% of deoxyadenosine and 10% of deoxyguanosine incorporated into nucleic acid were found in DNA. Phosphorylation of both deoxyadenosine and deoxyguanosine was catalyzed by a cell lysate of giardia when nucleoside kinase co-substrates were included in the assay but not when phosphotransferase co-substrates were present. The absence of detectable ribonucleotide reductase, the failure to incorporate purine nucleobases and nucleosides into DNA to any significant extent, the ready incorporation of deoxynucleosides into DNA, and the demonstration of a purine deoxynucleoside kinase suggest that giardia are dependent on the salvage of exogenous deoxynucleosides.

Production of highly labeled adenoviruses.

A method is described for increasing the incorporation of radioactive thymidine into adenovirus deoxyribonucleic acid by the use of amethopterin. In addition, a modified procedure is presented for the preparation of highly purified adenoviruses. This procedure, which employs enzymatic digestion of cellular debris, obviates the necessity for fluorocarbon treatment of crude virus suspensions, and routinely provides excellent recovery of virus.