A Bayesian phase I–II clinical trial design to find the biological optimal dose on drug combination

In recent years, combined therapy shows expected treatment effect as they increase dose intensity, work on multiple targets and benefit more patients for antitumor treatment. However, dose -finding designs for combined therapy face a number of challenges. Therefore, under the framework of phase I–II, we propose a two-stage dose -finding design to identify the biologically optimal dose combination (BODC), defined as the one with the maximum posterior mean utility under acceptable safety. We model the probabilities of toxicity and efficacy by using linear logistic regression models and conduct Bayesian model selection (BMS) procedure to define the most likely pattern of dose–response surface. The BMS can adaptively select the most suitable model during the trial, making the results robust. We investigated the operating characteristics of the proposed design through simulation studies under various practical scenarios and showed that the proposed design is robust and performed well.</p

CC5A/PBS induces A3G and A3F expression in human oral keratinocyte (OKF6) and human vaginal VK2 epithelial cells.

<p>(<b>A</b>) CC5A/PBS (5 µg) was used to treat OKF6 cells. The cells were harvested at the indicated time points, and qRT-PCR was used to measure the relative expression of A3F and A3G mRNA. The expression levels shown represent the ratio of CC5A/PBS treated cells compared to the untreated cells at each given time point. (<b>B</b>) Different doses (0–5 µg) of CC5A/PBS were used to treat OKF6 cells for 72 h, and A3G expression was measured by Western-blot analysis. (<b>C</b>) VK2 cells were treated with 40 µg CC5A/PBS or vehicle for the time periods shown. qRT-PCR was used to measure the relative expression of A3F and A3G mRNA in these cells. The expression levels shown represent the ratio of the levels found in CC5A/PBS treated cells compared to untreated cells at each time point. (<b>D</b>) Different doses (0–40 µg) of CC5A/PBS were used to treat VK2 cells for 48 h. A3G protein expression was measured by Western-blot analysis.</p

A3G knockdown compromised the effect of CC5A in reducing HIV infectivity.

<p>pX330-A3G1 and pX330-A3G2 (3 µg each) were transfected into 1×10⁶ THP-1 cells using Neon Transfection System (Invitrogen). Three days post transfection, the transfected THP-1 cells were cloned by limited dilution method. Single clones were screen by Western-blot and qRT-PCR for A3G expression analysis. A3GKD was selected as A3G knockdown cell line for further experiment. A3G expression was tested by Western-blot (A) and qRT-PCR (B) analysis. THP-1/IIIB and THP-1 A3GKD/IIIB were treated with CC5A or PBS to measure the effects of CC5A on reducing HIV infectivity in THP-1 and THP-1 A3GKD cell line (B).</p

CC5A/PBS and sonicated CC5A do not cause cytotoxicity in THP-1 cells.

<p>The cytotoxicity of CC5A/PBS and sonicated CC5A treatment was measured by a Live/Dead Cell Vitality Assay Kit (Invitrogen). THP-1 cells were treated with PBS, 40 µg CC5A/PBS and 25 µg sonicated CC5A for 16 h then subjected to the Live/Dead Cell Vitality Assay Kit analysis.</p

A3F and A3G are regulated through the MEK1/2 dependent pathway.

<p>(<b>A</b>) Prior to treatment with CC5A/PBS for 16 h, THP-1 cells were pre-treated by DMSO alone or the ERK1/2 inhibitor U0126 in DMSO for 1 h. The induction of A3G and A3F was measured by qRT-PCR. (B) Lysates from THP-1 cells pre-treated DMSO or U0126 in DMSO, following by CC5A/PBS (40 µg) are subjected to Western blot analysis. Phosphorylated ERK1/2 and ERK1/2 were probed by anti-p-ERK1/2 and anti-ERK2, respectively.</p

The active CC5A molecule inhibits HIV-1 replication. (A) THP-1/IIIB cells were treated with PBS or CC5A/PBS for 16 h.

<p>Post-treatment, THP-1 cells were washed twice by PBS and cultured for 24 h. The cells were then washed with PBS and cultured for another 24 h. The culture supernatants were collected for infectivity assays. The amount of input virus for the infectivity assay was normalized by p24 ELISA. (B) THP-1 cells were pre-treated overnight with CC5A/PBS or PBS before IIIB infection. After 3 h of infection, the cells were washed twice with PBS and put back into culture for eight days. Viral samples were collected every other day and viral replication was monitored by p24 assay.</p

CC5A enhances HIV G to A hypermutation rate. HIV from CC5A or PBS treated THP-1 culture supernatants were used to infect SupT1 cells.

<p>The infected SupT1 cells were washed twice with PBS then incubated at 37°C, 5% CO2 for 16 h. HIV cDNA was isolated using DNeasy DNA isolation kit (Qiagen). A 650 bp DNA fragment covering a portion of nef, U3 and R of HIV-1 was amplified by PCR. The PCR products were cloned into the TOPO TA-cloning vector. The nucleotide sequences of individual clones from each infected culture sample were determined. Statistical significance was determined by Mann Whitney U test using GraphPad Prism software. Mann Whitney U test was used to calculate p-value.</p

CC5A/PBS contains a small active heat-stable molecule, which may be covalently conjugated to the cell wall.

<p>(A) CC5A cells were incubated in PBS at 37°C or 4°C respectively. After 4 h of incubation, CC5A/PBS was prepared as described in Material and Methods. The CC5A/37°C and CC5A/4°C were used to treat THP-1 and the relative expression of A3G mRNA was measured by qRT-PCR. (B) CC5A cells were boiled in 2% SDS for 1 h. After being extensively washed with PBS, the SDS-treated CC5A cells were incubated in PBS at 37°C for 4 h for the preparation of CC5A/SDS. CC5A/SDS was used to treat THP-1 cells. The relative expression of A3G mRNA was measured by qRT-PCR. (C) After CC5A cells were treated with 2% SDS, the cells were sonicated to collect the CC5A extract as described in Material and Methods. The sonicated CC5A molecules were used to treat THP-1 cells. The relative expression of A3G mRNA was measured by qRT-PCR. (D) CC5A/PBS (100 µg) was subjected to a D-Salt Polyacrylamide Desalting Column (with a molecular weight cut-off of 6 kD). Two standards are shown by arrows: myoglobin (17 kD) and vitamin b12 (1.3 kD); 1.5 ml fractions were collected. The A3G induction response to eluate samples was measured by qRT-PCR. In all the qRT-PCR assays, the expression of A3G mRNA from untreated THP-1 cells was set as one.</p

A molecule derived from oral <i>Streptococcus cristatus</i> induces APOBEC3F/G expression.

<p>(A) Cell extracts from NC3 (<i>Actinomyces naeslundii</i> NC-3), DL1 (<i>Streptococcus gordonii</i> DL1) and DH5a, CC5A (<i>S. cristatus</i> CC5A), A.V (<i>Actinomyces viscosus</i>), 33277 (<i>Porphyromonas gingivalis</i> 33277) and S.MU (<i>S. mutans</i> KPSK2) were used to treat THP-1 cells. THP-1 cells treated by PBS were used as a control. After 16 h of exposure, total RNA was isolated and the expression of A3G and A3F quantified by qRT-PCR. (B) CC5A and different strains of <i>S. cristatus</i> (pSH11b, pSH11a, CR311, CR3, and CH34110) were used to treat THP-1. The induction of A3G and A3F was measured by qRT-PCR. (C) Different doses (0–40 µg) of CC5A/PBS were used to treat THP-1 cells for 16 h. A3G and A3F expression was measured by qRT-PCR. (D) A3G and A3F expression following a 48 h exposure to 0–10 µg CC5A was tested by Western blot assay. Values shown in (A), (B) and (C) are given as means ± SD of three independent experiments compared to normalized to 1.0 for controls. The Western-blot data is representative of three independent experiments.</p