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FOLFOX is one of the most effective treatments for advanced colorectal cancer. However, cumulative oxaliplatin neurotoxicity often results in halting the therapy. Oxaliplatin functions predominantly via the formation of toxic covalent drug–DNA adducts. We hypothesize that oxaliplatin–DNA adduct levels formed in vivo in peripheral blood mononuclear cells (PBMC) are proportional to tumor shrinkage caused by FOLFOX therapy. We further hypothesize that adducts induced by subtherapeutic “diagnostic microdoses” are proportional to those induced by therapeutic doses and are also predictive of response to FOLFOX therapy. These hypotheses were tested in colorectal cancer cell lines and a pilot clinical study. Four colorectal cancer cell lines were cultured with therapeutically relevant (100 μmol/L) or diagnostic microdose (1 μmol/L) concentrations of [14C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin–DNA adduct level with accelerator mass spectrometry (AMS). Oxaliplatin–DNA adduct formation was correlated with oxaliplatin cytotoxicity for each cell line as measured by the MTT viability assay. Six colorectal cancer patients received by intravenous route a diagnostic microdose containing [14C]oxaliplatin prior to treatment, as well as a second [14C]oxaliplatin dose during FOLFOX chemotherapy, termed a “therapeutic dose.” Oxaliplatin–DNA adduct levels from PBMC correlated significantly to mean tumor volume change of evaluable target lesions (5 of the 6 patients had measurable disease). Oxaliplatin–DNA adduct levels were linearly proportional between microdose and therapeutically relevant concentrations in cell culture experiments and patient samples, as was plasma pharmacokinetics, indicating potential utility of diagnostic microdosing.

Supplementary Figure S1 showing correlation between CRC cell line oxaliplatin sensitivity and oxaliplatin-DNA area under the adduct curve

IntroductionThe COVID-19 pandemic brought an urgent need to discover novel effective therapeutics for patients hospitalized with severe COVID-19. The ISPY COVID trial was designed and implemented in early 2020 to evaluate investigational agents rapidly and simultaneously on a phase 2 adaptive platform. This manuscript outlines the design, rationale, implementation, and challenges of the ISPY COVID trial during the first phase of trial activity from April 2020 until December 2021.Methods and analysisThe ISPY COVID Trial is a multi-center open label phase 2 platform trial in the United States designed to evaluate therapeutics that may have a large effect on improving outcomes from severe COVID-19. The ISPY COVID Trial network includes academic and community hospitals with significant geographic diversity across the country. Enrolled patients are randomized to receive one of up to four investigational agents or a control and are evaluated for a family of two primary outcomes—time to recovery and mortality. The statistical design uses a Bayesian model with “stopping” and “graduation” criteria designed to efficiently discard ineffective therapies and graduate promising agents for definitive efficacy trials. Each investigational agent arm enrolls to a maximum of 125 patients per arm and is compared to concurrent controls. As of December 2021, 11 investigational agent arms had been activated, and 8 arms were complete. Enrollment and adaptation of the trial design is ongoing.Ethics and disseminationISPY COVID operates under a central institutional review board via Wake Forest School of Medicine IRB00066805. Data generated from this trial will be reported in peer reviewed medical journals.Trial registration numberClinicaltrials.gov registration number NCT04488081Strengths and limitations of this studyThe ISPY COVID Trial was developed in early 2020 to rapidly and simultaneously evaluate therapeutics for severe COVID-19 on an adaptive open label phase 2 platformThe ISPY COVID Adaptive Platform Trial Network is an academic-industry partnership that includes academic and community hospitals spanning a wide geographic area across the United StatesOf December 2021, 11 investigational agent arms have been activated on the ISPY COVID Trial PlatformThe ISPY COVID Trial was designed to identify therapeutic agents with a large clinical effect for further testing in definitive efficacy trials—limitations to this approach include the risk of a type 2 error

A photochemical treatment (PCT) method to inactivate pathogens in platelet concentrates has been developed. The system uses a psoralen, amotosalen HCl, coupled with ultraviolet A (UVA) illumination.Three sequential clinical trials evaluated viability of PCT platelets prepared with a prototype device. Posttransfusion recovery and lifespan of (111)Indium-labeled autologous 5 day-old platelets in healthy subjects was assessed. In the first study, 23 subjects received transfusions of autologous PCT and/or control platelets. In a second study, 16 of these subjects received PCT platelets processed with a Compound Adsorption Device (CAD) (PCT-CAD) to reduce patient exposure to residual amotosalen. In the third study, the effect of gamma-irradiation on PCT platelets was studied. Data from control transfusions from Study A were used for paired comparisons in the latter 2 studies.Mean PCT-CAD platelet recovery for the 16 subjects with paired data was 42.5 +/- 8.7% versus 50.3 +/- 7.7% for control platelets, mean difference of 7.8% (p0.01). Mean lifespan for PCT-CAD platelets was 4.8 days (+/-1.3) versus 6.0 days (+/-1.2) for control platelets, mean difference of 1.3 days (p0.01). Platelet recovery and lifespan were similar to PCT-CAD for PCT without CAD treatment and PCT-CAD with gamma-irradiation.Viability of 5 day-old PCT platelets was less than for control platelets. However, both were within ranges reported for 5 day-old platelets.

The activity of sialytransferase with regard to the glycoprotein substrates asialofetuin and asialo-ovine submaxillary mucin was determined in normal, pathological control, and cystic fibrosis liver homogenates. Cystic fibrosis and pathological livers have about 40% of the average normal specific activity for sialytransferase. Several properties of cystic fibrosis sialytransferase were investigated and compared to those of the normal liver enzyme (Alhadeff et al. 1977). The pH optima curves were similar, but cystic fibrosis sialyltransferase appears to be more thermolabile than the normal liver enzyme. Isoelectric focusing studies revealed that the three most basic forms of sialyltransferase which are found in normal livers are deficient or absent in most cystic fibrosis liver. The data suggest that altered glycoprotein-sialyltransferases may be present in cystic fibrosis livers, probably a secondary effect due to general liver pathology.

The chemical relationship of the seven forms of human liver alpha-L fucosidase has been studied by isoelectric focusing of neuraminidase- and sialytransferase-treated preparations of alpha-L-fucosidase. Neuraminidase treatment leads to a decrease in the activity of the more acidic forms (IV-VII) and a concimitant increase in the activity of the more neutral forms (I-II). Incubation of the neuraminidase-treated fucosidase (forms I-III) with radiolabelled cytidine monophosphate-N-3H-acetylneuraminic acid and an enriched preparation of sialytransferase devoid of fucosidase activity led to regeneration of the more acidic fucosidase isoenzymes (IV-VII) with the same isoelectric points and in nearly the same proportion as before neuraminidase treatment. These experiments suggest that the isoenzymes of human liver alpha-L-fucosidase are related, at least in part, by sialic acid residues. The seven isoenzymes of purified human liver alpha-L-fucosidase have been separated by preparative isoelectric focusing and characterized kinetically and immunochemically. Differences in Michaelis constants (Km's) and pH optimum curves were found for some of the isoenzymes. All seven isoenzymes were immunoprecipitated using the IgG fraction of anti-alpha-L-fucosidase antiserum suggesting that the presence of sialic acid residues does not affect the antigenicity of the forms of alpha-L-fucosidase.

Sialyltransferase ( CMP -N- acetylneuraminate: d -galactosyl-glycoprotein N- acetylneuraminyltransferase , EC 2.4.99.1) has been studied using the two glycoprotein substrates asialofetuin and asialo-ovine submaxillary mucin (A-OSM) in fresh and frozen human adult and fetal livers. Enzymatic activity for both substrates is stimulated by Mg2+ (10–25 mM) and Triton X-100 (0.4–0.6%, v/v). Freeze-thaw, heat denaturation, stability on storage, additive and substrate competition studies suggest the presence of at least two forms of human liver glycoprotein sialyltransferase. Subcellular fractionation studies indicate that the major parts of both sialyltransferase activities are associated with membraneous fractions but that a greater percentage of A-OSM than asialofetuin activity is associated with these fractions. Subcellular fractionation studies using O.8% (v/v) Triton X-100 in the extracting buffer greatly diminishes the percentage of both asialofetuin and A-OSM sialyltransferase activity found in the mitochondrial and microsomal pellets. Kinetic characterization of sialyltransferase in a liver homogenate, resuspended 48 200 × g pellet and 48 200 × g supernatant suggested the possibility of two asialofetuin sialyltransferases. The pH optimum for homogenate and supernatant enzymes with respect to asialofetuin was 7.2 with a suggested second optimum at 8.5. The pellet enzyme had only one optimum at pH 7.2. Michaelis constants with regard to asialofetuin were 0.08, 0.06 and 0.22 mM in homogenate, resuspended pellet and supernatant, respectively. The homogenate had a Km of 5 μM for CMP-N-acetylneuraminic acid (CMP-NANA). Isoelectric focusing indicated that at least eight isoelectric forms of sialyltransferase with pI values ranging from pH 5.0 to 8.6 are present in the homogenate; several isoelectric forms were preferentially associated with the pellet or supernatant. All the data suggest at least two forms of liver glycoprotein sialyltransferase. One form is thermolabile, loses activity on freezing and storage, and is associated with membraneous fractions and A-OSM activity. The second form is less thermolabile, more stable to freezing and storage, less associated with membraneous fractions and associated more with asialofetuin activity. Furthermore, kinetic characterization of sialytransferase activity in liver homogenate, resuspended pellet and supernatant suggests the presence of two asialofetuin sialyltransferases.