Mechanistic Multiscale Pharmacokinetic Model for the Anticancer Drug 2',2'-difluorodeoxycytidine (Gemcitabine) in Pancreatic Cancer.

The aim of this work is to build a mechanistic multiscale pharmacokinetic model for the anticancer drug 2',2'-difluorodeoxycytidine (gemcitabine, dFdC), able to describe the concentrations of dFdC metabolites in the pancreatic tumor tissue in dependence of physiological and genetic patient characteristics, and, more in general, to explore the capabilities and limitations of this kind of modeling strategy. A mechanistic model characterizing dFdC metabolic pathway (metabolic network) was developed using in vitro literature data from two pancreatic cancer cell lines. The network was able to describe the time course of extracellular and intracellular dFdC metabolites concentrations. Moreover, a physiologically-based pharmacokinetic model was developed to describe clinical dFdC profiles by using enzymatic and physiological information available in the literature. This model was then coupled with the metabolic network to describe the dFdC active metabolite profile in the pancreatic tumor tissue. Finally, global sensitivity analysis was performed to identify the parameters that mainly drive the interindividual variability for the area under the curve (AUC) of dFdC in plasma and of its active metabolite (dFdCTP) in tumor tissue. From this analysis, cytidine deaminase (CDA) concentration was identified as the main driver of plasma dFdC AUC interindividual variability, whereas CDA and deoxycytidine kinase concentration mainly explained the tumor dFdCTP AUC variability. However, the lack of in vitro and in vivo information needed to characterize key model parameters hampers the development of this kind of mechanistic approach. Further studies to better characterize pancreatic cell lines and patient enzymes polymorphisms are encouraged to refine and validate the current model.

Determinants of the interindividual variability in serum cytidine deaminase activity of patients with solid tumours.

AIMS: Cytidine deaminase (CDA) activity in cancer patients' serum has been proposed as a predictive biomarker for efficacy and toxicity of nucleoside analogues. However, discrepant results about its predictive value have been reported due to the high interindividual variability in CDA activity. This study aimed at identifying determinants of this interindividual variability. METHODS: From December 2014 to November 2015, 183 patients were prospectively included. Serum CDA activity, biological and clinical characteristics as well as five common single nucleotide polymorphisms (SNPs) in the CDA gene (c.-451C > T, c.-92A > G, c.-33_-31delC, c.79A > C, c.435 T > C) were analysed. Associations between clinical characteristics, pharmacogenetic variants and CDA activity were univariately tested. P < 0.1-candidate variables were analysed through a multivariate analysis. The association between CDA activity and toxicity was assessed for the 56 gemcitabine-treated patients. Intraindividual variability in CDA activity was explored in six pancreatic cancer patients treated with gemcitabine. RESULTS: Median CDA activity was 3.97 U mg-1 (range 1.53-15.49 U mg-1 ). A univariate analysis showed that CDA activity was statistically associated with Eastern Cooperative Oncology Group performance status, mild or severe malnutrition, inflammatory syndrome, leucocyte count, neutrophil count, albumin, C-reactive protein and -c.-33_-31delC single nucleotide polymorphism. A multivariate analysis identified that only neutrophil count (P < 0.0001) and severe malnutrition (P = 0.0278) were independently associated with CDA activity. Low CDA activity (<2 U mg-1 ) was not statistically associated with severe gemcitabine-related toxicities (P = 0.16). A decrease in CDA activity was observed during the longitudinal follow-up of six pancreatic cancer patients treated with gemcitabine (P = 0.03). CONCLUSIONS: These results suggest that neutrophil count and malnutrition should be considered for the interpretation of pretherapeutic CDA activity.

IFN-α-mediated Base Excision Repair Pathway Correlates with Antiviral Response Against Hepatitis B Virus Infection.

Previous studies identified APOBEC deaminases as enzymes targeting hepatitis B virus (HBV) DNA in the nucleus thus affecting its persistence. Interferon (IFN)-α treated chimpanzees and hepatitis C patients showed elevated APOBEC expression. We thus hypothesized that the responses to IFN-α treatment of chronic hepatitis B (CHB) patients is influenced by IFN-induced base excision repair (BER). CHB-treatment naïve patients, patients treated with PEGylated IFN-α, and patients with sequential treatment of Entecavior and PEGylated IFN-α were recruited. Blood and liver biopsy samples were collected before treatment and at treatment endpoint. BER genes were assessed by quantitative RT-PCR. BER gene expression levels and IFN treatment responses were correlated in patient liver biopsies. APOBEC3A, -B, -C, -D/E, and-G mRNA levels were up-regulated in IFN-treated patients. APOBEC3A expression was significantly higher in IFN-responders than in non-responders. BER genes NEIL3 was down-regulated in IFN-treated patients. APOBEC3 and BER gene expression at treatment endpoints partially correlated with the corresponding absolute DNA level or degree of HBsAg and HBV DNA decline. Our study suggests that the expression of APOBEC3A positively correlates with IFN-treatment responses in CHB patients, while NEIL3 shows negative correlation. These genes may involve to IFN mediated viral suppression and serve as biomarkers for CHB disease management.

AID expression in peripheral blood of children living in a malaria holoendemic region is associated with changes in B cell subsets and Epstein-Barr virus.

The development of endemic Burkitt's lymphoma (eBL) is closely associated with Epstein-Barr virus (EBV) infection and holoendemic malaria infections. The role of EBV in the development of malignancy has been studied in depth, but there is still little known about the mechanisms by which malaria affects Burkitt's lymphomagenesis. Activation induced cytidine deaminase (AID) expression is necessary for the introduction of c-myc translocations that are characteristic of BL, but a link between AID and EBV or malaria is unclear. To determine whether frequency of malaria exposure leads to increased AID expression in peripheral blood mononuclear cells (PBMC) we examined two cohorts of children in western Kenya with endemic and sporadic malaria transmission dynamics. High frequency of malaria exposure led to increased expression of AID, which coincided with decreases in the IgM(+) memory B cells. In the children from the malaria endemic region, the presence of a detectible EBV viral load was associated with higher AID expression compared to children with undetectable EBV, but this effect was not seen in children with sporadic exposure to malaria. This study demonstrates that intensity of malaria transmission correlates with AID expression levels in the presence of EBV suggesting that malaria and EBV infection have a synergistic effect on the development of c-myc translocations and BL.

Selection of the best blood compartment to measure cytidine deaminase activity to stratify for optimal gemcitabine or cytarabine treatment.

Cytidine deaminase (CDA) plays a crucial role in the degradation of cytidine analogs, such as gemcitabine and cytarabine. Several studies showed that a low CDA activity is associated with more toxicity but a higher efficacy, while a high activity will lead to a lower efficacy but less toxicity. A stratified dosing strategy based on the relative CDA activity would increase efficiency. In order to predict these events, a reliable measurement of CDA with a validated method is crucial. We aimed to determine which phenotype assay would be most suitable; a spectrophotometric assay using cytidine as a substrate, or an HPLC assay using gemcitabine as a substrate. In serum and whole blood of 26 volunteers, both assays showed an excellent correlation (R>0.999), but not in plasma nor in red blood cells. Moreover, there was no difference between males and females. In conclusion, the spectrophotometric assay seems the most simple and cost-effective test. It should be performed in serum, while it should be normalized on protein content as measured by the Bicinchoninic Acid.

Activation-induced cytidine deaminase expression in CD4+ T cells is associated with a unique IL-10-producing subset that increases with age.

Activation-induced cytidine deaminase (AID), produced by the Aicda gene, is essential for the immunoglobulin gene (Ig) alterations that form immune memory. Using a Cre-mediated genetic system, we unexpectedly found CD4(+) T cells that had expressed Aicda (exAID cells) as well as B cells. ExAID cells increased with age, reaching up to 25% of the CD4(+) and B220(+) cell populations. ExAID B cells remained IgM(+), suggesting that class-switched memory B cells do not accumulate in the spleen. In T cells, AID was expressed in a subset that produced IFN-γ and IL-10 but little IL-4 or IL-17, and showed no evidence of genetic mutation. Interestingly, the endogenous Aicda expression in T cells was enhanced in the absence of B cells, indicating that the process is independent from the germinal center reaction. These results suggest that in addition to its roles in B cells, AID may have previously unappreciated roles in T-cell function or tumorigenesis.

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease.

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

Cytidine deaminase residual activity in serum is a predictive marker of early severe toxicities in adults after gemcitabine-based chemotherapies.

PURPOSE Anticipating toxicities with gemcitabine is an ongoing story, and deregulation in cytidine deaminase (CDA) could be associated with increased risk of developing early severe toxicities on drug exposure. PATIENTS AND METHODS A simple test to evaluate CDA phenotypic status was first validated in an animal model investigating relationships between CDA activity and gemcitabine-related toxicities. Next, relevance of this test as a marker for toxicities was retrospectively tested in a first subset of 64 adult patients treated with gemcitabine alone, then it was tested in a larger group of 130 patients who received gemcitabine either alone or combined with other drugs and in 20 children. Additionally, search for the 435 T>C, 208 G>A and 79 A>C mutations on the CDA gene was performed. Results In mice, CDA deficiency impacted on gemcitabine pharmacokinetics and had subsequent lethal toxicities. In human, 12% of adult patients experienced early severe toxicities after gemcitabine administration. A significant difference in CDA activities was observed between patients with and without toxicities (1.2 +/- 0.8 U/mg v 4 +/- 2.6 U/mg; P < .01). Conversely, no genotype-to-phenotype relationships were found. Of note, the patients who displayed particularly reduced CDA activity all experienced strong toxicities. Gemcitabine was well tolerated in children, and no CDA deficiency was evidenced. CONCLUSION Our data suggest that CDA functional testing could be a simple and easy marker to discriminate adult patients at risk of developing severe toxicities with gemcitabine. Particularly, this study demonstrates that CDA deficiency, found in 7% of adult patients, is associated with a maximum risk of developing early severe toxicities with gemcitabine.

Optimized blood sampling with cytidine deaminase inhibitor for improved analysis of capecitabine metabolites.

The 5FU prodrug capecitabine undergoes a 3-step enzymatic conversion, including the conversion of 5'DFRC into 5'DFUR by cytidine deaminase (CDA). The presence of CDA activity in blood led us to analyze the possible ex vivo conversion of 5'DFCR into 5'DFUR in blood samples. We thus examined the impact of the addition of a CDA inhibitor (tetrahydrouridine (THU) 1 microM final) in blood. Blood samples from 3 healthy volunteers were taken on tubes containing or not THU. Blood was spiked with 5'DFCR (20 microM final) (T0) and was maintained at room temperature for 2 h. Plasma concentrations of 5'DFRC and 5'DFUR were analyzed with an optimized HPLC assay. In the absence of THU, 5'DFUR was detectable as early as T0. The percent of 5'DFUR produced relative to 5'DFCR increased over time, up to 7.7 % at 2h. In contrast, the presence of THU totally prevents the formation of 5'DFUR. The impact of THU for preventing the conversion of 5'DFCR was confirmed by the analysis of blood samples from 2 capecitabine-treated patients. Addition of THU in the sampling-tube before the introduction of blood is thus strongly recommended in order to guarantee accurate conditions for reliable measurement of capecitabine metabolites in plasma, and thus faithful pharmacokinetic data.

Toxic death case in a patient undergoing gemcitabine-based chemotherapy in relation with cytidine deaminase downregulation.

Gemcitabine is an antimetabolite drug used in the treatment of various solid tumours, including lung, pancreatic or gynaecological cancers. Innovative combinational strategies (e.g. gemcitabine+capecitabine or gemcitabine+oxaliplatin) make gemcitabine an extensively prescribed drug now. Gemcitabine is characterized by a narrow therapeutic index, and its liver elimination depends upon a key enzymatic step, driven by cytidine deaminase (CDA). CDA is prone to gene polymorphism, including the 208A>G mutation, which can result in marked enzymatic deficiency with subsequent impact on drug exposure levels and related toxicities. We have developed a simple and inexpensive method to determine phenotypically CDA status in cancer patients, as an attempt to detect those at risk upon gemcitabine intake. Conjointly to genotypic investigations, this method was used to phenotype, in a retrospective setting, a female patient displaying extremely severe, and eventually lethal, toxicities after administration of a standard gemcitabine/carboplatin protocol. Phenotypic investigation showed a marked CDA deficiency (-75%) in this patient when compared with a reference, nontoxic population. Genetic studies undertaken next to screen mutations, possibly at the origin of this deficiency, showed heterozygosity for the 79A>C single-point mutation, whereas surprisingly the canonical CDA 208A>G polymorphism was not found. Taken together, this case report demonstrates, for the first time, that CDA downregulation can lead to toxic-death in patients exposed to gemcitabine. Besides, we showed here that our cost-effective and simple phenotypic approach should enable, in the future, the detection of deficient patients at risk upon gemcitabine administration.

Pharmacokinetics of gemcitabine in Japanese cancer patients: the impact of a cytidine deaminase polymorphism.

PURPOSE: Gemcitabine is rapidly metabolized to its inactive metabolite, 2',2'-difluorodeoxyuridine (dFdU), by cytidine deaminase (CDA). We previously reported that a patient with homozygous 208A alleles of CDA showed severe adverse reactions with an increase in gemcitabine plasma level. This study extended the investigation of the effects of CDA genetic polymorphisms on gemcitabine pharmacokinetics and toxicities. PATIENTS AND METHODS: Genotyping of CDA was performed by a direct sequencing of DNA obtained from the peripheral blood of Japanese gemcitabine-naïve cancer patients (n = 256). The patients recruited to the association study received a 30-minute intravenous infusion of gemcitabine at a dose of either 800 or 1,000 mg/m2, and eight blood samples were periodically collected (n = 250). Plasma levels of gemcitabine and dFdU were measured by high-performance liquid chromatography. Plasma CDA activities toward cytidine and gemcitabine were also measured (n = 121). RESULTS: Twenty-six genetic variations, including 14 novel ones and two known nonsynonymous single nucleotide polymorphisms (SNPs), were detected. Haplotypes harboring the nonsynonymous SNPs 79A>C (Lys27Gln) and 208G>A (Ala70Thr) were designated *2 and *3, respectively. The allelic frequencies of the two SNPs were 0.207 and 0.037, respectively. Pharmacokinetic parameters of gemcitabine and plasma CDA activities significantly depended on the number of haplotype *3. Haplotype *3 was also associated with increased incidences of grade 3 or higher neutropenia in the patients who were coadministered fluorouracil, cisplatin, or carboplatin. Haplotype *2 showed no significant effect on gemcitabine pharmacokinetics. CONCLUSION: Haplotype *3 harboring a nonsynonymous SNP, 208G>A (Ala70Thr), decreased clearance of gemcitabine, and increased incidences of neutropenia when patients were coadministered platinum-containing drugs or fluorouracil.

Cytidine deaminase in polymyalgia rheumatica and elderly onset rheumatoid arthritis.

Serum cytidine deaminase (CD) as a marker of inflammatory disease was assessed in 44 patients and 47 controls to differentiate polymyalgia rheumatica (PMR) from elderly onset rheumatoid arthritis (EORA). The patients were divided into four groups: PMR with and without synovitis and seropositive and seronegative EORA. No statistically significant differences were found when serum CD levels of seropositive EORA patients were compared with serum CD of PMR patients without synovitis, neither when serum CD levels of all PMR patients were compared with a seronegative EORA group, nor when serum CD levels of PMR patients with synovitis were compared with those with EORA. Nevertheless, statistically significant differences were detected between EORA's serum CD levels and the control group (p=0.023). This difference was 10% when comparing CD levels of PMR patients with the control group (p=0.070). We did not demonstrate that serum CD levels could be a useful tool to differentiate PMR from EORA, but these findings could nevertheless reflect the presence of an inflammatory disease.

Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation.

In B cells, somatic hypermutation (SHM) and class switch recombination (CSR) depend on the activation-induced cytidine deaminase (AID) gene product, although the precise mode of action of AID remains unknown. Because some chronic lymphocytic leukemia (CLL) B cells can undergo CSR without SHM, it constitutes a useful model to dissect AID function. In this work, we have studied AID expression, the presence of mutations in the preswitch mu DNA region, CSR, and the SHM in 65 CLL patients. Our results demonstrate that unmutated CLL B cells can constitutively express AID and that AID expression is associated with the presence of mutations in the preswitch region and in clonally related isotype-switched transcripts. They also demonstrate that in CLL without constitutive AID expression, AID induction on stimulation results in preswitch mutations and the CSR process. Our results show a dissociation between SHM and CSR in CLL and suggest that, in this disease, AID would require additional help for carrying out the SHM process.

Serum adenosine deaminase and cytidine deaminase activities in patients with systemic lupus erythematosus.

We evaluated serum total adenosine deaminase, its isoenzymes adenosine deaminase-1 and adenosine deaminase-2, and cytidine deaminase activities in 24 patients with active systemic lupus erythematosus, and in 26 healthy control subjects, and found the means +/- SD values to be 21.38 +/- 5.96 IU/l, 3.74 +/- 2.12 IU/l, 17.72 +/- 5.02 IU/l and 17.89 +/- 4.62 IU/l, respectively in the patients, and 14.97+/- 4.71 IU/l, 4.01 +/- 1.35 IU/l, 10.91 +/- 3.91 IU/l and 7.39 +/- 3.97 IU/l, respectively in the control subjects. When compared to the healthy controls, serum total adenosine deaminase, adenosine deaminase-2 and cytidine deaminase levels were significantly higher (p<0.001) in systemic lupus erythematosus patients, but the decrease of adenosine deaminase-1 level was not statistically significant (p>0.05). The increased adenosine deaminase-2 may be of macrophage origin. It closely correlated with clinical signs of active systemic lupus erythematosus. The membranes of polymorphonuclear neutrophils may be damaged, and cytidine deaminase may be released into serum. In conclusion, serum total adenosine deaminase, adenosine deaminase-2 and cytidine deaminase activities may serve as useful indicators for evaluating disease activity in patients with active systemic lupus erythematosus.

Bioactive cytidine deaminase, an inhibitor of granulocyte-macrophage colony-forming cells, is massively released in fulminant meningococcal sepsis.

Cytidine deaminase (CDD) catalyzes the hydrolytic deamination of cytidine, which thereby is converted to uridine. CDD is found in serum and different tissues, with particularly high concentrations in polymorphonuclear neutrophils (PMN). We measured the CDD levels in plasma from patients with systemic meningococcal disease. Thirty-seven patients had significantly higher plasma levels of CDD than did 29 healthy control subjects (P=.0001). CDD levels in plasma or serum increased from a median of 96 ng/mL in healthy control subjects to medians of 168 ng/mL in patients without persistent shock (n=23; P=.001) and 422 ng/mL in patients with fulminant meningococcal septicemia (n=14; P=.0001). In most patients with fulminant septicemia, CDD levels in plasma increased during the first 3-53 h after the initiation of therapy (P=.003). CDD alone had no immediate harmful effect when injected into mice during a 4-day period. CDD may modulate the stimulatory effect of colony-stimulating factors on PMN in patients.

Changes in biochemical markers of joint tissue metabolism in a randomized controlled trial of glucocorticoid in early rheumatoid arthritis.

OBJECTIVE: To determine the effects of low-dose prednisolone on joint tissue metabolism in early rheumatoid arthritis (RA). METHODS: In addition to a range of biochemical markers of cartilage, bone and synovial tissue turnover, levels of pro-matrix metalloproteinase 3 (pro-MMP-3), pro-MMP-1, and cytidine deaminase (CD) were measured in serum from 79 of 128 patients with early RA who took part in the Arthritis and Rheumatism Council Low-Dose Glucocorticoid Study. Serum concentrations of joint tissue metabolites on treatment and off treatment were compared using Student's t-test. RESULTS: Levels of the keratan sulfate epitope, 5D4, and glycosaminoglycan (GAG) were similar on and off treatment. However, the levels of synovium-derived markers, hyaluronate (HA) and N-propeptide of type III procollagen (PIIINP), were reduced by 23.9% (P < 0.01) and 25.2% (P < 0.001), respectively, during treatment with prednisolone. Serum osteocalcin (OC) was reduced by 25.8% (P < 0.001), while the levels of CD and pro-MMP-3 increased by 31.2% (P < 0.01) and 53.7% (P < 0.001) during prednisolone treatment compared with the off-treatment period. CONCLUSION: Low-dose prednisolone had no significant effect on markers of cartilage turnover (GAG, 5D4) in early RA, suggesting that early erosions do not involve cartilage surfaces. The reduction in the markers of bone turnover (OC) and synovial tissue turnover (HA and PIIINP) support the general view that prednisolone reduces synovitis and suppresses bone turnover.