[A preliminary study on the improved efficacy of mesalazine combined with vitamin D3 in ulcerative colitis].

Objective: To analyze the influence of vitamin D3 supplementation on the clinical efficacy of mesalazine in patients with ulcerative colitis (UC). Methods: From January 2015 to December 2020, patients with mild-to-moderate active UC were retrospectively and continuously enrolled, who accepted mesalazine treatment for at least 12 months at the Second Affiliated Hospital of Wenzhou Medical University. According to simultaneous supplement of vitamin D3 (125 IU/d), the patients were divided into study group and control group. Demographic and disease characteristics, serum 25-hydroxyvitamin D[25(OH)D] levels and other information were collected through retrieving hospital database. Student's t-test, Mann-Whitney U test and Chi-square test were applied for comparison of disease characteristics. The changes of modified Mayo scores[ΔMayo] and 25(OH)D[Δ25(OH)D] were compared before and after treatment by paired t-test, Wilcoxon signed rank test and Chi-square test. Multiple linear regression model was used to analyze the independent factors affecting ΔMayo and Δ25(OH)D, and variables with P-values less than 0.20 in the univariate analysis were allowed for further multivariate analysis. Results: A total of 74 UC patients (44 males, 30 females), with median age (range) 39.5 (20-76) years old, were analyzed and respectively assigned into study group (n=36) and control group (n=38). In study group, the average level of serum 25(OH)D was significantly increased at month 12 compared with that at baseline [(22.87±7.30) µg/L vs. (18.15±7.48) µg/L,P<0.001]. However, no significant elevation of serum 25(OH)D was found in control group [(19.17±8.49) µg/L vs. (19.82±9.47) µg/L,P=0.466]. Furthermore, there was a significant decrease of modified Mayo score [-3(-4.75, -1.25) vs.-2(-3.25, 0), P=0.034] and a higher clinical remission rate (55.6% vs. 28.9%, P=0.020) at month 12 in study group than those in control group. In addition, according to the baseline level of serum 25(OH)D before mesalazine treatment, 74 UC patients were divided into vitamin D deficiency group (n=38, serum 25(OH)D<20 µg/L) and non-deficiency group (n=36, serum 25(OH)D≥20 µg/L). At month 12 in vitamin D deficiency group, patients with vitamin D3 supplementation had a greater decline in modified Mayo score [-4(-5.75, -2) vs.-2(-4, 0), P=0.048] and a higher clinical remission rate (60.0% vs. 22.2%, P=0.019) compared with those without. Conclusions: In patients with mild-to-moderate active UC receiving mesalazine treatment, vitamin D3 supplementation may improve the clinical efficacy, especially in patients with vitamin D deficiency.

Arbuscular mycorrhizal fungi: Effects on secondary metabolite accumulation of traditional Chinese medicines.

Traditional Chinese medicine (TCM) has played a pivotal role in maintaining the health of people, and the intrinsic quality of TCM is directly related to the clinical efficacy. The medicinal ingredients of TCM are derived from the secondary metabolites of plant metabolism and are also the result of the coordination of various physiological activities in plants. Arbuscular mycorrhizal fungi (AMF) are among the most ubiquitous plant mutualists that enhance the growth and yield of plants by facilitating the uptake of nutrients and water. Symbiosis of AMF with higher plants promotes growth and helps in the accumulation of secondary metabolites. However, there is still no systematic analysis and summation of their roles in the application of TCM, biosynthesis and accumulation of active substances of herbs, as well as the mechanisms. AMF directly or indirectly affect the accumulation of secondary metabolites of TCM, which is the focus of this review. First, in this review, the effects of AMF symbiosis on the content of different secondary metabolites in TCM, such as phenolic acids, flavonoids, alkaloids and terpenoids, are summarized. Moreover, the mechanism of AMF regulating the synthesis of secondary metabolites was also considered, in combination with the establishment of mycorrhizal symbionts, response mechanisms of plant hormones, nutritional elements and expression of key enzyme their activities. Finally, combined with the current application prospects for AMF in TCM, future in-depth research is planned, thus providing a reference for improving the quality of TCM. In this manuscript, we review the research status of AMF in promoting the accumulation of secondary metabolites in TCM to provide new ideas and methods for improving the quality of TCM.

Effects of methylsulfonylmethane on growth performance, immunity, antioxidant capacity, and meat quality in Pekin ducks.

This study was conducted to determine the effect of methylsulfonylmethane (MSM) on growth performance, immune function, antioxidant capacity, and meat quality in Pekin ducks. A total of 960 female 1-day-old Pekin ducklings (53.3 ± 0.4 g) were randomly allotted to 3 treatments with 8 replicates of 40 birds, based on their body weight (BW). The experiment lasted 6 wks, and dietary treatments included a corn-soybean meal-based diet supplemented with 0%, 0.15%, and 0.3% MSM, that is, CON, MSM1, and MSM2, respectively. Growth performance, serum profiles, and meat quality were determined. During the period of days 22-42, BW gain (BWG) in MSM2 treatment was higher (P < 0.05) and feed-to-gain ratio (F/G) was lower (P < 0.05) than those of CON and MSM1 treatments. BW gain and final BW in MSM2 treatment were increased (P < 0.05) compared with CON and MSM1 treatments during the period of days 1-42. Serum activities of superoxide dismutase and glutathione peroxidase, total antioxidative capacity, and concentrations of interleukin-2 and interleukin-6 were higher (P < 0.05) in MSM2 than in CON treatment. Ducks in the MSM2 treatment group had lower (P < 0.05) serum malondialdehyde, interferon gamma, and tumor necrosis factor-α levels than those in the CON treatment group. The supplementation of MSM increased (P < 0.05) water-holding capacity and redness (a*) and decreased (P < 0.05) values for 2-thiobarbituric acid and drip loss on day 5. Ducks in the MSM2 treatment group had higher (P < 0.05) pH24h than those in the CON treatment group. Taken together, the inclusion of MSM (0.3%) increased final BW and BWG during periods of days 22-42 and days 1-42, reduced feed-to-gain ratio during the period of days 22-42, and resulted in positive effects on immunity, antioxidant capacity, and meat quality.

Clinical effectiveness of 3 days preoperative treatment with recombinant human erythropoietin in total knee arthroplasty surgery: a clinical trial.

AIMS: The purpose of study is to evaluate the effect and complication of preoperative short-term daily recombinant human erythropoietin (rhEPO) treatment for blood-saving in patients undergoing unilateral primary total knee arthroplasty (TKA). METHODS: This three-arm randomized clinical trial compared three different rhEPO-based treatment protocols for unilateral primary TKA. Group A: application of daily doses of rhEPO combined with iron supplement starting 3 days before surgery; Group B: application of daily doses of rhEPO combined with iron supplement starting the day of surgery; Group C: iron supplement alone. Perioperative hemoglobin (Hb) level gaps, total perioperative blood loss, reticulocyte levels and treatment-related complications were studied. RESULTS: A total of 102 patients were included (35, 35 and 32 patients in Groups A, B and C, respectively). Total blood loss (TBL) in Groups A, B and C was 490.84, 806.76 and 924.21 ml, respectively. Patients in Group A had a significant lower TBL than Groups B and C (A vs. B: P = 0.010; A vs. C: P < 0.001). There was no difference as for TBL between Groups B and C (P = 0.377). Group A patients had significant smaller Hb decline than Group C on the third and fifth postoperative day (P = 0.049, P = 0.037), as well as than Group B on the fifth postoperative day (P = 0.048). There was no difference as for Hb decline between Groups B and C. No difference was shown in levels of inflammatory biomarkers or blood-saving protocol-related complications among three groups. CONCLUSIONS: Daily dose of rhEPO combined with iron supplement administered 3 days before TKA procedures could significantly decrease perioperative blood loss and improve postoperative Hb levels, without significantly elevating risks of complication, when compared with admission of rhEPO on the day of surgery and iron supplement alone. Preoperative daily rhEPO treatment could be a more effective blood-saving protocol in TKA procedures.

Effects of absorbents on growth performance, blood profiles and liver gene expression in broilers fed diets naturally contaminated with aflatoxin.

OBJECTIVE: The study was conducted to evaluate the effects of the absorbent (a mixture of activated carbon and hydrated sodium calcium aluminosilicate) on growth performance, blood profiles and hepatic genes expression in broilers fed diets naturally contaminated with aflatoxin. METHODS: A total of 1200 one-day-old male chicks were randomly assigned to 6 treatments with 10 replicate cages per treatment. The dietary treatments were as follows: (1) control (basal diets); (2) 50% contaminated corn; (3) 100% contaminated corn; (4) control + 1% adsorbent; (5) 50% contaminated corn + 1% absorbent; (6) 100% contaminated corn + 1% absorbent. RESULTS: During d 1 to 21, feeding contaminated diets reduced (p&lt;0.05) body weight (BW), average daily gain (ADG) and average daily feed intake (ADFI), but increased (p&lt;0.05) feed-to-gain ratio (F/G). The absorbent supplementation increased (p&lt;0.05) BW, ADG and ADFI. There were interactions (p&lt;0.05) in BW, ADG and ADFI between contaminated corn and absorbent. Overall, birds fed 100% contaminated diets had lower (p&lt;0.05) final BW and ADG, but higher (p&lt;0.05) F/G compared to those fed control diets. The absorbent addition increased (p&lt;0.05) serum albumin concentration on d 14 and 28 and total protein (TP) level on d 28, decreased (p&lt;0.05) alanine transaminase activity on d 14 and activities of aspartate aminotransferase and alkaline phosphatase on d 28. Feeding contaminated diets reduced (p&lt;0.05) hepatic TP content on d 28 and 42. The contaminated diets upregulated (p&lt;0.05) expression of interleukin-6, catalase (CAT) and superoxide dismutase (SOD), but downregulated (p&lt;0.05) glutathione S-transferase (GST) expression in liver. The absorbent supplementation increased (p&lt;0.05) interleukin-1ß, CAT, SOD, cytochrome P450 1A1 and GST expression in liver. There were interactions (p&lt;0.05) in the expression of hepatic CAT, SOD and GST between contaminated corn and absorbent. CONCLUSION: The results suggest that the naturally aflatoxin-contaminated corn depressed growth performance, while the adsorbent could partially attenuate the adverse effects of aflatoxin on growth performance, blood profiles and hepatic genes expression in broilers.

Transcriptome analysis reveals corresponding genes and key pathways involved in heat stress in Hu sheep.

Heat stress (HS) seriously affects animal performance. In view of global warming, it is essential to understand the regulatory mechanisms by which animals adapt to heat stress. In this study, our aim was to explore the genes and pathways involved in heat stress in sheep. To this end, we used transcriptome analysis to understand the molecular responses to heat stress and thereby identify means to protect sheep from heat shock. To obtain an overview of the effects of heat stress on sheep, we used the hypothalamus for transcriptome sequencing and identified differentially expressed genes (DEGs; false discovery rate (FDR) < 0.01; fold change > 2) during heat stress. A total of 1423 DEGs (1122 upregulated and 301 downregulated) were identified and classified into Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heat stress triggered dramatic and complex alterations in gene expression in the hypothalamus. We hypothesized that heat stress induced apoptosis and dysfunction in cells and vital organs and affected growth, development, reproduction, and circadian entrainment via the calcium signaling pathway, which influences ribosome assembly and function. Real-time PCR was used to evaluate the expression of the genes regulating important biological functions or whose expression profiles were significantly changed after acute heat stress (FDR < 0.01; fold change > 4), and the results showed that the expression patterns of these genes were consistent with the results of transcriptome sequencing, indicating that the credibility of the sequencing results. Our data indicated that heat stress induced calcium dyshomeostasis, blocked biogenesis, caused ROS accumulation, impaired the antioxidant system and innate defense, and induced apoptosis through the P53 signaling pathway activated by PEG3, decreased growth and development, and enhanced organ damage. These data is very important and helpful to elucidate the molecular mechanism of heat stress and finally to find ways to deal with heat stress damage in sheep.

Preoperative vitamin D deficiency increases the risk of postoperative cognitive dysfunction: a predefined exploratory sub-analysis.

BACKGROUND: Vitamin D is important for maintaining physiological functions including cognition and its deficiency is associated with the occurrence of cognitive impairment. This study was to explore the association between preoperative vitamin D status and the occurrence of postoperative cognitive dysfunction (POCD) in elderly patients undergoing major surgery. METHODS: This was a predefined exploratory sub-analysis of one-centre data from a randomized controlled trial. In all, 123 elderly (≥ 65 years) patients who were scheduled to undergo major cancer surgery were recruited. Serum 25-hydroxyvitamin D concentration was measured before surgery. In total, 59 nonsurgical control subjects with comparable age and education level were also enrolled. A battery of neuropsychological tests was administered the day before and the 7th day after surgery in patients or at the same time interval in control subjects. POCD was diagnosed according to the ISPOCD1 definition. RESULTS: 71.5% (88/123) of elderly patients had vitamin D deficiency (serum 25-hydroxyvitamin D concentration < 12 ng/ml) before surgery; 24.4% (30/123) of them developed cognitive dysfunction at 1 week after surgery. After adjusting for confounding factors, high preoperative serum 25-hydroxyvitamine D concentration was related to a decreased risk of POCD (odds ratio [OR]: 0.829, 95% confidence interval [CI]: 0.708-0.971; P = 0.020), whereas preoperative vitamin D deficiency was associated with an increased risk of POCD (OR: 8.427, 95% CI: 1.595-44.511; P = 0.012). CONCLUSIONS: Vitamin D deficiency is prevalent in elderly patients undergoing major cancer surgery and increases the risk of early POCD development. Whether prophylactic vitamin D supplementation can reduce POCD in the elderly deserves further study.

Influences of Copper/Zinc-Loaded Montmorillonite on Growth Performance, Mineral Retention, Intestinal Morphology, Mucosa Antioxidant Capacity, and Cytokine Contents in Weaned Piglets.

The effects of copper/zinc-loaded montmorillonite (Cu/Zn-Mt) on growth performance, mineral retention, intestinal morphology, mucosa antioxidant capacity, and cytokine contents in weaned piglets were investigated in the present study. One hundred eight piglets weaned at 21 ± 1 days of age (Duroc × Landrace× Yorkshire; average initial weight of 6.36 kg) were allotted to three treatments for 2 weeks. The three treatments were as follows: (1) control group: basal diet; (2) Cu/Zn-Mt group: basal diet + 39 mg/kg Cu and 75 mg/kg Zn as Cu/Zn-Mt; (3) Cu + Zn + Mt group: basal diet + mixture of CuSO4, ZnSO4, and Mt (equal amount of Cu, Zn, and Mt to the Cu/Zn-Mt group). Each treatment had six pens of six piglets. The results showed that as compared with the control group and the Cu + Zn + Mt group, Cu/Zn-Mt supplementation increased (P < 0.05) the average daily gain and the gain/feed ratio; Cu/Zn-Mt supplementation increased (P < 0.05) the Cu and Zn concentrations in serum, jejunum, and ileum mucosa, villus height, the ratio of villus height to crypt depth, and the activities of SOD, GSH-Px, and IL-10 levels, and decreased the malondialdehyde concentrations in the jejunum and ileum, and intestinal IL-1ß, IL-6, and TNF-α levels. Moreover, supplementation with the mixture of CuSO4, ZnSO4, and Mt had no effect on the growth performance, but increased the mucosa Cu and Zn concentrations, intestinal morphology, antioxidant capacity, and immune function in the duodenum, while it had no effect on the above indexes in the jejunum and ileum. The results indicated that Mt could be used as a controlled carrier for Cu and Zn, which made Cu/Zn-Mt have better biological activities in the intestine than the mixture of Cu, Zn, and Mt.

[Biological evaluation of three-dimensional printed co-poly lactic acid/glycolic acid/tri-calcium phosphate scaffold for bone reconstruction].

Objective: To biologically evaluate the three-dimensional(3D) printed co-poly lactic acid/glycolic acid/tri-calcium phosphate(PLGA/TCP) scaffold which could be used for repairing oral and maxillofacial bone defects, and to provide experimental evidence for its further research and clinical application. Methods: PLGA/TCP scaffolds were fabricated using low temperature rapid prototyping technique. Micro-CT and scanning electron microscope(SEM) were used to characterize the surface morphology. MC3T3-E1 cells were seeded onto the scaffold and stained with the rhodamine phalloidin and calcein acetomethoxy. After that, confocal laser scanning microscope was exploited to observe the features and viability of the cells. Moreover, the cells were co-cultured with the extract of PLGA/TCP and complete medium, respectively. The proliferation capability of the cells was assessed by the cell counting kit-8 (CCK-8) on the 1st, 2nd, and 3rd day. The PLGA/TCP scaffolds incorporated with recombinant human bone morphogenetic protein-2(rhBMP-2) of 0, 30, 60 µg(i.e. blank control group, low-dose group and high-dose group) were implanted into the latissimus dorsi muscle of the rats, and 6 weeks later, the samples were harvested to estimate the volume and pattern of new bone. Results: The 3D printed PLGA/TCP scaffold possessed a regular and well-defined porous stereo-structure with porosity of (73±3)%. Micro-CT and SEM showed that pore size were (379±32) and (453±29) µm respectively, and distance between layers were (452± 24) and (415±25) µm, and cylinder diameter were (342±24) and (350±28) µm. It also exhibited excellent cell adhesion and growth ability on the exterior and inner surface through rhodamine phalloidin and calcein acetomethoxy staining. The CCK-8 test demonstrated that the absorbance value of extract group on the 1st and 2nd day(0.51±0.08 and 0.63±0.09) were significantly higher than those in the blank control group(0.39± 0.05 and 0.53±0.05)(P<0.05), while there was no significant difference between the extract group(0.67±0.06) and the blank control group(0.68±0.04)(P>0.05) on the 3rd day. For in vivo test, there was obvious ectopic new bone formation on the PLGA/TCP scaffold incorporated with rhBMP-2, and this was demonstrated using the histological examination and micro-CT. The bone formation in the low-dose group was similar to the shape of the pre-implanted 3D printed scaffold, while much diversity was revealed in the high-dose group duo to over osteogenesis which was validated by the examinations of gross observation, histology and micro-CT. Conclusions: Customized PLGA/TCP scaffolds can be manufactured by 3D printing technique. The scaffold showed an excellent biocompatibility and ectopic osteogenesis when incorporated with rhBMP-2. However, further research is needed to validate it's effect on repairment of the oral and maxillofacial bone defects.

Clinical application of remifentanil in local anesthesia for tumor resection in functional brain area.

OBJECTIVE: The aim of this study was to investigate clinical application of remifentanil in local anesthesia for resection of tumors in functional brain area. PATIENTS AND METHODS: Twenty-four patients were randomly divided into two groups: control group and remifentanil group. In remifentanil group remifentanil was infused intravenously with micro pump in 0.05-0.1 µg·kg-1·min-1. The hemodynamic changes and complications during operation were monitored. RESULTS: The satisfactory degree for surgical procedure was evaluated. The surgery of two groups could be completed in a conscious state. Mean artery pressure (MAP), heart rate (HR) in remifentanil group during opening or closing skull or intra- cranial period were significantly lower than control group (p < 0.05). There were no conspicuous complications in two groups such as respiratory depression, nausea, vomitting and dysphoria. Satisfaction rate of remifentanyl group was significantly higher than control group (p < 0.05). CONCLUSIONS: Awake brain tumor surgery could be completed in rational use of remifentanil on the base of good local anesthesia, and hemodynamics were stable and the patients were well tolerated.

Effect of You Gui Wan on mouse sperm fertilising ability in vivo and in vitro.

This study is to explore whether YGW has an impact on sperm fertilising ability in mice. Twenty male mice were randomly divided into two groups. In vivo experiments, one group of animals were orally administrated with YGW decoction and another group administered with saline for 14 days. Afterwards, the animals were mated with their female partners. Percentages of retrieved zygotes were then compared. In vitro experiments, in vitro fertilisation (IVF) assay, sperm acrosome reaction and acrosin activity were used to compare sperm fertilising ability between the two groups. The YGW-treated group had a significantly higher percentage of zygotes than the saline controls (P = 0.005). The IVF rates induced by spermatozoa from the herb-treated mice were also significantly higher than those from the control animals (P = 0.015). The sperm acrosin activity of the herb-treated group was significantly higher than that of the saline-treated group (P = 0.048), although there was no significant difference in testicular weight, sperm count and sperm motility. These data suggest that YGW decoction has a significant effect on normal sperm fertilising ability both in vivo and in vitro, which may be due to, at least in part, increments in the sperm acrosin activity.

Photoprotection in the green tidal alga Ulva prolifera: role of LHCSR and PsbS proteins in response to high light stress.

Ulva prolifera, an intertidal macroalga, has to adapt to wide variations in light intensity, making this species particularly rewarding for studying the evolution of photoprotective mechanisms. Intense light induced increased non-photochemical quenching (NPQ) and stimulated de-epoxidation of xanthophyll cycle components, while DTT-treated samples had lower NPQ capacity, indicating that the xanthophyll cycle must participate in photoprotection. In this work, we found that the PsbS-related NPQ was maintained in U. prolifera. According to analysed gene expression, both LhcSR and psbS were up-regulated in high light, suggesting that these two genes are light-induced. LHCSR and PsbS proteins were present at different light intensities and accumulated under high light conditions, and PsbS concentrations were higher than LHCSR, showing that the NPQ mechanism of U. prolifera is more dependent on PsbS protein concentration. Moreover, the level of both LHCSR and PsbS proteins was high even in the darkness, and neither the transcript level nor protein content of LhcSR and psbS genes varied significantly following short-term exposure to intense light. These findings suggest that this alga can modulate NPQ levels through regulation of the xanthophyll cycle and concentrations of PsbS and/or LHCSR.

Excitatory amino acids, TNF-alpha, and chemokine levels in synovial fluids of patients with active arthropathies.

The aim of this study was to assess the synovial fluid (SF) neurotransmitter excitatory amino acid (EAA) levels, including glutamate (Glu) and aspartate (Asp), in the context of SF levels of other amino acids, TNF-alpha and chemokines from patients with active arthropathies. The SF was collected from patients with active rheumatoid arthritis (RA), gout, or osteoarthritis (OA). The SF samples were analysed for levels of neurotransmitters glutamate and aspartate, tumour necrosis factor-alpha (TNF-alpha), Regulated upon Activation Normally T-cell Expressed and Secreted (RANTES), macrophage inhibitory factor-1 alpha (MIP-1alpha) and interleukin 8 (IL-8). SF WBC counts were also determined. Correlations between SF EAA, TNF-alpha and chemokines were determined by the Pearson product-moment correlation. Primary cultures derived from SF from active RA and gout patients were incubated with added l-glutamate, to assess if exposure to Glu could increase TNF-alpha levels. There were significant elevations in SF EAA, SF TNF-alpha and SF RANTES in RA patients compared to gout or OA patients. Significant correlations between SF EAA and SF RANTES, MIP-1alpha and IL-8 levels were seen, and SF EAA and SF TNF-alpha or SF WBC levels approached significance. Addition of exogenous neurotransmitter glutamate significantly increased TNF-alpha levels in primary cell cultures derived from RA and gout patients. The SF neurotransmitter EAA levels significantly correlated to selected SF chemokine levels, in clinically active RA, gout and OA patients, independent of disease. Added Glu resulted in significantly increased TNF-alpha levels in primary synovial cell cultures. These data expand the relationship of SF neurotransmitter EAA levels to SF cytokines and chemokines in patients with clinically active arthritis, and suggest that neurotransmitters Glu and Asp contribute to peripheral inflammatory processes.

Flavonol glycosides from Elaeagnus lanceollata (Elaeagnaceae).

Two new flavonol glycosides were isolated together with two known glycosides and an ester from the leaves of E. lanceollata Warb. Their structures were characterized by spectroscopic methods.

Minor methylated pyranoamentoflavones from Calophyllum venulosum.

Two new biflavonoids, pyranoamentoflavone 7-methyl ether (1) and pyranoamentoflavone 4'-methyl ether (2), have been isolated from the leaves of Calophyllum venulosum. The structures of these two new compounds were elucidated by spectroscopic data.

Synergistic antitumor activity of irinotecan in combination with 5-fluorouracil in rats bearing advanced colorectal cancer: role of drug sequence and dose.

The basis for current clinical trials in the treatment of colorectal cancer with the combination of irinotecan (CPT-11) and 5-fluorouracil (FUra) with or without leucovorin (LV) is their proven activity as single agents, their different mechanisms of action, and lack of CPT-11 cross-resistance to previous FUra/LV treatment. The role of drug dose and administration sequence in this combination was studied in vivo using a rat colon tumor model (Ward colon carcinoma); we administered CPT-11 and FUra by i.v. push once a week for four consecutive weeks (weekly x 4), a clinically relevant schedule. The maximum tolerated doses (MTDs) of CPT-11 and FUra administered as single agents were 100 mg/kg/week for both agents. Three different combination administration sequences were evaluated: (a) CPT-11 administered simultaneously with FUra (sequence I); (b) FUra administered 24 h before CPT-11 (sequence II); and (c) CPT-11 administered 24 h before FUra (sequence III). When combining the two drugs at 50% of their respective MTD, the antitumor efficacy was sequence dependent with 62, 38, and 95% complete tumor regression rate for sequences I, II, and III, respectively. For sequences I and II, dose escalation to 75% of the MTD for each drug was paralleled by reversible host toxicity with no significant increase in the antitumor activity of the combination. With sequence III, however, the combination was lethal in 100% of treated animals when the doses of both drugs were at 75% of the MTD or higher. With the sequential combination of CPT-11 followed 24 h later by FUra (sequence III), the high complete tumor regression rate (cure) could be maintained, even when the dose of CPT-11 was reduced to 12.5% of the MTD as long as the doses of FUra was kept at 50 -75 % of the MTD. The data demonstrate that the antitumor activity and toxicity of combining CPT-11 with FUra is highly sequence dependent and that a sequence of CPT-11 preceding FUra is superior with a significant increase in the therapeutic index over the other sequences tested. In addition, the data also demonstrate that toxicity associated with high dose of CPT-11 can be eliminated without loss of the antitumor efficacy by reducing the dose of CPT-11 to at least 50% of its MTD, whereas the dose of FUra is kept at 50-75 % of its MTD.

New drugs in therapy of colorectal cancer: preclinical studies.

For years, 5-fluorouracil (5-FU) was the only chemotherapeutic agent for the treatment of patients with advanced colorectal cancer. Based on laboratory data, modulation of 5-FU by leucovorin (LV) was proven to be an active alternative. In addition, a number of 5-FU prodrugs and antifolate antimetabolites became available for preclinical and clinical evaluation. With the 5-FU prodrugs, the overall aim was to improve the therapeutic efficacy and selectivity of 5-FU and to provide an oral form of therapy. In preclinical systems, several of the 5-FU prodrugs, eg, capecitabine, uracil/ ftorafur (UFT)/LV, and S- , are active and offer significant therapeutic advantages over 5-FU/LV. A direct and specific new thymidylate synthase (TS) inhibitor, Tomudex (raltitrexed, ZDI694; Zeneca Pharmaceuticals, Macclesfield, UK), is active in several preclinical and clinical settings. The major focus of this report will be on the preclinical development of selected fluoropyrimidine prodrugs.

Rationale for treatment design: biochemical modulation of 5-fluorouracil by leucovorin.

Preclinical in vitro and in vivo results have demonstrated the conditions required for optimal modulation of 5-FU activity by LV. The ability to increase intracellular concentrations of higher chain length polyglutamates was a function of duration of longer exposure to LV rather than the dose. In rats bearing advanced colorectal tumors, the role of LV dosage was more clearly evident with the weekly 5-FU treatment schedule than with the daily schedule. Phase III clinical trials in patients with advanced colorectal cancer demonstrated that low-dose and high-dose LV (daily x 5) and weekly high-dose LV schedules yielded similar response rates with different toxicity profiles. A phase III trial demonstrated significant therapeutic advantages for a bimonthly schedule of high-dose LV over a monthly schedule of low-dose LV. Taken together, these results provide insight into LV biomodulation, but the optimal conditions for these regimens for individual patients remain undetermined. To date it has not been possible to identify the optimal conditions for modulation of 5-FU by LV in individual patients with advanced colorectal cancer, and response rates are comparable. A regimen that offers the opportunity to manage treatment-induced toxicity is recommended. With diarrhea being the primary dose-limiting toxicity with the weekly 5-FU and high-dose LV (manifested during the 2-3 weeks of treatment), management of toxicity can be achieved by delaying treatment, by dose reduction, and/or by treatment with octreotide47 without compromising efficacy. In contrast, with the daily x 5 schedule, multiple toxicities (mucositis [stomatitis], diarrhea, neutropenia, and hand and foot syndrome) are manifested regardless of the dose of LV administered. An additional advantage to the weekly schedule is that it provides the opportunity to use 5-FU/LV treatment in sequence or combination with other drugs, such as topoisomerase I inhibitors (CPT-11), antifolates (methotrexate, trimetrexate), and platins (oxaliplatin).

Thymidylate synthase inhibitors in cancer therapy: direct and indirect inhibitors.

PURPOSE AND METHODS: Although fluoropyrimidines, in particular, fluorouracil (5-FU) and fluorodeoxyuridine (FdUrd), are active alone and in combination with other agents in a variety of human malignancies, therapeutic selectivity, resistance, and efficacy have been a major limitation in cancer therapy. Preclinical and clinical results in advanced and adjuvant colorectal cancers confirmed that the therapeutic efficacy of fluoropyrimidines, with thymidylate synthase (TS) as a primary target, can be improved significantly with leucovorin (LV) modulation. With the recognition that TS is an important therapeutic target, direct and specific inhibitors have been developed and are under intensive preclinical and clinical evaluation, primarily in patients with colorectal cancer, with demonstrable activity. The direct TS inhibitors have been shown to be potent, with a high level of specificity under therapeutic conditions for TS. This includes ZD1694, AG337, and LY231514. To date, although the therapeutic activity of both direct and indirect inhibitors of TS is similar, differences in the magnitude and profile of toxicity have been observed. A phase III comparative evaluation of a direct inhibitor of TS (ZD1694) with an indirect inhibitor (5-FU/LV) has been completed and showed similar activity but reduced toxicity in favor of ZD1694. RESULTS: Recognition that greater than 95% of the injected dose of 5-FU is rapidly inactivated by dihydropyrimidine dehydrogenase (DPD) to therapeutically inactive products, but with toxicity to normal tissues, led to the development of inhibitors of this enzyme with the aim to modify the therapeutic index of 5-FU. Several inhibitors in combination with 5-FU are under preclinical and clinical evaluation, including uracil and 5-chloro-2,4-dihydroxy pyridine, as modulators of 5-FU derived from its prodrug tegafur and 5-ethynyluracil as a modulator of 5-FU. CONCLUSION: In this review, an update of the present status of direct and indirect inhibitors of TS is discussed, as well as the future prospect for new drugs alone and in combination.