Biomedical subjects

Junichi Kurebayashi

Publications and source records attributed to Junichi Kurebayashi.

At least 19 recordsLinked to original sources

PubMed2006

Additive antitumor effect of concurrent treatment of 4-hydroxy tamoxifen with 5-fluorouracil but not with doxorubicin in estrogen receptor-positive breast cancer cells.

PURPOSE: The sequential addition of tamoxifen (TAM) to chemotherapy seems superior to its concurrent addition in patients with breast cancer. This study was conducted to clarify the hypothesis that there are differential interactions among TAM and chemotherapeutic agents. METHODS: Estrogen receptor (ER)-alpha-positive or -negative breast cancer cells were treated with 4-hydroxy TAM (4OHT), 5-fluorouracil (FU) and/or doxorubicin (Dox). Changes in the expression levels of genes related to sensitivity and resistance to TAM, 5-FU or Dox were tested. RESULTS: Concurrent treatment of 4OHT with 5-FU but not with Dox additively inhibited the growth of ER-alpha-positive cells. 5-FU did not change the expression levels of any tested genes related to either sensitivity or resistance to TAM. Although Dox did not change the expression levels of any genes related to the sensitivity to TAM, Dox significantly increased the expression levels of some genes related to TAM resistance, Eph A-2, ER-beta, Fos and vascular endothelial growth factor. 4OHT significantly decreased thymidilate synthase (TS) activity. CONCLUSIONS: Although the antitumor effect of concurrent 4OHT and 5-FU was additive, that of concurrent 4OHT and Dox was less than additive in ER-alpha-positive cells. The increased expression of genes related to TAM resistance by Dox might be responsible for the interaction. Decreased TS activity by 4OHT might increase the antitumor activity of 5-FU. These findings may provide a preclinical rationale for concurrent use with 5-FU and TAM.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2006

Combined measurement of serum sialyl Lewis X with serum CA15-3 in breast cancer patients.

BACKGROUND: Serum CA15-3 has been one of the most reliable tumor markers used in monitoring breast cancer patients; however, its sensitivity in detecting metastases is limited. To increase its sensitivity, the combined measurement of other tumor markers with CA15-3 was investigated. METHODS: Serum CA15-3, carcinoembryonic antigen (CEA) and sialyl Lewis X (CSLEX) were simultaneously measured in a prospective series of 455 postoperative breast cancer patients with or without metastasis. The diagnostic parameters sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for detecting metastases were compared. The correlation of values between pairs of tumor markers was analyzed. The efficacy of combined measurement of two different tumor markers was also evaluated. RESULTS: The sensitivity for detecting metastases was 61.5, 56.9 and 52.3%; specificity was 97.2, 93.6 and 96.2%; PPV was 78.4, 59.7 and 69.4%; NPV was 93.8, 92.9 and 92.4%; and accuracy was 92.1, 88.8 and 89.9% for CA15-3, CEA and CSLEX, respectively. The values for CA15-3 were significantly correlated with those for CEA (P < 0.001) but not those for CSLEX. The combined measurement of CSLEX and CA15-3 increased the sensitivity by 17.0% but that of CEA and CA15-3 increased the sensitivity by only 10.8%. All diagnostic parameters for the combined measurement of CSLEX and CA15-3 were higher than those for the combined measurement of CEA and CA15-3. CONCLUSIONS: These findings suggest that CSLEX may be more useful than CEA in combination with CA15-3 in monitoring breast cancer patients. The results of this study suggest that CSLEX may be more useful than CEA in combination with CA15-3 in monitoring breast cancer patients.

Adult↗

PubMed2006

Additive antitumor effects of gefitinib and imatinib on anaplastic thyroid cancer cells.

PURPOSE: Anaplastic thyroid cancer (ATC) is one of the most aggressive malignancies. Although multidisciplinary treatments have been introduced, patients with this disease rarely survive longer than 1 year. These findings prompted us to investigate the antitumor activity of molecular targeting agents in thyroid cancer cells. METHODS: Two tyrosine kinase inhibitors, gefitinib and imatinib, were tested in a poorly differentiated thyroid cancer cell line, KTC-1, and two ATC cell lines, KTC-2 and KTC-3. RESULTS: All cell lines expressed not only a target molecule of gefitinib, HER1, but also a cognate receptor, HER2. They also expressed target molecules of imatinib, c-ABL and platelet-derived growth factor receptors at various levels. Both agents had modest antitumor activity in these cell lines. Combined treatment with gefitinib and imatinib led to an additional antitumor effect. Each agent induced apoptosis and their combined treatment enhanced apoptosis associated with the down-regulation of antiapoptotic proteins, Bcl-2 and Bcl-xL. Moreover, their combined treatment additionally inhibited the growth of KTC-3 xenografts in nude mice. CONCLUSIONS: These are the first findings to suggest that both gefitinib and imatinib have antitumor activity against ATC cells and that their combined use has greater activity than either drug alone.

Animals↗

PubMed2006

[Biomarkers, tumor markers and prognostic markers in breast cancer].

Biomarkers are measured in the management of breast cancer patients for the following purposes: (1) early detection, (2) monitoring of advanced breast cancer patients, (3) prediction of prognosis, and (4) prediction of therapeutic response. Summarized results investigated by the Study Group of the Japanese Breast Cancer Society in 2001 concerning the present status of tumor marker measurement in Japan and usefulness of tumor markers for the evaluation for therapeutic response are presented. Significance of two prognostic markers, vessel invasion and HER2 status were discussed at the 9th St Gallen International Consensus Meeting in 2005. Current status, clinical significance, problems and future directions on predictive markers for response to endocrine therapy and cytotoxic chemotherapy are also discussed.

Biomarkers↗

PubMed2006

[Evaluation of safety and efficacy for bi-weekly Docetaxel and 5'-DFUR combination therapy in patients with advanced or recurrent breast cancer--Phase I study].

BACKGROUND: A clinical trial of Docetaxel was performed by tri-weekly administration in patients with advanced or recurrent breast cancer. However, careful observation is necessary for outpatients because serious neutropenia often occurs during the therapy. A bi-weekly schedule is recommended since weekly administration requires more visits to the hospital. Docetaxel is proved to express synergistic efficacy in combination with 5'-DFUR by induced dThdPase in vivo. But there are no clinical trials to evaluate efficacy of bi-weekly Docetaxel and 5'-DFUR combination therapy. PURPOSE: To evaluate safety, the recommended dose of Docetaxel and the efficacy of biweekly Docetaxel and 5'-DFUR combination therapy. PATIENTS AND METHODS: Patients with advanced or recurrent breast cancer within 1 regimen of prior chemotherapy and without prior use of both Docetaxel and 5'-DFUR were enrolled. 5'-DFUR was orally administered by 600 mg/day. Docetaxel was intravenously given for at least 2 cycles (8 weeks) by 30 mg/m(2) for level 1, 40 mg/m(2) for level 2 and 50 mg/m(2) for level 3. At each level with 3 cases enrolled,the maximum tolerated dose (MTD) level was defined as that in which 2 or 3 cases showed dose limiting toxicity (DLT). The recommended dose was defined as the dose before MTD level. Therapeutic safety was evaluated by analyses of adverse events with the recommended dose. RESULTS: MTD was in level 3 and the recommended dose of Docetaxel was 40 mg/m(2) of level 2. No DLT was observed in level 2, and this combination therapy seemed safe and feasible for outpatients. In addition, all 6 cases for whom therapeutic efficacy was evaluated expressed a clinical response.

Administration, Oral↗

PubMed2006

[Therapeutic efficacy of capecitabine on advanced and recurrent breast cancer with special reference to time to progression].

We investigated 29 patients with advanced and recurrent breast cancers who underwent capecitabine therapy in the department. Patients'backgrounds: 41-89 years of age (median, 57 years of age). Advanced breast cancers, 5; recurrent breast cancers, 24. PS< or =2 in 18 cases and PS 3< or =in 11 cases. Eighty-six percent of patients were positive for ER and/or PgR. Multiorgan metastases occurred in 22 cases; bone metastases, 22 cases; lymph node metastases, 12 cases; skin metastases, 11 cases; lung metastases, 10 cases. The rate of patients who received chemotherapy was 93%, and the rate of those who received endocrinotherapy was 90%. Therapeutic response rate was CR in 1 case, PR in 5 cases, long SD in 5 cases, SD in 10 cases and PD in 8 cases, indicating a response rate of 20.7% and a clinical benefit rate of 37.9%. Time to progression (TTP) was 1-15 months (the median time, 4 months). Overall survival time (OS) was 2-23 months (median length, 12 months). OS was significantly longer in patients who had therapeutic effects than in patients with no such effects. TTP was significantly longer in patients who had therapeutic effects and in those who had longer SD than in patients with no such effects. OS was significantly longer in patients who had TTP of 6 months or longer. Clinical benefit (presence vs. absence) and PS (< or =2 vs. 3< or =) were independent factors affecting TTP. Capecitabine is expected to prolong the length of survival in patients who are able to continue treatment for 6 months or longer.

Adult↗

PubMed2006

[Postoperative adjuvant therapy for premenopausal patients with breast cancer].

The age peak of breast cancer morbidity is younger in Japan in comparison with that in Western countries. This results in a higher incidence of premenopausal patients in Japan. There are specific concerns on the postoperative adjuvant therapy for premenopausal patients, such as its influences on pregnancy, delivery and lactation, and acute and chronic adverse events related to earlier ovarian dysfunction. International guidelines such as the recommendations by the St. Gallen consensus conference provide clinicians with useful information on the risk assessment for recurrence and treatment selection of postoperative adjuvant therapy. Recommendations on postoperative adjuvant therapy for premenopausal breast cancer patients are presented according to the guidelines and evidence established by clinical trials. On-going clinical trials to resolve unanswered questions on the adjuvant therapy are also reviewed. Finally, future perspectives on the adjuvant therapy are discussed.

Adult↗

PubMed2005

Resistance to endocrine therapy in breast cancer.

Endocrine therapy is the treatment of choice for patients with breast cancer expressing estrogen receptor (ER) and/or progesterone receptor. The efficacy of endocrine therapy is well established in the prevention, adjuvant and metastatic settings. However, either de novo or acquired resistance is frequently observed. Much effort has been made to elucidate the mechanisms of action underlying resistance to endocrine therapy in breast cancer, and several possible explanations have been suggested. Our previous studies have indicated that combined treatment with an antiestrogen, fulvestrant, and an inhibitor of the HER2 signaling pathway, trastuzumab, or an inhibitor of the HER1 signaling pathway, gefitinib, leads to an additive antitumor effect in breast cancer cells expressing ER and HER2 or HER1, respectively. It has also been suggested that the HER1 or HER2 signaling pathway is upregulated during the development of antiestrogen-resistant growth in breast cancer cells. These findings suggest that signal transduction inhibitors are effective for the treatment of antiestrogen-resistant breast cancer. A hypoxic microenvironment has been shown to promote malignant progression in cancer cells. Our previous study and others have suggested that hypoxia posttranscriptionally reduces ER expression and decreases sensitivity to hormonal agents in breast cancer cells. Our preliminary study has also shown that a hypoxic cytotoxin, tirapazamine, increases ER expression in breast cancer xenografts. Differential antitumor activity of tirapazamine on tumor cells under normoxic or hypoxic conditions may cause this phenomenon. These findings suggest that hypoxic cytotoxins may retard the development of endocrine resistance induced by hypoxia. Molecular mechanisms responsible for endocrine resistance in breast cancer are reviewed and possible therapeutic strategies against this resistance are discussed.

Antineoplastic Agents, Hormonal↗

PubMed2005

Retrospective analysis of predictive factors for recurrence after curatively resected papillary thyroid carcinoma.

PURPOSE: This retrospective study analyzes the predictive factors after curative surgery for papillary thyroid carcinoma (PTC). METHODS: We analyzed 386 patients who underwent a curative operation for PTC in our hospital between 1977 and 1997, subject to the inclusion criteria. RESULTS: According to univariate analysis, pathological lateral cervical lymph node involvement (P < 0.0001), dedifferentiation of the tumor (P < 0.002), male sex (P < 0.0001), a large tumor (P < 0.005), and an age of over 50 years (P < 0.05) were significant factors. Cox's proportional hazard model showed that a man (P < 0.05), aged over 50 years (P < 0.05), who had a large primary tumor (P < 0.05) with dedifferentiation (P < 0.05), and pathological lateral cervical lymph node metastasis (P < 0.005) was more likely to have recurrence of PTC. CONCLUSIONS: Determining whether lymph node metastasis exists could be useful for predicting recurrence in patients who have undergone curative resection of PTC.

Adolescent↗

PubMed2005

[Cancer prevention].

The 12 th Oncology Forum discussed the progress and future strategy of cancer prevention in Japan. The National Cancer Center has established a research center for screening focusing on the most common six cancer, stomach, lung, liver, colon, breast and uterus cancer. The program so far had a cumulative detection rate of 3.3%, which is high,and may reflect the selection of subjects. Screening and chemoprevention is also being investigated in prostate cancer, but the issues centre on how to make this widely available. High risk subjects can also be identified for breast cancer. Obesity and family history are especially important. In colorectal cancer studies are evaluating different diets, but general application is not yet possible and the infrastructure to implement any general screening and prevention does not exist. Development of pharmaceutical treatments for prevention is difficult because of the need for very safe treatments, and also because of the length of time needed to carry out studies. Overall, cancer prevention is still in evolution. New approaches are needed, and new infrastructure will be needed at a government level to implement this.

Breast Neoplasms↗

PubMed2004

Inhibition of HER1 signaling pathway enhances antitumor effect of endocrine therapy in breast cancer.

Epidermal growth factor receptor (EGFR)/HER1 is expressed at high levels in at least 20% of breast cancers. This high expression correlates with a poor prognosis in patients with breast cancer. Experimental and clinical findings suggest that aberrant activation of tyrosine receptor kinases, such as HER1 pathway, play a causal role in the development of antiestrogen resistance in breast cancer. Recent preclinical and clinical evidence shows that inhibition of growth factor signaling pathways suppresses the growth of malignant cells without serious toxicities. To test the hypothesis that inhibition of the HER1 signaling pathway enhances the antitumor effect of endocrine therapy, a promising signal transduction inhibitor (STI) of HER1 tyrosine kinase, gefitinib, and an estrogen receptor (ER) antagonist, fulvestrant, were administered to human breast cancer cells. Our experimental results have revealed that gefitinib additively enhances the antitumor effect of fulvestrant in estrogen receptor (ER)-positive breast cancer cells under estrogen-supplemented conditions. An additive increase in the protein expression level of a cyclin-dependent kinase inhibitor, p21 may play a key role of this additive cytostatic effect. The rationale and future perspectives of the combined use of STIs with endocrine therapy in breast cancer are discussed.

Antineoplastic Agents↗

PubMed2004

Significance of serum tumor markers in monitoring advanced breast cancer patients treated with systemic therapy: a prospective study.

OBJECTIVE: The significance of serum tumor markers in monitoring advanced breast cancer patients is still controversial. To clarify this issue, the Tumor Marker Study Group of the Japanese Breast Cancer Society conducted a prospective study. METHODS: Patients with advanced breast cancer who were treated with systemic therapy between January and December 2002 were recruited from five collaborative institutes in Japan. The patients were monitored every four weeks using three serum tumor markers, CEA, CA 15-3 and NCC-ST-439 during the therapy. RESULTS: Findings from 108 eligible patients were analyzed. The pretreatment positivity rates were 51.9% for CEA, 50% for CA 15-3, and 34.3% for NCC-ST-439. The changes in each marker level at 8 and 12 weeks but not at 4 weeks after the start of therapy seemed to correlate with the response to therapy in pretreatment marker-positive patients but not in negative patients. The Cox proportional hazard model revealed a greater than 20% reduction in CEA, CA 15-3 or NCC-ST-439 levels at 4, 8 and/or 12 weeks after the start of therapy to be an independent predictive factor for longer time-to-progression (TTP) in pretreatment marker-positive patients. CONCLUSION: This prospective study supported the findings obtained from our previous retrospective study that in pretreatment marker-positive patients 1) the changes in serum tumor marker levels after the start of therapy correlate with the response to therapy; and 2) a greater than 20% reduction in the tumor marker levels was a favorable predictive factor for TTP during systemic therapy. When the pretreatment serum level of these markers is over the respective cut-off value, sequential measurement of them may be useful for evaluating the efficacy of treatment as well as monitoring the outcome of patients with advanced breast cancer.

Adult↗

PubMed2004

Establishment of enzyme-linked immunosorbent assays for thymidylate synthase and dihydropyriminide dehydrogenase in cancer tissues.

Thymidilate synthase (TS) is a major target of 5-fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the degradation of 5-FU. Intratumoral activity and expression levels of both enzymes are suggested to be predictive markers for response to 5-FU-based chemotherapy in cancer patients. Several different methods are used to measure intratumoral levels of TS and DPD. We developed new enzyme-linked immunosorbent assays (ELISAs) for these enzymes. New ELISAs were produced using anti-TS and anti-DPD polyclonal antibodies obtained using recombinant human TS and purified DPD from pig liver, respectively. Intra-assay coefficients of variations (CVs) were less than 5% in both ELISAs. Inter-assay CVs tested using the extract from 20 breast cancer specimens were 1.5-24.2% for TS-ELISA and 0.4-7.2% for DPD. Importantly, TS and DPD levels measured by the respective ELISAs closely related to TS activity (r(2)=0.8492) and DPD activity (r(2)=0.7666), respectively, measured by the respective substrate assays. To investigate the correlations between clinicopathological characteristics and intratumoral TS and DPD levels, data on 52 breast cancer patients were analyzed. Estrogen receptor (ER)-negative tumors had significantly higher TS and DPD levels than ER-positive tumors. Progesterone receptor (PgR)-negative tumors showed a significantly higher DPD level than PgR-positive tumors. There was no significant correlation of the TS or DPD levels with other clinicopathological factors in this preliminary study. Further studies are warranted to investigate predictive and prognostic values of intratumoral TS and DPD levels in various malignancies using these ELISAs.

Breast Neoplasms↗

PubMed2004

[Anti angiogenesis].

Based on presentations on the basic concepts and scientific rationale of anti-angiogenic approaches to cancer therapy and the possible applications in the area of prostate cancer, gastrointestinal cancer, lung cancer and breast cancer it is easy to conclude that development of anti-angiogenic approaches into clinical therapies is extremely challenging. It is now well established that cancer growth is increased by angiogenic factors and that inhibition of angiogenesis decreases growth and metastatic potential. Anti-angiogenic effect can be obtained through interference with multiple targets. Further development of new strategies involving such novel cancer therapies requires wide reaching development of translational research abilities. However, for moving new therapies into the clinic same rigorous criteria need to be applied as is done for traditional therapies. Angiogenesis appear to be a critical factor for development of prostate, gastric, lung and breast cancers. Development of new anti-angiogenic treatment modalities might become very important in these diseases. A critical requirement for the successful clinical development will be the development of imaging techniques that can help evaluate the effect on blood vessel functionality. Such surrogate markers of anti-angiogenic effect will be essential for optimising molecules and doses.

Angiogenesis Inhibitors↗

PubMed2004

[Retrospective analysis on efficacy and toxicity of paclitaxel-containing treatments in patients with advanced or recurrent breast cancer].

Our department recently began using paclitaxel in treating patients with breast cancer. Retrospective analysis was conducted to clarify its clinical usefulness. Forty-one patients with metastatic breast cancer were treated with paclitaxel between November 2000 and September 2002. Hospital records of the patients, except for one unsensored patient, were retrospectively reviewed. Characteristics of the patients were as follows: age, 36-81 Y (median, 56); 8 stage IV and 32 recurrent diseases; most frequent dominant site of metastasis was the liver (22 patients, 55%); number with previous chemotherapy was 0-5 (median, 2); anthracycline-based treatment and docetaxel treatment were previously performed in 21 (53%) and 15 (38%) patients, respectively; weekly dose of paclitaxel was 30-150 mg/body (median, 100); and total dose administered was 600-6, 480+ mg/body (median, 1,820). Objective response and clinical benefit rates were 35% and 80%, respectively. Median duration of response, time-to-progression and overall survival were 27+, 33+ and 41.5 weeks, respectively. Common adverse events were sensory neuropathy (45%) and nausea/vomiting (37.5%). Most were graded as 1 or 2. Various agents, such as hormonal agents and trastuzumab, were administered with paclitaxel in 26 patients (65%). No significant difference was observed in efficacy or toxicity among patients treated with paclitaxel alone or paclitaxel plus other agents. Paclitaxel seems to be a feasible, safe and active agent for patients with metastatic breast cancer.

Adult↗

PubMed2004

[Biomarkers in breast cancer].

Biomarkers are measured in the management of breast cancer patients for the following purposes. (1) Early detection of breast cancer: blood tumor markers such as CA 15-3 are useless for this detection because of a low sensitivity. Proteomics profiling has recently been investigated using blood or nipple aspirate fluid for the detection. Measurement of CEA and HER 2 in abnormal nipple discharge has been approved for diagnosis of breast cancer in Japan. (2) Monitoring of breast cancer patients: serum tumor markers are routinely measured for early detection of recurrent diseases, evaluation of therapeutic response and monitoring outcome of patients by a majority of breast cancer experts in Japan. Study results investigated by the Study Group of the Japanese Breast Cancer Society in 2001 are presented with regard to the questionnaire survey on the present status of tumor marker measurement and the clinical study on usefulness of tumor markers for the evaluation for therapeutic response. (3) Prognostic factors: new biomarkers have been investigated to select patients at high risk for distant metastases, which could not be selected by classic prognostic factors. Three prognostic factors (UPA/PAI-1, cyclin E, gene profiling), which were discussed at the 8th St. Gallen International Consensus Meeting last year, are mainly discussed. (4) Predictive factors for therapeutic response: hormone receptors (HR) have been used as reliable predictive factors for response to endocrine therapy. Other biomarkers have been investigated to select patients with tumors HR-positive but unresponsive to endocrine therapy. Current status, clinical significance, problems and future directions on predictive factors for response to cytotoxic chemotherapy are also discussed.

Biomarkers, Tumor↗

PubMed2004

[Therapeutic effects of Anastrozole in patients with advanced and recurrent breast cancer].

We reviewed therapeutic effects and harmful side effects in 33 patients with advanced or recurrent breast cancer who underwent treatment with Anastrozole 1 mg/day in our department. The patients ranged in age from 40 to 83 years old (median, 59). The Performance Status was 0-2, and there was 1 case of advanced breast cancer and 32 cases of recurrent breast cancer. The duration of disease was from 5 to 233 months (median, 50 months). The estrogen and/or progesterone receptor-positive rate was 72.7%. Metastatic sites were in multiple organs in 9 cases, in the lung only in 1 case, in bone only in 12 cases, and in soft tissue only in 10 cases. First-line therapy was used in 10 cases, second-line therapy in 6 cases, and above third-line therapy in 17 cases. There was a complete response in 3 cases, partial response in 5 cases, no long change in 13 cases, no change in 9 cases, and progressive disease in 3 cases. The response rate was 24.3%, The response period ranged from 2 to 22 months (median, 8 months), and clinical benefit was achieved in 63.7%. The clinical benefit rates for first-line were 60%, second-line 83.3% and above third-line therapy 58.8%. The response rate for patients with breast cancer resistant to Anthracyclines and/or Taxanes was 20%. Time-to-progression ranged from 2 to 28 months (median, 11 months), and overall survival ranged from 7 to 30 months (median, 15 months). The most frequent harmful side effects were rise in total cholesterol, general fatigue, hot flashes and arthralgia (9.1%). In this study, we confirmed the availability and safety of Anastrozole, which was suggested to be a useful drug in salvage therapy for patients having resistance to Anthracyclines and/or Taxanes, not only but also useful as a first- or second-line therapy.

Adult↗

PubMed2004

[Tumor markers in breast cancer].

A number of breast cancer-orientated tumor markers have been utilized for various purposes in clinical practice in Japan. Their purposes are as follows: early detection of primary tumors, prediction of disease extent and prognosis, early detection of recurrent diseases, evaluation of therapeutic responses, monitoring patient outcome, and differential diagnosis of metastatic tumors of unknown origin. The clinical significance of the measurement of tumor markers for these purposes is overviewed in this article. In addition, the results are summarized for two clinical studies conducted by the Tumor Marker Study Group of Japanese Breast Cancer Society (Chairperson: Dr. Kurebayashi), a questionnaire survey on the current status of tumor markers in breast cancer in Japan and a multi-institutional study on the utility of tumor markers for evaluation of therapeutic responses. Future directions of clinical and experimental research for breast cancer-orientated tumor markers are also discussed.

Antigens, Tumor-Associated, Carbohydrate↗