Isoth erma l Titration Calorime try in the student lab

合集下载

等温滴定量热法

结合位点数不同，平衡常数的物料平衡的表达公式是不同的
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characteristics
▪ 它通过高灵敏度、高自动化的微量量热仪连续、准确地监测和记录一个变化过程的量热曲线，原位、在线和无损伤地同时提供热力学和动力学信息，它已经成为鉴定生物分子间相互作用的首选方法。
等温滴定微量热法
(IsothermalTitrationCalorimetry,IT C)
ITC
Isothermal titration calorimetry (ITC) is used to measure the heat adsorbed or released during changes in the composition of a system undergoing a titration process. 等温滴定量热技术（ITC）是一种监测由结合成分的添加而起始的任何化学反应的热力学技术，即用一种反应物滴定另一种反应物，随着加入滴定剂的数量的变化，测量反应体系温度的变化
During the 2000’s, isothermal titration calorimetry was widely employed in the design and discovery of drugs and in addition to this, in this last decade an isothermal titration calorimeter with open cell was employed for the study of liquid mixtures .
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沙利度胺的传说

The International Journal of Biochemistry&Cell Biology39(2007)1489–1499ReviewThe thalidomide sagaMagda Melchert a,∗,Alan List ba Mofﬁtt Cancer Center and Research Institute,12902Magnolia Drive,SRB-4,Tampa,FL33612,USAb12902Magnolia Drive,SRB-4,Tampa,FL33612,USAAvailable online30January2007AbstractOver the past50years,thalidomide has been a target of active investigation in both malignant and inﬂammatory conditions. Although initially developed for its sedative properties,decades of investigation have identiﬁed a multitude of biological effects that led to its classiﬁcation as an immunomodulatory drug(IMiD).In addition to suppression of tumor necrosis factor-alpha(TNF-␣), thalidomide effects the generation and elaboration of a cascade of pro-inﬂammatory cytokines that activate cytotoxic T-cells even in the absence of co-stimulatory signals.Furthermore,vascular endothelial growth factor(VEGF)and betaﬁbroblast growth factor (bFGF)secretion and cellular response are suppressed by thalidomide,thus antagonizing neoangiogenesis and altering the bone marrow stromal microenvironment in hematologic malignancies.The thalidomide analogs,lenalidomide(CC-5013;Revlimid)and CC-4047(Actimid),have enhanced potency as inhibitors of TNF-␣and other inﬂammatory cytokines,as well as greater capacity to promote T-cell activation and suppress angiogenesis.Both thalidomide and lenalidomide are effective in the treatment of multiple myeloma and myelodysplastic syndromes for which the Food and Drug Administration granted recent approval.Nonetheless,each of these IMiDs remains the subject of active investigation in solid tumors,hematologic malignancies,and other inﬂammatory conditions.This review will explore the pharmacokinetic and biologic effects of thalidomide and its progeny compounds.©2007Elsevier Ltd.All rights reserved.Keywords:Thalidomide;Immunomodulatory agent;Angiogenesis;Lenalidomide;CC-4047Contents1.Introduction (1490)2.Pharmacokinetic properties (1490)3.Pharmacodynamic effects (1491)3.1.Immunomodulation (1491)3.2.T-cell activation (1492)3.3.Angiogenesis (1492)3.4.Bone marrow microenvironment (1493)3.5.Anti-tumor and apoptotic mechanisms (1493)4.Role of IMiDs in cancer therapy (1493)4.1.Multiple myeloma (1493)4.2.Myelodysplastic syndromes (1493)5.Conclusion (1496)References (1496)∗Corresponding author.Tel.:+18137453163;fax:+18137453071.E-mail addresses:Melcheme@mofﬁ(M.Melchert),ListAF@mofﬁ(A.List).1357-2725/$–see front matter©2007Elsevier Ltd.All rights reserved.doi:10.1016/j.biocel.2007.01.0221490M.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–14991.IntroductionThalidomide(Thalomid TM,Celgene Corp,Warren, NJ)is a synthetic glutamic acid derivative which was ini-tially developed in the early1950s as an anticonvulsant for the treatment of epilepsy.Following a lack of sufﬁ-cient efﬁcacy as an anti-epileptic,it was eventually mar-keted as a sleep aid and was also widely used as an anti-emetic during pregnancy(Randall,1990).Prior to its use in gravid women,the teratogenic effects of thalidomide in animals or humans were unknown.However,by1962, two published reports by William McBride in Australia and Widukind Lenz in Germany emerged describing limb and bowel malformations in children born to moth-ers who were exposed to thalidomide during pregnancy (Lenz,1962;McBride,1961).Use of this agent dur-ing theﬁrst trimester of gestation led to alarming rates of phocomelia,defects in long bones,absence of auri-cles,cleft lip,and cardiac and gastrointestinal anomalies. While thalidomide had not received approval by the Food and Drug Administration(FDA)in the United States (US)at this time due to concerns of neurotoxicity,the widespread international use of this drug as a sedative was eventually halted during the early1960s.The pharmacological effects of thalidomide extended beyond its neurosedative effects,and for that reason, it was subsequently investigated in a number of der-matologic,rheumatologic,and malignant diseases.In 1965,theﬁrst report of clinical activity in erythema nodosum leprosum(ENL)was serendipitously discov-ered by a dermatologist from Israel,Jacob Sheskin (Sheskin,1965).Thalidomide was found to be effec-tive in reducing fevers,night sweats,and improving skin lesions in a patient with ENL.However,it was not until1998that thalidomideﬁrst received approval by the FDA for the treatment of ENL(Pearson&Vedagiri, 1969;Sheskin,1965;Waters,1971).More recently, thalidomide was approved for the treatment of multi-ple myeloma in May2006,and has reported efﬁcacy in a wide spectrum of malignant and non-malignant dis-eases.Access to this agent in the US is restricted,and requires participation in the System for Thalidomide Education and Prescription Safety(STEPS)program to prevent adverse outcomes related to teratogenicity (Zeldis,Williams,Thomas,&Elsaved,1999).Thalidomide and its analogs are immunomodula-tory drugs(IMiDs)that exhibit a multitude of biologic effects on cytokine and cell-mediated responses.These effects are largely responsible for the clinical efﬁcacy seen in conditions such as lupus erythematosis,apt-hous ulcers that occur in human immunodeﬁciency virus and Behcet’s disease,ENL,graft versus host dis-ease,and cancer(Hamuryudan et al.,1998;Jacobson et al.,1997;Kumar,Witzig,&Rajkumar,2004;Parker et al.,1995;Pearson&Vedagiri,1969).Thalidomide is the most widely studied of the IMiDs;however, the newer structural analogs,CC-5013(lenalidomide, Revlimid TM,Celgene Corp.,Warren,NJ)and CC-4047 (Actimid TM),promise even greater activity for clinical development.These two analogs of thalidomide were developed in the mid-1990s with enhanced potency and a relatively favorable toxicity proﬁle.This review will summarize the pharmacokinetic and biologic properties of thalidomide and its derivatives.2.Pharmacokinetic propertiesThalidomide(␣[N=Phthalimido]glutarimide)con-tains a glutarimide moiety with a single chiral center and is formulated as a racemic mixture of two active enantiomers,S(−)and R(+)(see Fig.1).Initially,the S(−)isoform was thought to be the enantiomer pri-marily responsible for the teratogenic effects and the R(−)isoform for the sedative properties.Puriﬁcation of the R(−)enantiomer was attempted in order to optimize its clinical application,but was eventually not found to be technically feasible secondary to the rapid interconversion of isomers under physiologic con-ditions.Furthermore,both forms were found to be teratogenic in the New Zealand rabbit model,and thus it remains a racemic mixture(Eriksson,Bjorkman,Roth,& Fig.1.Adapted with permission from Macmillan Publishers Ltd:Nature Reviews Cancer,2004(Bartlett et al.,2004);(a)the chemical structure of thalidomide;(b)alteration of the structure of thalidomideby adding an amino(NH2−)group at the4position of the phthaloyl ring to generate the IMiDs CC-5013and CC-4047.For CC-5013,one of the carbonyls(C O)of the4-amino-substituted phthaloyl ring has been removed.M.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–14991491Hoglund,2000;Fabro,Smith,&Williams,1967).Poor water solubility led to the development of thalidomide exclusively as an oral agent,with peak plasma con-centrations of the drug detected at3–6h(Chen et al., 1989).Upon ingestion,thalidomide undergoes sponta-neous non-enzymatic hydrolytic cleavage into more than 12different metabolites.Thalidomide and its metabo-lites are quickly eliminated in the urine,with a mean elimination half-life of approximately5h.The pharma-cokinetic properties of thalidomide in the presence of renal or hepatic dysfunction are largely unknown.Dos-ing with thalidomide is disease-dependent,with daily oral schedules of50–800mg,demonstrating efﬁcacy in a variety of disease processes.Over the last decade,second-generation IMiD com-pounds have been developed by chemical modiﬁcation of the structural backbone of thalidomide in order to enhance immunomodulatory potency and minimize the dose-limiting neurotoxic effects.Both lenalidomide and CC-4047are4-amino-gultaramide derivatives of thalidomide in which an amino group was added to the fourth carbon of the phthaloyl ring of the parent com-pound(see Fig.1)(Bartlett,Dredge,&Dalgleish,2004; Teo et al.,2003).Both agents also exist as a racemic mixture of the active S(−)and R(+)forms.Like thalido-mide,they are both administered in daily oral dosing every21–28days of monthly cycles.Renal elimination predominates,and caution is recommended in dosing of patients with impaired creatinine clearance.The ter-minal half-life of lenalidomide and CC-4047after oral administration are reported as3and7h,respectively (Bartlett et al.,2004;Schey et al.,2004).In contrast to thalidomide in which somnolence,constipation,and peripheral neuropathy are common dose-limiting toxic-ities,both lenalidomide and CC-4047lack signiﬁcant neurosedative toxicity.Dose-limiting neutropenia and thrombocytopenia are the most common toxic sequelae, while venous thromboembolism has been reported with use of all three agents.3.Pharmacodynamic effects3.1.ImmunomodulationThalidomide and its derivatives are potent immunomodulators with biologic effects on both cytokine stimulation and cell-mediated immunity(see Fig.2).One of the key mediators responsible for the anti-inﬂammatory responses seen with the use of IMiD agents is tumor necrosis factor-alpha(TNF-␣). Thalidomide enhances degradation of TNF-␣mRNA, thus suppressing this pro-inﬂammatory cytokinerelease Fig.2.Reproduced with permission from Journal of Clinical Oncol-ogy,2004(Kumar et al.,2004).Proposed mechanism of action of thalidomide in cancer illustrated using myeloma as an example. Thalidomide inhibits angiogenesis,enhances effects of the immune system,inhibits binding of tumor cells to stroma,and inhibits various cytokines.Thaliomide may also have direct effects on the tumor.NK: natural killer;TNF:tumor necrosis factor;IL-6:interleukin-6;VEGF: vascular endothelial growth factor;NF-kB:nuclear factor kappa B. from endotoxin-stimulated monocytes and macrophages (Moreira et al.,1993).The effect on TNF-␣is felt to be largely responsible for the clinical beneﬁt seen in patients with inﬂammatory conditions such as ENL,who have high endogenous TNF-␣production that is suppressed following thalidomide treatment (Sampaio et al.,1993).In addition,both lenalidomide and CC-4047have up to a50,000-fold higher potency than thalidomide as inhibitors of cytokine generation, including suppression of endotoxin-induced TNF-␣secretion(Bartlett et al.,2004;Corral et al.,1999a). Activation of the transcription factor nuclear factor kappa B(NF-kB),a key regulator of TNF-␣and inter-leukin(IL)-8production,is blocked after thalidomide exposure through inhibition of the inhibitor of kappa B (IkB)kinase(Keifer,Guttridge,Ashburner,&Baldwin, 2001).However,the cellular response to IMiD agents is quite complex and lineage and ligand speciﬁc,with evidence that TNF-␣generation is actually enhanced in the setting of T-cell activation.This is demonstrable in1492M.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–1499vitro with upregulation of TNF-␣production by CD4+ and CD8+T-lymphocytes stimulated by anti-CD3 (Marriott et al.,2002).Furthermore,an increase in TNF-␣serum concentration has been reported upon exposure to IMiD agents in early-phase trials involving solid tumors and inﬂammatory dermatologic diseases such as toxic epidermal necrolysis(Bartlett et al.,2004; Wolkenstein et al.,1998).While the biologic effects of TNF-␣modulation by the IMiD agents may be central to the therapeutic bene-ﬁts seen in a variety of inﬂammatory conditions,there are several other cytokines that are similarly affected by this class of agents that may play a signiﬁcant role in immune modulation.Generation of pro-inﬂammatory enzymes and cytokines,such as cyclooxygenase-2(COX-2), interleukin-1beta(IL-1␤),transforming growth fac-tor(TGF)-␤,and IL-6are suppressed upon exposure to IMiDs,and contribute to the activation of the T-cell receptor(TCR),particularly in the T H1subset (Bartlett et al.,2004;Corral et al.,1999a;Musto,2004). Analogous to TNF-␣modulation,IL-12secretion is sup-pressed by IMiDs when monocytes are stimulated by lipopolysaccharide and is enhanced in the setting of T-cell stimulation(Corral et al.,1999a,1999b).Expansion of both T-cells and NK-cells is promoted by IL-12secre-tion and thus IMiD agents have the potential to be useful adjuncts in the development of cancer vaccines and other immunotherapuetic approaches(Trinchieri,1998).Fur-thermore,IL-12stimulates interferon-␥(IFN-␥)produc-tion and both cytokines have demonstrable anti-tumor activity and anti-angiogenic-activity(Beatty&Paterson, 2001;Qin&Blankenstein,2000;Trinchieri,1998). 3.2.T-cell activationThe immune response to foreign antigens is a highly regulated process requiring the presentation of major histocompatability(MHC)-bound peptides on antigen presenting cells(APCs)to the TCR.Acces-sory molecules,such as B7on APC and CD28on the T-cell surface,provide secondary signals that are vital to the promotion of T-cell responsiveness.These interactions lead to the subsequent activation and pro-liferation of T-cells followed by a cascade of cytokine and cellular responses.Thalidomide and its deriva-tives are able to enhance the CD8+T-cell response in the absence of these secondary co-stimulatory sig-nals(Haslett,Corral,Albert,&Kaplan,1998;Mueller, Jenkins,&Schwartz,1989).In vitro data of primary human T-cells suggest that thalidomide enhances IL-2 mediated T-cell proliferation and IFN-␥production via the TCR complex.The effect of thalidomide on T-cell expansion is dose-dependent and occurs even at low lev-els of CD3stimulation.Furthermore,thalidomide affects the balance between T-helper(Th)-1and-2subsets at least in part through cytokine modulation of IL-4,IL-5,and IFN-␥(McHugh et al.,1995).In the presence of thalidomide,mitogen-and antigen-stimulated human peripheral blood mononuclear cells preferentially induce the Th-2subtype,which corresponds with maximum enhancement of IL-4production.Thalidomide derivatives are also potent co-stimulators of T-cells that enhance activation of CD8+ T-cells in vitro(Stirling,2001).Upon exposure to CMV and inﬂuenza virus matrix proteins,both CC-5013and thalidomide were shown to enhance CD8+cytokine production and cytotoxic activity(Haslett,Hanekom, Muller,&Kaplan,2003).CC-4047has demonstrated durable,tumor-speciﬁc Th-1type immunity in mice tumor xenograft models(Dredge,Marriott,Todryk et al.,2002).These preclinical investigations have suggested a potential role for the use of IMiD agents in the development of tumor vaccine adjuncts and as modulators of immune response in the setting of defective CD4+mediated immunity such as HIV and graft versus host disease.3.3.AngiogenesisIn the early1990s,thalidomide wasﬁrst reported to exhibit potent anti-angiogenic properties that were thought to contribute to the teratogenic effects of limb bud malformations observed in offspring of thalidomide exposed gravid mothers(D’Amato,Loughnan,Flynn, &Folkman,1994).Eventually,the effect of IMiDs on vasculogenesis was recognized as a mechanism of poten-tial clinical beneﬁt in the treatment of malignancies in which neoangiogenesis is a conserved feature of the malignant phenotype.Vascular endothelial growth fac-tor(VEGF)and betaﬁbroblast growth factor(bFGF)are potent stromal mitogens that are produced in excess in a variety of malignancies,including multiple myeloma and myeloid disorders.Paracrine and autocrine secre-tion of VEGF causes proliferation of multiple myeloma cell lines and has also been shown to promote self-renewal of leukemia progenitors(Bellamy et al.,2001; Gupta et al.,2001).Secretion of both VEGF and bFGF from tumor and bone marrow stromal cells is sup-pressed upon exposure to IMiDs,resulting in reduced endothelial cell migration and adhesion(D’Amato et al.,1994;Dredge,Marriott,Macdonald et al.,2002; Lentzsch et al.,2003).Both thalidomide and CC-4047 have been shown to suppress the induction of VEGF in co-cultures of multiple myeloma cell lines and boneM.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–14991493marrow stromal cells(Gupta et al.,2001).Additionally, thalidomide inhibits bFGF-induced angiogenesis in vivo using a rabbit cornea micropocket assay(D’Amato et al.,1994).In murine lymphoma and rectal carcinoma cell line xenografts,IMiDs decrease tumor microvessel formation(Dredge,Marriott,Macdonald et al.,2002; Lentzsch et al.,2003).The latter anti-angiogenic effect is independent of TNF-␣suppression or endothelial cell proliferation,and appears to be dose-or concentration-dependent.Furthermore,lenalidomide has been shown to attenuate AKT-dependent phosphorylation,resulting in a reduction of bFGF-induced endothelial cell migra-tion(Dredge,Marriott,Macdonald et al.,2002;Dredge et al.,2005).3.4.Bone marrow microenvironmentThe pro-inﬂammatory responses within the bone marrow microenvironment are thought to play a sup-portive or tumor nurturing role in many hematologic diseases,including multiple myeloma and myeloid dis-orders.TNF-␣induces expression of IL-6secretion by bone marrow stromal cells and is markedly upregulated upon adhesion of multiple myeloma cells to bone mar-row stromal cells(Gupta et al.,2001;Witzig,1999). The adhesion molecules,ICAM-1,LFA-1,and VCAM-1,are similarly affected in the bone marrow milieu (Miyamoto et al.,1995).IL-6promotes the prolifera-tion of multiple myeloma cell lines and inhibits Fas-and dexamethasone-induced apoptosis in vitro.Both thalidomide and CC-4047decrease upregulation of IL-6in co-cultures of bone marrow stromal and multiple myeloma cells,suggesting an additional mechanism of therapeutic beneﬁt in multiple myeloma.Lenalido-mide induces activation of cell adhesion molecules and increases␤1integrin-mediated adhesion to extracel-lular matrix proteinﬁbronectin(Dredge et al.,2005; Miyamoto et al.,1995).3.5.Anti-tumor and apoptotic mechanismsIndependent of the immune-modulating activities, IMiDs have direct anti-proliferative activity in hema-tologic malignancies.The IMiDs,as a class,induce concentration-dependent inhibition of proliferation in multiple myeloma cell lines and primary myeloma cells that are resistant to standard chemotherapy(Lentzsch et al.,2003).Effects on apoptosis are evident at multiple levels of death receptor signaling,including potentia-tion of TNF-related apoptosis-inducing ligand(TRAIL), inhibition of apoptosis protein-2,increased sensitivity to Fas induction,and upregulation of caspase-8activa-tion(Keifer et al.,2001;Mitsiades et al.,2002).G0/G1 cell cycle arrest or apoptosis in leukemia cell lines and other hematologic malignancies occurs with lenalido-mide exposure and is mediated,at least in part,by A-dependent mechanisms(Dredge et al.,2005).Further-more,lenalidomide has preferential anti-proliferative activity against a5q mutant cell line(MUTZ-1)with corresponding induction of expression of genes encoded at the5q locus(Gandhi et al.,2004).These preclinical ﬁndings have been conﬁrmed in clinical trials involving patients with myelodysplastic syndromes(MDS).4.Role of IMiDs in cancer therapyThalidomide and its analogs have therapeutic potential in a wide spectrum of diseases given their multifaceted pharmacologic effects.The known immunomodulatory and anti-angiogenic properties of IMiDs provided the impetus to investigate these agents in the treatment of both hematologic malignancies and in solid tumors.Numerous early-phase trials in solid tumors have shown activity in prostate cancer,breast cancer,Kaposi’s sarcoma,renal cell cancer,melanoma, neuroendocrine tumors,hepatocellular carcinoma,lung cancer,and gliomas(Kumar et al.,2004).Response rates have been promising in some;however,further studies are needed to elucidate the true magnitude of thera-peutic beneﬁt in solid tumors.In addition,the IMiDs are attractive agents for the treatment of both myeloid and lymphoid malignancies with reported activity in non-Hodgkin’s lymphoma,acute myeloid leukemia,and myeloﬁbrosis with myeloid metaplasia(Kumar et al., 2004).However,the earliest focus of clinical investiga-tions involved multiple myeloma and MDS and thus will be discussed in further detail.4.1.Multiple myelomaThe use of IMiDs in the treatment of multiple myeloma has recently emerged as the standard of care following multiple reports of efﬁcacy in both front-line therapy and in relapsed or refractory dis-ease(Glasmacher et al.,2006;Rajkumar,Blood,Vesole, Fonseca,&Greipp,2006;Singhal et al.,1999;Tosi, Zamagni,&Cellini,2004).In May2006,thalidomide was approved by the US FDA for the treatment of newly diagnosed multiple myeloma in combination with dex-amethasone.The approval was based on a phase III multicenter trial performed by the Eastern Coopera-tive Oncology Group(ECOG)in which patients were randomized to four cycles of dexamethasone40mg on days1–4,9–12,17–20of28-day cycles either1494M.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–1499Table1Summary of MM-009and MM-010,phase III trials of CC-5013in multiple myelomaMM-009MM-010North American trial International trialCC-5013+Dex Dex alone CC-5013+Dex Dex alone TTP(weeks)60.120.753.420.6 Overall RR(%)61.222.85821.7CR(%)26.5 4.113.6 4.0 Dex:dexamethasone;TTP:time to progression;RR:response rate;CR:complete response.alone or with200mg daily of thalidomide(Table1) (Rajkumar et al.,2006).Paraprotein responses in serum and urine monoclonal protein levels were observed in63%of patients treated on the combination arm compared to41%of patient receiving dexamethasone alone(p=.0017).Toxicities were signiﬁcant,with17% of patients developing deep venous thrombosis which prompted the recommendation for anticoagulation dur-ing combination therapy.Other adverse events included rash,bradycardia,and neuropathy.As a consequence,the thalidomide/dexamethasone combination is a common induction regimen for use as initial therapy in multiple myeloma and prior to autologous transplant.Thalidomide has also enjoyed success in the salvage setting,with multiple phase I and II trials demon-strating efﬁcacy either alone with response rates of 25–35%,or in combination with dexamethasone with response rates of50–60%(Glasmacher et al.,2006; Tosi et al.,2004).Combination chemotherapy trials with thalidomide have yielded variable rates of suc-cess in relapsed and refractory disease when applied in conjunction with dexamethasone,cisplatin,adriam-cyin,cyclophosphamide,etoposide,clarithramycin,and melphalan(Coleman et al.,2002;Kyriakou et al.,2005; Lee et al.,2003;Srkalovic,Elson,Trebisky,Karam, &Hussein,2002).Finally,thalidomide has also shown beneﬁt in the maintenance setting.The Intergroupe Fran-cophone du Myelome(IFM)recently reported the results of trial involving597patients who were randomized after autologous stem cell transplantation to three of the treatment strategies,i.e.,either(A)no maintenance, (B)pamidronate maintenance,or(C)thalidomide and pamidronate maintenance treatment until disease pro-gression(Attal et al.,2006).A complete or very good partial response was achieved in55%of patients in arm A,57%in arm B,and67%in arm C(p=0.03).The 3-year post-randomization probability of event-free sur-vival was improved with the thalidomide combination maintenance with36%of patients in arm A,37%in arm B,and52%in arm C remaining without evidence of disease progression(p<.009).The4-year probabil-ity of survival from the date of diagnosis was improved in the thalidomide/pamidronate maintenance arm(77% versus74%versus87%;p<.04).The initial dose of thalidomide was400mg daily,with39%of patients dis-continuing treatment secondary to adverse effects at a median of8months.Thus,while toxicities remain sub-stantial,thalidomide is an effective agent in the treatment of myeloma in all stages of the disease.Of particular importance,maintenance therapy with thalidomide in this trial did not increase the risk of thromboembolic complications,suggesting that thrombogenic potential may be highest when thalidomide is administered dur-ing induction therapy when tumor burden is high or when combined with agents that have intrinsic thrombogenic potential.Lenalidomide was subsequently approved by the FDA in June2006for combination treatment with dex-amethasone for relapsed or refractory multiple myeloma. Based on encouraging data from several phase I and II trials,two phase III randomized,multicenter trials (MM-009,US and MM-010,Europe)were initiated comparing lenalidomide and dexamethasone combi-nation treatment with dexamethasone and placebo in patients with multiple myeloma who had received at least one prior therapy(Weber et al.,2006).Interim results were reported at the American Society of Clinical Oncology Meeting in2006.Lenalidomide was adminis-tered in a daily25mg dose for21days in combination with dexamethasone pulse dose treatment of40mg on days1–4,9–12,and17–20in a28-day cycle.Major reductions in serum paraprotein concentration were reported in51–53%of the lenalidomide/dexamethasone group and were statistically superior to those seen in patients receiving dexamethasone alone(OR 5.5 [3.9,9.1];p<.0001).The median time to progression (TTP)in MM-009compared favorably:37weeks in the lenalidomide/dexamethasone arm versus19.9weeks for dexamethasone alone(HR=0.356[0.257,0.494]; p>.0001).Toxicities were manageable;however,grade 3/4neutropenia and thrombocytopenia were experienced in27%and17%of patients,respectively,with a7.8% incidence of deep vein thrombosis.Importantly,the adverse events commonly seen with thalidomide,such asM.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–14991495neuropathy,constipation,and sedation,were infrequent in these trials.Other toxicities include included rash, gastrointestinal symptoms,myalgias,and pulmonary embolism.Among patients receiving the lenalidomide combination in the MM-009study,thrombo-embolic events were reported in20of87patients(23%)who received concomitant recombinant erythropoietin ther-apy and in4of83patients who did not receive erythropoietin(5%)(Knight,DeLap,&Zeldis,2006). In the group that received placebo and dexametha-sone,thrombosis occurred in5of67patients(7%) who received concomitant erythropoietin and in1of 103patients(1%)who did not receive erythropoietin. Multivariate analysis of both studies combined showed an independent correlation between the development thrombotic events and treatment with the combination of lenalidomide and high-dose dexamethasone or treatment with concomitant erythropoietin.Of particular interest, thrombotic events were not reported among23patients who received aspirin or salicylates during theﬁrst month of treatment,as compared with52of668patients who did not receive aspirin or salicylates.A recent study showed a reduction in the thrombosis rate with the administration of aspirin in myeloma patients receiving a combination of thalidomide and anthracycline,sug-gesting that thrombotic risk with the IMiDs may be reduced when used in combination therapy by avoid-ance of recombinant erythropoietins that have inherent thrombotic potential,or prophylaxis with aspirin or alter-nate strategies(Baz et al.,2005).The latter approach is a subject of current investigation in an ECOG study.Dis-tribution of lenalidomide,like its parent compound,is also highly regulated given concerns for possible ter-atogenic effects.All patients prescribed this drug must participate in the Rev-Assist program prior to receiving medication.A phase I,dose-escalation study of CC-4047in multiple myeloma has also been reported with demon-strated safety and efﬁcacy(Schey et al.,2004).The drug was well tolerated,with the main dose-limiting toxic-ities being neutropenia and venous thrombosis with a maximum tolerated dose of2mg/day.A25%or more reduction in paraprotein was seen in67%of patients,and 54%achieved greater than a50%reduction.In support of T-cell activation as possible mechanistic role,increased serum IL-2receptor and IL-12levels were detected in patients receiving CC-4047.4.2.Myelodysplastic syndromesIMiDs were initially targeted as potential agents for the treatment of MDS based on their immunomodula-tory and anti-angiogenic features.Theﬁrst report of the use of thalidomide in MDS involved a trial of83 patients and included biologic correlates designed to assess angiogenic and inﬂammatory responses(Raza et al.,2001).Thalidomide was initiated at100mg daily and titrated upwards to a dose of400mg daily as tolerated. At12weeks of treatment,only61%of patients were able to complete the planned course of treatment sec-ondary to dose-limiting toxicities.Only13%of patients experienced a major erythroid response,and platelet or neutrophil improvements were uncommon.How-ever,erythroid responses were durable,with a median duration of306days.Thalidomide has since been inves-tigated in multiple trials at doses of200–1000mg daily in the treatment of both low-and high-risk MDS,with either hematologic improvement or partial responses reported in approximately20–60%of patients(Bouscary et al.,2005;Moreno-Aspitia et al.,2006;Zorat et al.,2001).At these doses,tolerance of thalidomide is discouraging,with a high frequency of dose-limiting fatigue,constipation,neuropathy,and sedation.For this reason,enthusiasm for development of thalidomide in the treatment of MDS has been tempered.In contrast,lenalidomide has shown remarkable activ-ity in the treatment of MDS with toxicities that compare favorably to thalidomide.The initial report on the safety and efﬁcacy of lenalidomide in MDS included 43patients with symptomatic anemia who had either failed treatment with erythropoietin(EPO)or were poor candidates for beneﬁt from EPO therapy(frequent transfusions(>2per month)and/or high endogenous ery-thropoietin serum concentration(>500mU/ml))(List et al.,2005).Patients were randomized to one of three dos-ing schedules:25mg daily,10mg daily,or10mg/day for21of ing the International Work-ing Group(IWG)criteria,56%of patients experienced durable erythroid responses and20of32patients who previously required RBC support became transfusion independent.Furthermore,10of12patients with an isolated interstitial deletion of chromosome5(5q31.1) experienced an erythroid response compared to57%of patients with a normal karyotype or12%of patients with other chromosomal abnormalities.Patients with 5q31.1had particular beneﬁt with a longer duration of transfusion independence.Both cytogenetic responses and decreased medullary myeloblast percentage were noted in patients responding to lenalidomide,and were more pronounced in patients with chromosome5q31.1 deletion.The dose-limiting toxicities were related to myelosuppression,with grade3or greater neutrope-nia(58%)and thrombocytopenia(50%)necessitating dose reductions or treatment interruption in47–77%of。

李铁教授经方治疗阿狄森氏病1例经验总结

・96・中国实用医药2019年10月第14卷第28期China Prac Med.Oct2019,Vol.14,No.28•临床案例•李铁教授经方治疗阿狄森氏病1例经验总结汪莉李铁周正国【摘要】阿狄森氏病在中医历代医家未见系统论述，但结合本病临床表现，李铁教授认为,其与“黑疸”、“女劳疸”、“虚劳”等有类似之处。

患者在发病后期可出现全身性黑色素沉着，尤以脸部出现最为明显。

虽然本病的病因病机可以从外感六淫，邪气久积而向元阳不足、命门火衰转化，从肾论治为治疗本病之大法。

临床以和缓为主，以经方八珍汤加减，辅助以益气养阴、化湿解毒等法。

以调和阴阳，改善患者免疫。

在众多交杂的病证中先理胃气，养胃阴，收到了较好的临床效果。

【关键词】阿狄森氏病；女劳疸；黑疸D0I:10.14163/ki.11-5547/r.2019.2&053Professor Li Tie's experience in the treatment of Adison's disease WANG Li,LI Tie,ZHOU Zheng-guo.Dalian Integrated Traditional Chinese and Western Medicine Hospital,Dalian116011,China[Abstract]Adison's disease has not bfcen systematically discussed by physicians in the past dynastiesof traditional Chinese medicine,but according to its clinical manifestations,Professor Li Tie believes that it hassimilarities with"black jaundice","coital jaundice"and"consumptive disease".Patients may have systemicmelanin deposition in the later stage of the disease,especially in the face.Although the etiology and pathogenesisof this disease can be transformed from six exogenous pathogenic factors,chronic accumulation of evil Qi tokidney-yang insufficiency and life-gate fire deficiency,and the treatment of this disease from kidney is the mainmethod.Palliation is the main method in clinic,with the addition and subtraction of the classical prescriptionBazhen Decoction,supplemented by the methods of invigorating Qi and nourishing yin,removing dampness anddetoxification,in order to regulate Yin and Yang and improve the immunity of patients.In many mixed diseasesand syndromes,first regulating stomach qi and nourishing stomach yin have received good clinical effect.[Key words】Adison's disease;Coital jaundice;Black jaundice辽宁省名医李铁教授善治杂症，作者自2014年至今，已经侍诊其左右5载。

我喜欢植物迷迭香英语作文

Title: My Love for the Versatile Herb, RosemaryAmong the myriad of aromatic plants that grace our gardens and kitchens, one stands out to me as a true favorite – rosemary (Rosmarinus officinalis). This resilient herb, with its needle-like leaves and enchanting fragrance, has woven itself into the fabric of my life, becoming not just a culinary staple but also a symbol of health, resilience, and memories.The Enchanting AromaAt first glance, rosemary may seem unassuming, with its dark green, almost-black leaves and spiky appearance. But it is when you brush past its foliage or inhale deeply near a bush that its true magic is revealed. The fragrance of rosemary is pungent yet refreshing, with hints of pine and citrus that evoke feelings of warmth and comfort. This scent has the power to transport me to Mediterranean shores, where the herb grows wild and abundant.Culinary DelightsOne of the primary reasons I adore rosemary is its versatility in the kitchen. Its robust flavor pairs beautifully with a wide range of dishes, from hearty roasts and stews to delicate salads and breads. Adding a sprig or two of fresh rosemary to a potof simmering soup or braising meat can transform an ordinary meal into a culinary masterpiece. Its pungent essence infuses food with a depth of flavor that is both savory and aromatic, making it a staple in my culinary repertoire.Health BenefitsBeyond its culinary uses, rosemary is also renowned for its numerous health benefits. Traditionally, it has been used to aid digestion, boost memory, and even ease respiratory issues. The herb is rich in antioxidants, which help protect cells from damage and promote overall wellness. Whether consumed as a tea, infused in oil for topical use, or simply enjoyed as a culinary herb, rosemary is a valuable addition to any health-conscious lifestyle.Symbolic SignificanceMoreover, rosemary holds significant symbolic meaning across cultures. In ancient times, it was believed to enhance memory and stimulate the mind, leading to its association with wisdom and longevity. In modern times, it is often given as a token of friendship and loyalty, symbolizing remembrance and the bonds that connect us. This symbolism adds an extra layer ofmeaning to my love for the herb, reminding me of the cherished relationships in my life.Ease of CultivationPerhaps one of the most appealing aspects of rosemary for me is its ease of cultivation. This hardy herb thrives inwell-drained soil and prefers full sun, making it an ideal addition to any garden or indoor herb garden. It is relatively drought-tolerant and requires minimal care, making it alow-maintenance plant that even novice gardeners can enjoy. ConclusionIn conclusion, my love for rosemary stems from its enchanting aroma, culinary versatility, health benefits, symbolic significance, and ease of cultivation. This versatile herb has become an integral part of my life, bringing joy to my kitchen, promoting wellness, and reminding me of the beauty and complexity of nature. As I continue to nurture my rosemary plants and incorporate them into my daily life, I am grateful for the many ways this remarkable herb enriches my world.。

千层酥英语作文300字

千层酥英语作文300字Mille-feuille: A Culinary Masterpiece.Amidst the vast culinary tapestry of the world, where flavors dance and textures intertwine, lies a pastry that has captivated hearts and palates for centuries—the mille-feuille. This exquisite creation, also known as a Napoleon or custard slice, is a testament to the boundless artistry of pastry chefs and the timeless allure of culinary ingenuity.Origins and Etymology.The mille-feuille traces its roots back to the early19th century, emerging in France as a beloved delicacy. Its name, which translates to "a thousand leaves," aptly describes the pastry's most striking characteristic—the multitude of wafer-thin layers that form its delicate structure. Each layer is crafted with precision, creating an ethereal lightness that belies the richness of the dish.Ingredients and Assembly.At its core, the mille-feuille consists of three principal elements: puff pastry, pastry cream, and a decadent glaze.The puff pastry is the foundation upon which this masterpiece is built. It is a labor-intensive dough, demanding patience and skill to create its signature flaky texture. The dough is rolled and folded numerous times, creating countless layers that, when baked, expand into a symphony of crispy, buttery goodness.The pastry cream, al so known as crème pâtissière, provides a velvety smoothness to complement the crunchy pastry. It is typically flavored with vanilla, but variations can incorporate other spices or extracts to cater to different palates.Finally, the mille-feuille is crowned with a shimmering glaze, often made from a combination of sugar, water, andlemon juice. This glaze seals the pastry, protecting itfrom moisture while adding a touch of sweetness and shine.Flavor and Texture.The mille-feuille is an orchestra of flavors andtextures that harmoniously combine to create anunforgettable culinary experience.The crispy, flaky layers of puff pastry provide a delightful crunch, while the smooth, creamy pastry cream melts in the mouth. The combination of these textures creates a playful contrast that keeps the taste buds engaged.The flavors of the mille-feuille are equally captivating. The buttery richness of the puff pastry balances the sweetness of the pastry cream, while thesubtle tang of the glaze adds a finishing touch of elegance.Variations and Adaptations.Over the years, the classic mille-feuille has inspired countless variations and adaptations. Some popular variations include:Mille-feuille au Chocolat: This variation incorporates chocolate into the pastry cream, creating a decadent treat for chocolate lovers.Mille-feuille aux Fruits: This variation features fresh fruit, such as raspberries or strawberries, layered between the pastry and cream. The fruit adds a refreshing burst of flavor and color.Mille-feuille Salé: This savory variation uses a different dough and filling, such as cheese or vegetables, to create a delicious appetizer or main course.Presentation and Appeal.The mille-feuille is not only a culinary delight but also a feast for the eyes. Its towering layers and delicate glaze are visually striking, making it a centerpiece of anydessert table.Traditionally, the mille-feuille is served in individual portions, cut into rectangular or triangular shapes. However, some pastry chefs create larger versions, showcasing their artistry and creativity.Legacy and Enduring Popularity.The mille-feuille has stood the test of time, captivating taste buds and inspiring pastry chefs for generations. Its timeless appeal lies in its perfect balance of texture, flavor, and visual beauty.Today, the mille-feuille remains a beloved dessert enjoyed worldwide. Whether savored as a simple afternoon treat or as the grand finale of a special occasion, this culinary masterpiece continues to enchant and delight all who encounter it.。

小学上册第11次英语第2单元真题试卷

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EcologyC. BiologyD. Genetics17.Vinegar is an example of an __________.18.What is the opposite of ‘happy’?A. GladB. JoyfulC. SadD. Cheerful19.I have a __________ in my class. (朋友)20.The ________ loves to play.21.The ______ (青蛙) can leap great distances with its strong legs.22.What do we call the scientific study of the earth and its features?A. BiologyB. ChemistryC. GeographyD. HistoryC23.I like to ride my __________ when the weather is nice. (自行车)24.The park is ______ (close) to my house.25.What do you call the process of cooking food in water?A. BoilingB. BakingC. FryingD. Grilling26.The __________ (历史的反思) helps societies grow.27.I see a _____ (cat/dog) in the garden.28.I like _____ (吃) pizza.29._____ (小草) can grow in cracks in the pavement.30.The capital city of Haiti is __________.31.The ________ was a time of great artistic and scientific advancement in Europe.32.The __________ of an animal can vary greatly between species.33.I enjoy _______ (体验) different cultures.34.How many hearts does an octopus have?A. OneB. TwoC. ThreeD. FourC35.We are having ______ for lunch today. (pizza)36.smart growth) focuses on sustainable urban development. The ____37.What do you call the sweet food made from nuts and sugar?A. NougatB. CandyC. PralineD. FudgeB38.The chemical symbol for aluminum is __________.39.The __________ can reveal the historical context of geological formations.40.The _______ helps nourish the soil.41.I like to ______ after school.42. A _____ (植物文化交流) fosters appreciation for diversity.43.Which fruit is yellow and curved?A. AppleB. BananaC. CherryD. Grape44.The process of separating a solid from a liquid is called _______. (过滤)45.The Sun is primarily made of hydrogen and ______.46. ________ (画画) after school. She love47.The __________ (历史的探索) encourages curiosity.48.Asteroids can be found in the ______ belt.49.__________ (化学反应物) must be carefully measured for successful experiments.50.What is the opposite of "fast"?A. QuickB. SlowC. SpeedyD. RapidB51.The first successful airplane flight was achieved by the ______ (莱特兄弟).52.The _____ (金鱼) swims in its bowl.53.What do we call the process of water turning into vapor?A. EvaporationB. CondensationC. PrecipitationD. Filtration54.We have a ______ (丰富的) schedule for school events.55.My teacher’s name is _______ (李老师), and she is very _______ (友好).56.I eat ______ (水果) every day.57.My brother collects ____ (coins) from around the world.58.What is 20 ÷ 4?A. 4B. 5C. 6D. 759.The chemical symbol for selenium is ______.60.What is the largest continent?A. AfricaB. AsiaC. North AmericaD. South America61.What do you call a young vulture?A. ChickB. EagletC. CubD. Hatchling62.What is the main ingredient in a fruit salad?A. VegetablesB. FruitC. NutsD. Grain63.Objects that are far away appear ______ (smaller).64.The grapes are ___ (purple).65.The ______ (植物的利用方式) is diverse and impactful.66.I love to ______ (在阳光下) play outside.67.The garden has many _______ that blossom beautifully all year round.68.The __________ (历史的变化) is a constant theme.69.What do we call a person who studies plants?A. BiologistB. BotanistC. ZoologistD. EcologistB70.I like to ___ (play) outside.71.My pet rabbit loves to hide in ______ (草丛).72.What is the capital of Mozambique?A. MaputoB. BeiraC. NampulaD. TeteA73.We can _______ together to finish the project.74.I built a spaceship with my ________ (玩具名称).75.The ________ (invitation) is for a party.76.Who wrote "Romeo and Juliet"?A. Mark TwainB. Charles DickensC. William ShakespeareD. J.K. RowlingC77. 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麻烦麻里英语作文In the vast ocean of language, English composition stands as a fascinating and often challenging endeavor. It is a skill that requires not only a grasp of grammar and vocabulary but also an understanding of the subtle nuances that make written English engaging and effective.The Art of Crafting Sentences:The backbone of any English composition is the sentence. A well-crafted sentence can convey a wealth of information, emotion, and persuasion. It is important to vary sentence length and structure to keep the reader engaged. Short sentences can be used for emphasis, while longer sentences can provide detailed explanations or paint a vivid picture.Building a Coherent Structure:A successful composition is not just a collection of sentences; it is a well-organized structure that guides the reader through a clear narrative or argument. An effective essay will have a clear introduction, body paragraphs that develop the main ideas, and a conclusion that summarizes the points made.The Power of Vocabulary:The richness of English vocabulary is a tool that can elevate the quality of writing. Using a wide range of words not only makes the composition more interesting but also allows for more precise expression of thoughts and ideas. Synonyms andantonyms can be used to avoid repetition and to add depth to the writing.The Role of Grammar:While it is true that grammar is not the most exciting aspect of English composition, it is crucial for clarity and correctness. Proper use of tenses, articles, and prepositions ensures that the reader can follow the writer's train of thought without confusion.The Importance of Proofreading:No composition is complete without a thorough proofreading process. This final step is where the writer can catch and correct any grammatical errors, awkward phrasings, or inconsistencies in the argument. It is also an opportunity to refine the language and ensure that the composition reads smoothly.Overcoming Writer's Block:Sometimes, the blank page can be daunting. To overcomewriter's block, one can start by brainstorming ideas, creating an outline, or even writing a rough draft without worrying about perfection. The key is to get the ideas flowing and then refine them in subsequent drafts.The Joy of Revision:Finally, the process of revision is not a chore but an opportunity to improve the composition. Each draft can bring new insights and improvements, making the final product a reflection of the writer's best efforts.In conclusion, English composition is a multifaceted skill that requires practice, patience, and a keen eye for detail. It is through the careful crafting of sentences, the thoughtful organization of ideas, and the diligent proofreading that a writer can create a composition that is both compelling and correct.。

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Teachers and student guide to Isothermal TitrationCalorimetry (ITC)BackgroundThis is a supplementary material to a paper in Journal of Chemical Education in which experimental procedures and practical advice on the two experiments described in the paper are given. A teacher can use this material as a base from which he/she develops a student’s guide, or give the whole guide to the students.We have used these titration experiments in courses in isothermal calorimetry for PhD-students with mixed backgrounds (physical chemists, civil engineers, biologists etc.) in which the experiments are started before lunch and the injections made during the afternoon during breaks in the lectures. As ITC experiment demands some skill and concentration for the experiment to be successful, they are probably best suited to PhD-students used toexperimental work or to upper-division undergraduates doing projects in which they can try the technique a few times before doing the final measurements.Isothermal calorimetry and ITCGenerally, isothermal calorimetry is the measurement of heat and thermal power (heatproduction rate) at constant temperature. Such measurements are of interest in many fields of physics, chemistry and biology as almost all processes produce heat. This technique can be seen as the most fundamental calorimetric technique as it directly measures the heatproduction rate of any type of process. Note that there are a multitude of other experimental techniques being called “calorimetry”. Some other common ones are the solution calorimeter, the bomb calorimeter and the differential scanning calorimeter (DSC).Titration experiments – ITC, isothermal titration calorimetry – are a common use ofisothermal calorimetry. ITC is usually made by injecting small aliquots (typically 1-10 µL) of the solution of one component into a solution of another component with the help of aprogrammable syringe pump. The thermal powers peaks that results from the reaction/binding are then integrated to give the heats produced by the reactions following each injection.Isothermal calorimetry is a fundamental way of studying all types of reactions. The thermal power is proportional to the rate of reaction and the produced heat is proportional to the amount that has reacted (the extent of reaction):H dtdn P ∆= (1) H t n n Q ∆⋅−−=))()0(((2)Here, n (mol) is the amount of reactant, P (W) is the thermal power at time t (s), ∆H (J/mol) is the reaction enthalpy, and Q (J) is the heat produced from time zero to time t (note that ∆H isalways negative for exothermic processes; for such processes P and Q are here defined as positive). These two fundamental equations connect the kinetic concepts rate (d n/d t) and extent of reaction (n(t)/n(0)) with the thermodynamic concepts thermal power (P) and heat (Q) that are measured by the isothermal calorimeter. Isothermal calorimetry can thus be used both in kinetic and thermodynamic studies. In the present experiments we will measure thermal powers P and integrate them to get the heats Q that are used to evaluate enthalpies and binding constants.You are going to use an instrument with four calorimeters housed in an insulated box. The instrument is not actively thermostated and the quality of the results depends to some extent on the temperature stability of the environment. Therefore, do not open the instrument more than necessary.Each calorimeter (Fig. 1) has two heat flow sensors placed on an aluminium heat sink. These are thermocouple plates (“Peltier devices”) that are mostly used for cooling electronics. In this application we use them the opposite way by measuring the weak Seebeck-effect voltages that are proportional to the heat flow between the sample and the heat sink (as both heat flow and voltage are proportional to the temperature difference over a thermocouple plate). Note that the temperature differences are so low (typically in the order of 0.01 K) in a well designed experiment that the situation is essentially isothermal (the term “isothermal calorimetry” does not imply that the sample is at thermodynamically isothermal conditions, but only that the sample temperature is constant enough so that one gets the same result as one would get from a truly isothermal experiment).Figure 1. One calorimetric unit with two glass vials with reusable plastic caps. Heat sink (A), sample heat flow sensor (B), sample vial holder (C), reference heat flow sensor (D) and reference vial holder (E). The heat flow sensors (placed between vial holders and aluminium heat sink) are connected so that the reference signal is subtracted from the sample signal.Each calorimeter has one sample and one reference position (Fig. 1). In the reference vial holder an inert sample should be placed to balance the heat flows in the calorimeter and reduce the noise caused by temperature fluctuations. In the calorimeters that you are going to use vials with 20 ml water have been placed as references.On each heat flow sensor an aluminum vial holder with an inner diameter only slightlysmaller than the used vials is placed. One heat flow sensor is used for the sample and the other for an inert reference and the measured signal is the difference between these two signals. In this lab you are going to use 20 ml glass vials that are closed with reusable polyethylene caps.The four calorimeters are placed in a box made from polystyrene foam covered withaluminum plates on the inside. In the top parts of aluminum and insulation eight holes are made above the eight vial holders. During this experiment these holes should be filled with plugs made from soft packaging foam.The voltage signals from the four calorimeters are collected by a multchannel logger on its most sensitive range. This datalogger connects (and gets its electrical power) from the USB connection to a computer.The calorimeter has been calibrated and calibration coefficients, baseline values and time constants are given later in this guide. Note that it is necessary in all applications to subtract the baseline (the signal when no heat is produced) as this is not necessarily zero. The calibration coefficient (ε in units of W/V) and the baseline (U 0 in units of V) should be used in the following way:)(0U U P −=ε(3)Here, U (V) is the output voltage from the calorimeter and P (W) is the non-Tian-corrected thermal power (see below).The signal from the calorimeter is delayed compared to the actual heat production because of the thermal inertia of the sample. You will therefore need to make a correction with the Tian equation (named after the Frenchman Albert Tian who developed isothermal calorimeters in the 1920s):dtdP P P c τ+= (4)Here, P (W) is the thermal power. Note that the derivative will generate noise so you need to use a noise reduction procedure afterwards (built into the MATLAB software described below).Experiment 1: Acid-base titrationIn this experiment you are going to titrate a strong base (NaOH) into a strong acid (HCl) to determine the enthalpy of neutralization (H 3O + + OH - → 2H 2O). Follow the generalinstructions below with the 1 M NaOH-solution in the syringe and about 10 g of the 0.025 M HCl-solution in the ampoule. Make injections of about 65 µL until there is no more heat produced when you make an injection. Wait for a new baseline after each injection (or – ifyou will use the Tian correction – until the signal has decreased significantly towards the baseline).CautionBe careful with the chemicals. Of special concern is that if a syringe needle gets blocked high pressure will build up inside a syringe and the needle may pop of the syringe, releasing NaOH(1M). Wear eye and skin protection. The chemicals can be disposed in the sink together with plenty of water.After the measurement the data is collected from the computer and the result from each calorimeter is multiplied by the calibration coefficient and corrected for the thermal inertia of the sample by the Tian equation. The evaluation is then done by integrating each peak using baselines before and after each peak, and then calculating the neutralization enthalpy in two ways:1.From each of the first heats (that should be similar) by assuming that the reaction goesto completion after each step (the equilibrium constant is very high).2.From the sum of all the measured heats and the total amount of acid.The evaluation is done with the software described in the section Evaluation Software. In this experiment (if done with the concentrations and volumes suggested above) there is no need to make a correction for the dilution heat, as this is small (see Appendix 3).Experiment 2: Ba2+ to 18-crown-6 titrationIn this experiment you are going to titrate barium ions to the solution of the cage molecule 18-crown-6 into which one barium ion (or other similar ions) can bind (“binding” is weak bonding, i.e., with a comparatively low equilibrium constant). It is thus a model reaction for, e.g., protein-ligand binding. In this experiment you are going to determine the equilibrium constant and the binding enthalpy for this process.You should perform the experiment twice: first with injections of 1 M Ba2+ into 0.02 M 18-crown-6, and secondly a separate experiment with injection of Ba2+ into water. This makes it possible to correct for the enthalpy of dissolution of the rather strong BaCl2 solution (see evaluation).For each experiment follow the general instructions below with the Ba2+-solution in the syringe and about 10 g of the 18-crown-6-solution in the ampoule. Make injections of about 65 µL until the peak heights have decreased to less than 5% of the height of the first peak (at least 6 injections).CautionBe careful with the chemicals. Of special concern is that if a syringe needle gets blocked high pressure will build up inside a syringe and the needle may pop of the syringe, releasingBaCl2(1M). Wear eye and skin protection. The chemicals can be disposed in the sink together with plenty of water.After the measurement the data is collected from the computer and the result form each calorimeter is multiplied by the calibration coefficient and corrected for the thermal inertia ofthe sample by the Tian equation. The evaluation is done by integrating each peak using baselines before and after each peak to get the heats in each peak (from each injection). This should be done for both the main experiment and for the dilution experiment. The heats from the dilution experiments should then be subtracted from the corresponding heats from the binding experiment and used to calculate the binding constant and the binding enthalpy with the software bind1to1.m (see section on Evaluation software). Appendix 1 describes how the evaluation of the binding constant and binding enthalpy is done.The isothermal titration calorimeterThe instrument that you are going to use contains four isothermal calorimeters that measure the heat production rate (thermal power) in small samples. The output from each calorimeter is a voltage that you will convert to thermal power by a calibration coefficient. The calibration coefficients of the calorimeters of the instrument that you are going to use are:Calorimeter 1 ____6.324__________ mW/mVCalorimeter 2 ____6.418__________ mW/mVCalorimeter 3 ____6.186__________ mW/mVCalorimeter 4 ____6.333___________mW/mVNOTE: Green text should be changed to parameters relevant to your equipment.You will also need to use a time constant. If you use about 10 g of solution the time constant will be about 340 s (the same for all four calorimeters).The instrument contains a stirring device that will set stirring magnets in the ampoules in motion. Therefore you should place a stirring magnet in each ampoule that you are going to titrate into. Make sure that the magnet is placed on the same side as the stirring device, so that it moves.For a measurement to be successful you need to have a reference sample with approximately the same heat capacity as the sample. Use ampoules with about 10 g water.Note that the calorimeters are sensitive instrument and to get good results you need to handle them carefully.Titration accessoriesTo perform the titrations you need injection pumps, syringes, syringe needles and ampoules with guide tubes. The ampoules are plastic ampoules. In the lids of each ampoule is a rubber stopper with a guide tube through which the thin injection needle will run. The injection needle has a connector so that it can be fastened on the 1 mL plastic syringe, which is placed in the syringe pump.Note that standard glass ampoules are unsuitable for this experiment as they give a too low first peak (lower than the second peak), possibly because Ba2+ will bind to the glass surface. Polyethylene ampoules do not give this problem.The ampoules should be placed in the measurement ampoule holders, so that the guide tubes come out of the instrument. Place some soft foam in the holes of instrument so that the instrument is protected from the ambient temperature variations.Caution: If there is a risk that anyone can hurts his/her eyes the guide tubes, place a tape flag on the upper end of them to make them clearly visible.Injections are made by turning he handle on the syringe pump either full turns or half turns. You have to calibrate each syringe and syringe pump before the measurements. Do this by filling the syringes with water and ejecting the water on a balance. Do for example ten turns on the pump and measure how much water that is ejected. Then you can calculate a calibration coefficient for each syring+pump (µL/mm) that you can use to calculate how many turns you need to turn to get the injected volume you want.Caution: handle the syringe needle with care so that it does not hurt anyone’s eyes.General notesHere are a few points that you should have in mind when performing these measurements: Make sure that all syringes, needles and ampoules are clean.Treat the syringe needle with great care. It is thin walled and will easily get bent, which will make it much more difficult to get it through the guide tube.The solutions have already been made. Do not take solution directly from these flasks;instead pour some of the solution into a smaller flask and then use this solution.Make sure that there are no air-bubbles in the syringes (can be removed by knocking on syringes, e.g., with a plastic pen).To make sure that you have the correct concentration in the whole system, fill a syringe and then empty it through the syringe needle, before filling it a second time to make the measurement,Before putting down the syringe needles into the calorimeter through the guide tubes, eject some solution to make sure that you have solution all the way to the needle tip.Wait a few seconds and clean the needle tip by stroking the needle tip against a hardsurface. Do not use an absorbing material like paper as you may then empty the tip of the needle.The amounts of the reacting substances are determined from their molarity and the used volumes. The volume solution in the ampoule is determined by weighing (thesolutions in the ampoules are dilute, so their densities can be assumed to be the same as water).Make sure that the stirring magnet is on the same side as the moving magnet on the stirring device when the ampoules are charged, so that it will move and stir thesolution.You can either wait until you have a baseline between each peak (will take about 60 min), or inject more frequently (for example every 20 or 30 min) and later make aTian correction to separate the peaks (see Evaluation software section below).Measurement procedureThis is the procedure for each calorimeter (here charging into the ampoule by mass is described as the solutions are dilute; one can of course also work by volume):1.Weigh the correct mass of solution into ampoule. Note exact mass. Also note theapproximate height of the solution.2.Place a stirring magnet in the ampoule, close it with a stopper with a guide tube andplace in calorimeter.3.Make a reference (without guide tube, but with the same mass of water as you havesolution in the sample ampoule).4.Start the computer data logging.5.Fill the syringe and needle with the base.6.After baseline has been attained (usually takes 30-60 min), insert the needle to thebottom of the ampoule, then pull it up so that the needle tip is positioned in the upperpart of the solution.7.Make an injection. You should see a peak from the heat produced.8.Wait 15-20 min.9.Repeat steps 7 and 8.Evaluation softwareWe have supplied the following MATLAB programs that our students use to evaluate theITC-measurements:newfile.m Enters data into the workspaceTian.m Applies the calibration coefficient and corrects for the thermal inertia of the samplefindheats.m Lets you mark baselines, makes a first order baseline fit, and then lets you mark heats, so that it can integrate them.bind1to1.m Evaluates ITC-measurements (1-to-1 binding)theotitr.m Calculates the heats from an ideal ITC measurement. Can be used to optimize ITC measurements.You also need to place the following two programs in the same catalogue as the other programs (or on the path): closest.m and PLW2ML.m.Each program has a help text. Here is an example how you can use them to evaluate a Ba2+ to 18-crown-6 measurement (binding experiment in channel 1 and dilutilon experiment in channel 2; 6.322 and 6.186 are calibration coefficients; 340 is the time constant; the input arguments of bind1to1 are concentrations and volumes):[t,U]=newfile;[t,P1]=Tian(t,U(:,1),6.322,340);Qbind=findheat(t,P1);[t,P2]=Tian(t,U(:,2),6.186,340);Qdil=findheats(t,P2);[k,DeltaH]=bind1to1(Qbind-Qdil,0.02003,10.034,1.004,65.3e-6);Note that U is a matrix with all data measured, but that Tian and findheats will work only on vectors, i.e., data from one calorimeter. Therefore U(:,1) etc. is used to pick out one column from the matrix U.Possible variationsThe acid-base experiments can be done with other acids and bases.The binding experiment can be done with other ions than barium, for example with strontium, calcium and magnesium.The binding experiment can also be done at several temperatures. This is how ITC is often used, but it may be difficult to work at different temperatures with the unthermostated student calorimeter.Reference valuesHere are literature values of the measured properties:Acid-base titrationEnthalpy of neutralization: -56.1 kJ mol-1Binding of Ba2+ to 18-crown-6Binding enthalpy: -21.3 kJ mol-1Binding constant: 5900 L mol-1Gibbs energy: -21.3 kJ mol-1Entropy change: 25.6 J mol-1 K-1Reference:Wadsö, I. and Goldberg, R. N. (2001). Standards in isothermal microcalorimetry (IUPAC) technical report). Pure Appl. Chem. 73(10): 1625-1639.Appendix 1: Evaluating a 1:1 binding experimentThe binding of Ba 2+ to 18-crown-6 is a model reaction, similar to that of, e.g., ligands binding to proteins. 18-crown-6 is a cage molecule into which one Ba 2+-ions fits. The binding reaction can thus be written (with 18-crown-6 called A, Ba 2+ called B, and the complex of A and B called C):C B A ⇔+ (5) The equilibrium constant is then:(6) The heat produced can be calculated as:H n Q C ∆⋅= (7)Note that the heats Q should be corrected for the heat of dissolution. Integrate peaks of both the Ba 2+ into 18-crown-6 and the Ba 2+ into water, and subtract the latter from the former for each injection number.The experiment consists of a number of titrations that result in measured heats. It is possible to evaluate both K and ∆H from such a measurement if it is performed in such a way that the measured heats show a decreasing sigmoid curve when plotted against the injection number. The evaluation can be made by finding K and ∆H that will generate the best fit to themeasured data. This can either be made with a numerical optimization tool that minimizes the sum of the squared errors:2)(mea i calc i Q Q −Σ(8) or by trial and error. In either case one needs to calculate calc i Q for each titration step by solving the following equation system for each titration step:V n n n K n i n n n n n C B A B C B AC A ⋅=⋅⋅∆⋅=+=+0(9-11)This is done by inserting n A and n B from Eqs. 9-10 into Eq. 11 and solving the resulting second order equation for n C . This gives the following second order equation for n C :0)(002=⋅⋅+⋅−+−inj B A C inj B A i C n i n n n i n K V n (12)This is solved for each titration step by the standard method for solving a second orderequation 0012=++k n k n C C :240211k k k n C −±−= (13)One can then calculate the heats with:H n n Q i C i C calc i ∆−=−)(1 (14)The above method can also be used for other reaction equations, for example A+2B C by rewriting Eqs. 1-2 and 5-7 with the appropriate expressions. The equation that has to be solved will have the order of the sum of the stochiometric coefficients (for example order 3 for A+2B C). Appendix 2. A Note on NaOH(aq) dilutionAs is mentioned above, it is not necessary to do a dilution experiment for the acid-base titration experiment. The reason for this is that the enthalpy of NaOH(aq) shows a complex behavior with a minimum and a maximum, and at the present concentrations in the syringe and in the ampoule after injections the enthalpy is approximately the same (See the figure below with data from Wagman et al., J. Phys. Chem. Ref. Data, Suppl. 11 (Sl) (1982) 1.-470-469.5-469-468.5-468-467.5-467log10(NaOH mol/L)∆H f o/ k J /m o l。