[原创] 请版主辟专栏讨论铜结合剂治疗癌症

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铜是癌组织自建血管与正常细胞争夺养料, 疯狂生长不可或缺的. 铜结合剂可以有效的减少人体各组织铜的积存, 进而降低或停止癌组织自建血管的能力, 饿毙癌细胞, 达到治疗癌症的目的.

常用的用来降低人体各组织铜的积存的药物有青霉胺(D-Penicillamine); 曲恩汀(trientine); 锌盐; DMSA,四硫钼酸铵(Ammonium tetrathiomolybdate)等.

请用过此方法的战友们与我们分享这方面的经验. 也请药物专家提供这类药物的知识供各位参考.

 

 

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以下是引用球在2008-8-30 9:07:06的发言：

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http://www.ebiotrade.com/newsf/2002-9/L200291292031.htm

 

近五年来，密西根大学综合癌症中心的医师注意到，一项早期的药物临床试验中大有可为的结果，这种药物降低了癌症患者血液中的铜含量。
    　　
    现在，新的U-M实验室之研究结果确切地告诉他们，这种实验性药物如何作用，并显示它们战胜癌症的潜力。研究结果发表于最新一期的Cancer Research期刊中，指出可利用抗血管新生的方法致疗癌症。
   
    该篇文章描述药物-tetrathiomolybdate，或TM-如何使肿瘤细胞无法发送讯号刺激新血管的生成。藉由保持铜的低含量和阻断NFkB的讯息传递路径，研究人员相信，TM可阻断血管新生或血管生成，使肿瘤无法增长和扩散。
   
    血管新生被认为许多癌症常见的起源，使肿瘤增生并移转至身体其它部位。U-M团队研究TM利用四种小鼠和细胞的实验方法，以研究抗血管新生潜力。他们明确地表示，TM抑制了肿瘤在植入人类乳癌细胞之小鼠体内的增生，使大鼠动脉细胞新血管的生成远离癌症倾向；减少刺激血管生成之关键讯息分子的释放；并避免植入人类乳癌细胞之小鼠之肿瘤生成。
   
    资深作者内科副教授，也是U-M乳癌和卵巢癌风险评估计划的指导者Sofia D. Merajver 医学博士指出“经过比较后，发现这些结果支持TM临床试验的初步结果，并指出铜的减少可以抑制肿瘤之血管新生，使不良影响降至最低。”她发现TM导致的铜含量减少，更甚于仅透过饮食所减少的铜含量。
   
    Merajver已协助领导TM在U-M的癌症临床和实验室研究调查，包括最近的晚期乳腺癌患第二阶段试验。TM已经在U-M和其它研究中心进行摄护腺癌、乳癌、头及颈部的癌症、多发性骨髓瘤、肝癌、间皮细胞瘤和其它恶性肿瘤患者。
   
    此药物最初由George Brewer医学博士研发作为医疗用途，他是U-M之Morton and Henrietta Sellner人类遗传学教授，专门研究铜含量过多所造成的罕见遗传性疾病：威尔森氏症。
   
    由硫和钼所制成的TM可以和血液中的铜结合，并与称为白蛋白的蛋白质结合，进行螯合作用。三个组成分的复合体之后便由身体排除。
   
    TM在U-M健康系统的一般临床研究中心拯救了许多威尔森氏症患者的生命，去除损坏他们脑部和肝脏并害死患者的铜。
   
    自从U-M于90年代开始成功地治疗威尔森氏症，U-M的研究开始揭露铜在血管新生中所扮演的角色-身体的正常过程及癌症中未受控制的血管新生。研究人员发现铜对于各种"生长因子"是很重要的，可使细胞成为新血管的一部分。
   
    这些发现激励Merajver和Brewer组成团队进行TM抗癌的实验室研究。而导引出各种癌症末期患者的第一阶段试验，结果发表于2000年1月的Clinical Cancer Research期刊。
   
    这项试验的目的是测试TM的安全性和降低癌症患者同含量的能力。但是它显示出少数铜含量被减少至五分之一的患者之肿瘤稳定三个月以上的证据。
   
    从那时候起，U-M研究人员进行TM的临床和基础研究。目前，许多患者参加U-M综合癌症中心的第二阶段试验。同时，Merajver和她的团队继续进行基本的实验室研究，以了解TM如何发挥它的抗血管新生作用。
   
    在新研究中，他们使用二种动物模型：一种是将人类乳癌细胞移转至小鼠体内，另一种方法是特殊育种的小鼠，并确定它们在一岁时都会罹患癌症。
   
    结果是令人震惊的。接受人类乳癌细胞”异体移植”的小鼠，TM使它们的肿瘤减小至未接受TM小鼠肿瘤之69%。接受TM的小鼠肿瘤中只有稀稀落落的血管。
   
    至于"遗传上预先计划"乳癌的小鼠，预先给予TM后都未发生肿瘤。这具有统计上的显著意义，虽然当小鼠停止接受TM后，在二星期内均发生肿瘤。显微镜检显示，它们的乳房区域有"微肿瘤"存在，但是因为无血液供应而无法继续增生。
   
    研究也使用了二种体外，或细胞培养的方法。在其中一项，环状切开大鼠的主动脉 (一种可能形成新血管的组织)，浸泡于发炎的乳癌细胞培养液中。
   
    在另一项研究中，研究人员植入乳房细胞及带有一段只能由NFkB转录因子读取的基因序列之乳癌细胞核—以及可产生NFkB作用警报的DNA片段。在癌细胞培养时，NFkB的活性是乳房细胞培养的2.5倍，但是当添加TM后，NFkB的活性几乎降低癌细胞的2倍，如同正常细胞般。
   
    当研究人员观察构成NFkB分子组成蛋白质的基因时，他们发现以TM治疗可以显著地减少那些蛋白质的生产。当他们观察介白素(interleukin)及生长因素分子时，NFkB的转录通常可获得控制，TM存在时的含量也较低。 Merajver表示“看起来，至少有一部分的TM抗血管新生作用，是藉由抑制促进血管新生因子的释放，并抑制NFkB的活性。这是令人振奋的发现，因为 NFkB与癌症对于化疗和放射治疗的抵抗力有关。我们所观察的抑制作用，也建议TM在化学预防药物中，扮演一个重要的角色，可改变易受癌症影响者的基因。 ”
   
    摘自：华文生技网

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http://clincancerres.aacrjournals.org/cgi/content/full/6/1/1

 

Treatment of Metastatic Cancer with Tetrathiomolybdate, an Anticopper, Antiangiogenic Agent: Phase I Study¹

George J. Brewer, Robert D. Dick, Damanjit K. Grover, Virginia LeClaire, Michael Tseng, Max Wicha, Kenneth Pienta, Bruce G. Redman, Thierry Jahan, Vernon K. Sondak, Myla Strawderman, Gerald LeCarpentier and Sofia D. Merajver²

Departments of Human Genetics [G. J. B., R. D. D.], Internal Medicine [G. J. B., V. L., M. T., M. W., K. P., B. G. R., S. D. M.], Surgery [K. P., V. K. S.], and Radiology [G. L.], Clinical Research Center [D. K. G.], and Comprehensive Cancer Center [V. L., M. T., M. W., K. P., B. G. R., V. K. S., M. S., S. D. M.], University of Michigan Health System, Ann Arbor, Michigan 48109, and Department of Internal Medicine, University of California at San Francisco, San Francisco, California 94115 [T. J.]

	ABSTRACT

 
Preclinical and in vitro studies have determined that copper is an important cofactor for angiogenesis. Tetrathiomolybdate (TM) was developed as an effective anticopper therapy for the initial treatment of Wilson's disease, an autosomal recessive disorder that leads to abnormal copper accumulation. Given the potency and uniqueness of the anticopper action of TM and its lack of toxicity, we hypothesized that TM would be a suitable agent to achieve and maintain mild copper deficiency to impair neovascularization in metastatic solid tumors. Following preclinical work that showed efficacy for this anticopper approach in mouse tumor models, we carried out a Phase I clinical trial in 18 patients with metastatic cancer who were enrolled at three dose levels of oral TM (90, 105, and 120 mg/day) administered in six divided doses with and in-between meals. Serum ceruloplasmin (Cp) was used as a surrogate marker for total body copper. Because anemia is the first clinical sign of copper deficiency, the goal of the study was to reduce Cp to 20% of baseline value without reducing hematocrit below 80% of baseline. Cp is a reliable and sensitive measure of copper status, and TM was nontoxic when Cp was reduced to 15–20% of baseline. The level III dose of TM (120 mg/day) was effective in reaching the target Cp without added toxicity. TM-induced mild copper deficiency achieved stable disease in five of six patients who were copper deficient at the target range for at least 90 days.

[此贴子已经被作者于2008-8-30 13:55:29编辑过]

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	Patients and Methods

 
Patients.
Eighteen adults with metastatic solid tumors exhibiting measurable disease, life expectancy of 3 or more months, and at least 60% Karnofsky performance status were enrolled. We excluded patients with effusions or bone marrow involvement as the only manifestations of disease and those who had severe intercurrent illness requiring intensive management or were transfusion dependent. Patients had to have recovered from previous toxicities and had to meet the following requirements for laboratory parameters: (a) WBC 3,000/mm³; (b) absolute neutrophil count 1,200/mm³; (c) Hct 27%; (d) hemoglobin 8.0 g/dl; (e) platelet count 80,000/mm³; (f) bilirubin 2.0 mg/dl; (g) aspartate aminotransferase and alanine aminotransferase 4 times the upper limit of institutional norm; (h) serum creatinine < 1.8 mg/dl or calculated creatinine clearance 55 ml/min; (i) calcium < 11.0; (j) albumin 2.5 g/dl; (k) prothrombin time 13 s; and (l) partial thromboplastin time 35 s. Other requirements were demonstrable progression of disease in the previous 3 months after standard treatments such as surgery, chemotherapy, radiotherapy, and/or immunotherapy or progressive disease after declining conventional treatment modalities.

Treatment Schema: Doses and Escalation.
Three dose regimens were evaluated. All dose levels consisted of 20 mg of TM given three times daily with meals plus an escalating (levels I, II, and III) in-between meals dose given three times daily for a total of six doses/day. Loading dose levels I, II, and III provided TM at 10, 15, and 20 mg, three times daily between meals, respectively, in addition to the three doses of 20 mg each given with meals at all dose levels.

Baseline Cp was taken as the nearest Cp measurement to day 1 of treatment (including day 1) because blood was drawn before TM treatment from all patients. The target Cp reduction was defined as 20% of baseline Cp. Due to Cp assay variability of approximately 2% at this institution, a change of Cp to 22% of baseline was considered as achieving the desired reduction of copper. In addition, if the absolute Cp was less than 5 mg/dl, then the patient was considered as having reached the target Cp. No patient reached the 5 mg/dl target without also being at least 78% reduced from baseline. After reaching the target copper-deficient state, TM doses were individually tailored to maintain Cp within a target window of 70–90% reduction from baseline.

Six patients were to be enrolled at each dose level. After four patients were enrolled at level I, if one patient experienced dose-limiting toxicity (defined as Hct < 80% of baseline), two more patients were enrolled at level I. If no dose-limiting toxicity was observed, patients were enrolled at the next dose level. Treatment was allowed to continue beyond induction of target copper deficiency if the patients experienced a partial or complete clinical response or achieved clinical stable disease by the following definitions. Complete response is the disappearance of all clinical and laboratory signs and symptoms of active disease; partial response is a 50% or greater reduction in the size of measurable lesions defined by the sum of the products of the longest perpendicular diameters of the lesions, with no new lesions or lesions increasing in size. Minor response is a 25–49% reduction in the sum of the products of the longest perpendicular diameters of one or more measurable lesions, no increase in size of any lesions, and no new lesions; stable disease is any change in tumor measurements not represented by the criteria for response or progressive disease; progressive disease is an increase of 25% or more in the sum of the products of the longest perpendicular diameters of any measurable indicator lesions compared with the smallest previous measurement or appearance of a new lesion. Because copper deficiency is not a cytotoxic treatment modality, the patients who provide information about the efficacy of TM for long-term therapy in this population of patients with advanced cancer are primarily those who remained within the target Cp window of 20 ?10% of baseline for over 90 days without disease progression.

Monitoring of Copper Status.
A method was required to monitor copper status easily and reliably, so that the TM dose could be adjusted appropriately during this trial. With TM administration, serum copper is not a useful measure of total body copper because the TM-copper-albumin complex is not rapidly cleared, and the total serum copper (including the fraction bound to the TM-protein complex) actually increases during TM therapy (34, 35, 36) . The serum Cp level obtained weekly was used as a surrogate measure of total body copper status. Cp was measured by the oxidase method; the Cp measurements were made by nethelometry (differential light scattering from a colored or turbid case solution with respect to a control solution) using an automated system and reagents available commercially (Beckman Instruments, Inc., Fullerton, CA). The serum Cp level is controlled by Cp synthesis by the liver, which, in turn, is determined by copper availability to the liver (38) . Thus, as total body copper is reduced, the serum Cp level is proportionately reduced. The serum Cp level is in the range of 20–35 and 30–65 mg/dl for normal controls and cancer patients, respectively. Our objective was to reduce Cp to 20% of baseline and to maintain this level, within a window spanned by 20 ?10% of baseline Cp, with typical Cp values in the range of 7–12 mg/dl. Because there appears to be no untoward clinical effects from this degree of copper reduction, we have termed this level of copper deficiency "chemical copper deficiency." The first indication of true clinical copper deficiency is a reduction in blood cell counts, primarily anemia, because copper is required for heme synthesis as well as cellular proliferation (36) . Thus, the copper deficiency objective of this trial was to reduce the Cp to 20% of baseline without decreasing the patient's Hct or WBC to below 80% of baseline value at entry.

Toxicity, Follow-Up, and Disease Evaluation.
Complete blood counts, liver and renal function tests, urinalyses, and Cp level were performed weekly for 16 weeks and then performed biweekly at the clinical laboratories of the University of Michigan Health System or at other affiliated certified laboratories. Physical examinations and evaluations of toxicity were carried out every 2 weeks for 8 weeks and then performed every 4 weeks for the duration of therapy. Toxicity was evaluated using the National Cancer Institute Common Toxicity Criteria. Extent of disease was evaluated at entry, at the point of achievement of copper deficiency (defined as Cp 20% of baseline), and every 10–12 weeks thereafter. CAT or magnetic resonance imaging was used as appropriate for conventional measurement of disease at all known sites and for evaluation of any potential new sites of disease. Angiogenesis-sensitive ultrasound with three-dimensional Doppler analyses was used in select cases as an adjunct to conventional imaging to evaluate blood flow to the tumors at different time points.

TM Preparation and Storage.
TM was purchased in bulk lots suitable for human administration (Aldrich Chemical Company, Milwaukee, WI). Because TM is slowly degraded when exposed to air (oxygen replaces the sulfur in the molecule, rendering it inactive; Refs. 34, 35, 36 ), it was stored in 100-g lots under argon. At the time a prescription was written, the appropriate dose of TM was placed in gelatin capsules by research pharmacists at the University of Michigan Health System. Previously, we had shown that TM dispensed in such capsules retained at least 90% of its potency for 8 weeks (34) . Thus, TM was dispensed to each patient in 8-week installments throughout the trial.

Measurement of Blood Flow.
Blood flow was measured by ultrasound in select patients with accessible lesions at the time they became copper deficient and at variable intervals of 8–16 weeks thereafter. Three-dimensional scanning was performed on a GE Logiq 700 ultrasound system, with the 739 L, 7.5 MHz linear array scanhead. The scanning and vascularity quantification techniques were as described previously by the authors (39 , 40) .

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	Results

 
Patient Characteristics
Eighteen eligible patients (10 males and 8 females) with 11 different types of metastatic cancer who had progressed through or (in one case) declined other treatment options were enrolled in the trial in the order in which they were referred. Six, five, and seven patients were enrolled at the 90, 105, and 120 mg/day drug levels, respectively, following the protocol dose escalation schema. One patient originally assigned to the 105 mg/day level was removed early to pursue cytotoxic chemotherapy, due to rapid progression of disease. This same patient was later retreated at the 120 mg/day level for a longer duration; thus, he is counted only at the 120 mg/day level for the analyses. The average age was 59 years; the average baseline Cp was 47.8 mg/dl, which is elevated with respect to the normal level, reflecting the patients' disease status. Table 1 summarizes the patient characteristics for each dose level.

Toxicity
There were no cardiac, pulmonary, GI, renal, hepatic, hematological, infectious, skin, mucosal, or neurological toxicities observed for Cp levels at or above 20% of baseline. Mild (>80% of baseline Hct) reversible anemia was observed in four patients with Cp levels between 10–20% of baseline. Two of these patients had been treated with cytotoxic chemotherapy, and two patients had evidence of extensive bone marrow involvement with their disease at the time of entry into the trial. Although in the latter two cases, the anemia was most likely due to causes other than treatment, TM was temporarily discontinued until Hct was restored to acceptable levels with a transfusion of 2 units of packed RBCs. In one patient, it is very likely that the copper deficiency caused by TM produced the anemia. Stopping administration of the drug allowed the Hct to recover within 5–7 days without the need for transfusion; at the patient’s request, TM was restarted at a lower dose, without further complications of anemia. Several patients experienced transient, occasional sulfur-smelling burping, within 30 min of TM ingestion. No additional toxicities of any type were observed with long-term maintenance of mild clinical copper deficiency over 8–15 months. Of note, no evidence of GI or other mucosal bleeding or impaired healing of minor trauma were observed with long-term therapy. One premenopausal patient with extensive metastatic renal cancer experienced normal menstrual periods during TM therapy, including 2.5 months of observation while she was copper deficient with Cp < 20% of baseline.

Cp as a Surrogate Measure of Copper Status
Fig. 1 shows the response of Cp as a function of time on TM therapy, expressed as the ratio of Cp at time t to baseline Cp level for each patient enrolled at the 90, 105, and 120 mg/day dose levels. Increasing the in-between meals dose from 10 mg three times daily to 15 or 20 mg three times daily had no significant effect on the rate of decrease of the Cp level, reaching a level of 50% baseline at a mean of 30 days (median = 28 days). The response of Cp to TM therapy as a function of time exhibited only minor fluctuations; when TM was discontinued, a rapid rise in Cp was observed within 48 h.

 

Four patients were removed from study due to progression of disease before achieving the target Cp of 20% of baseline, whereas the remaining 14 patients achieved the target Cp level. Because all 14 patients who achieved the target Cp level wished to remain on study, they were allowed to do so, according to the protocol, as long as they did not exhibit disease progression or toxicity. The TM doses were adjusted in these patients to maintain the Cp level between 10–20% of baseline. These patients provide the preliminary evidence of the efficacy and long-term tolerance of this approach.

Dose Adjustments to Maintain Target Cp
TM doses were adjusted to maintain a Cp target level of 20% of baseline and to prevent absolute Cp values < 5 mg/dl. Due to the routine 7-day turn-around for the Cp test at our laboratory, these dose changes were made approximately 7–10 days after the blood for the Cp measurement was taken. After achieving the target Cp, the in-between meals dose was typically decreased by 20 mg. Further decreases of 15–30 mg were necessary during long-term therapy. A patient with metastatic chondrosarcoma secondary to radiation treatment for breast cancer on long-term therapy has stable disease after 12 months of copper deficiency, with stable quality of life. One biopsy-proven metastatic nodule on her third digit is easily measurable and has been stable. Other sites of suspected disease in the chest also remain stable. Interestingly, this patient has required only a minor adjustment to her TM dose from the initial loading dose level to maintain the target Cp throughout this relatively long period. Fig. 2, A and B , illustrates the Cp response to dose adjustments required for two more representative patients over approximately 100 days of therapy. Thus far, the patient in Fig. 2A has required only decreases in dose 60 days apart. Most patients have required both an increase and a decrease in dose during long-term therapy. For example, as shown in Fig. 2B , the TM dose was increased after day 100 to respond to an increase in Cp outside the target range. Overall, there was considerable individual variability in the dose adjustments required. In conclusion, the Cp response to TM therapy evaluated weekly is not brittle or subject to wide fluctuations.

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Measurement of Response of Metastatic Cancer to TM
Clinical Evaluation.
Although the patients received different initial loading doses of TM, the Cp maintenance window of 20 ?10% of baseline was used in all groups, regardless of the loading dose. Patients who maintained this degree of copper deficiency through tailored adjustments of the TM dose for over 90 days are likely to reflect the antiangiogenic activity of TM against their tumors. The period of 90 days is selected for two main reasons. First, TM is not cytotoxic to either cancer or endothelial cells and mainly impairs endothelial cell function and proangiogenic factor production. This mechanism of action is expected to have a very slow effect on the size of tumor masses. Second, as tumors sequester copper, the microenvironment of the tumor is expected to take a longer time to be rendered copper deficient. Table 2 summarizes the clinical course of the 18 patients.

 

Fourteen patients achieved the target copper deficiency before disease progression or other disease complications. Of these, eight patients either progressed within 30 days of achieving copper deficiency or have had stable disease for <90 days; it is unlikely that most of these tumors experienced an antiangiogenic environment long enough to evaluate clinical response to this type of therapy. In all patients removed from the protocol due to disease progression or choice and in one patient removed from the protocol due to the need for abdominal surgery to relieve a small bowel obstruction, much more rapid rates of progression of disease were noted clinically after discontinuation of TM therapy.

The remaining six patients experienced stable disease (five of six patients) or progression of disease at one site, with stable disease elsewhere (one of six patients). Two patients who have stable disease by standard criteria also experienced complete disappearance of some lung lesions and a decrease in the size of other lung lesions during observation periods at target Cp of 120 and 49 days. The five patients on long-term (>90 days) maintenance therapy with stable disease have been copper deficient for 120–413 days at the time of this analysis.

Radiological Evaluation.
Serial evaluations of tumor masses by conventional imaging with CAT scan or magnetic resonance imaging revealed that the radiographic appearance of certain masses changed significantly over time. In particular, areas of presumed central necrosis (corresponding to lower attenuation of the X-ray signal) were observed in a variety of tumor types, most notably renal cell cancer, angiosarcoma, and breast cancer. Seeking to evaluate the blood flow to the tumors as a function of time during copper deficiency on long-term TM therapy, lesions accessible to ultrasound were imaged with color flow three-dimensional ultrasound at the onset of copper deficiency and at 2–4-month intervals thereafter.

A representative example of the comparison between conventional CAT scan images and blood flow-sensitive three-dimensional ultrasound is depicted in Fig. 3 . Here, a rib metastasis from renal cell carcinoma is depicted when the patient reached target copper deficiency (Fig. 3, A and C) and 8 weeks later (Fig. 3, B and D) by these two complementary imaging modalities. Fig. 3, A and B , shows stable size of this lesion by CAT scan over time, although a more distinct region of probable central necrosis is observed in Fig. 3B . In comparison, the color pixel density shown in Fig. 3, C and D , is the fraction of image voxels within the margins of the mass filled with color flow signals. There has been a 4.4-fold decrease in blood flow to this mass over a period of approximately 8 weeks. In addition to the mass depicted in Fig. 3 , this patient had extensive disease in the chest, pelvis, and femurs.

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TM in Combination with Other Treatment Modalities
During the long-term maintenance of copper deficiency, additional treatment modalities were added to TM as deemed appropriate for the optimal management of the patients. A patient with previously untreated metastatic breast cancer is doing well with a good-to-excellent quality of life after 12 months of treatment. She had metastases in the paratracheal, posterior cervical, and retroperitoneal lymph node chains but had declined all cytotoxic therapy. The patient had stable disease for more than 6 months on TM treatment, when, due to a slight increase (less than 25% of baseline) in the bidimensional size of the paratracheal and retroperitoneal nodes, she began concurrent trastuzumab (Herceptin; Genentech) therapy after this drug became commercially available. This patient showed a rapid response to trastuzumab at all sites of disease: after one cycle, there was a clinical complete response in the neck; and after three cycles of trastuzumab, there was radiological confirmation of complete response at all previous sites of disease. The patient remains on TM, but the trastuzumab was discontinued after six doses. She continues to maintain her status as a complete responder on TM alone for more than 6 months after discontinuation of trastuzumab therapy. Because the complete response was achieved after the addition of trastuzumab therapy, this patient is classified as having only stable disease on TM on Table 2 .

Two patients with extensive angiosarcoma of the face and scalp achieved stable disease on TM. In one patient with severe chronic bleeding from an ocular lesion that threatened the orbit, IFN-2 was added to TM to attempt to enhance tumor response. Given the suggestion that, based on studies of progressing hemangiomas, the use of low-dose IFN may be efficacious for the treatment of hemangioma (41) , IFN- was administered to both of these patients at a dose of 500,000 units s.c. twice a day. Radiotherapy was also given to these two patients while on TM to attempt to control actively bleeding (but not progressing) lesions. Both patients had disease stabilization for >60 days, with one of these patients remaining with stable disease for over 5 months before discontinuation of therapy due to patient choice. No exacerbation of toxicity was observed by the addition of any of these treatment modalities to TM.

Discussion

This is the first human trial of induction and maintenance of copper deficiency with TM as an antiangiogenic therapy for cancer. In a group of patients with advanced cancer, we have demonstrated that TM is remarkably nontoxic when Cp is lowered to 10–20% of baseline levels for up to 17 months of treatment. The only drug-related toxicity observed was mild anemia, which was easily reversible with adjustment of the TM dose to bring the Cp level to the desired target. Despite the diverse roles that copper plays in essential biological processes including heme synthesis and superoxide dismutase and cytochrome function, no lasting significant adverse effects were observed on reduction of Cp to approximately 20% of baseline or to a range between 5 and 15 mg/dl. From our data, we surmise that this level of copper reduction constitutes the lower limit of chemical copper deficiency and the beginning of mild clinical copper deficiency, the first manifestation of which is mild anemia. Table 3 summarizes the stages of copper deficiency in humans and their clinical characteristics. This information was derived from studies of patients with Wilson’s disease, from occasional patients with chemical and clinical copper deficiency, and from copper-deficient small rodents. Note that as Cp is reduced below 5 mg/dl, it becomes an insensitive marker of the degree of copper deficiency. However, based on observations in humans with normal copper metabolism from this trial, we find that Cp is a sensitive and valid marker of copper status for levels above 5 mg/dl. This key finding allows the targeting of the antiangiogenic window of copper deficiency that appears to be required to slow or arrest tumor growth.