Recombinant human endostatin administered as a 28-day continuous intravenous infusion, followed by daily subcutaneous injections: a phase I and pharmacokinetic study in patients with advanced cancer

Annals of Oncology 16: 1695–1701, 2005 doi:10.1093/annonc/mdi318 Published online 12 July 2005

Original article

Recombinant human endostatin administered as a 28-day continuous intravenous infusion, followed by daily subcutaneous injections: a phase I and pharmacokinetic study in patients with advanced cancer A. H. G. Hansma, H. J. Broxterman, I. van der Horst, Y. Yuana, E. Boven, G. Giaccone, H. M. Pinedo & K. Hoekman Vrije Universiteit Medical Center, Department of Medical Oncology, Amsterdam, The Netherlands

Background: Endostatin is an endogenous collagen XVIII-fragment with anti-angiogenic properties and remarkable antitumor activity in mice. Preclinical data suggest that continuous low dose administration of endostatin is much more potent than intermittent dosing. The feasibility of this approach is tested in a phase I study. Patients and methods: We determined the safety and pharmacokinetic profile of 4-week continuous intravenous infusion of recombinant human (rh)-endostatin, followed after an interval of 1 week by twice daily subcutaneous injections in patients with advanced cancer. Thirty-two patients received rh-endostatin in six dosing cohorts, ranging from 3.75 mg/m2/day to 120 mg/m2/day. Serum endostatin pharmacokinetics, toxicity and antitumor response were determined. Results: A total of 160 cycles were delivered without significant toxicities. Pharmacokinetic analysis showed a linear increase of steady-state serum endostatin concentrations with dose (i.v. r2 = 0.96; s.c. r2 = 0.99) reaching 300–1000 ng/ml for the two highest doses, with considerable interpatient variation. The main pharmacokinetic values for both routes of administration were similar. The apparent steadystate concentration and AUC reached at 60–120 mg/m2/day were within the range expected to induce anti-angiogenic and antitumor effects based on preclinical tumor models. Although no objective responses were observed, two patients had long-lasting stable disease (defined as a tumor increase < 100%). Conclusion: rh-endostatin was safely administered both by continuous infusion and by twice daily subcutaneous injections up to 120 mg/m2/day. Predictable pK was seen in this dose range and the target endostatin levels were reached from 60 mg/m2/day and above. Key words: angiogenesis, endostatin, pharmacokinetics, phase I

Introduction Angiogenesis is the formation of new capillaries from existing blood vessels [1, 2]. The recognition of tumor angiogenesis as a therapeutically useful target [3] is based on experimental evidence that tumors are dependent on new blood vessel formation for growth and progression. Angiogenesis in the healthy adult is thought to be essential for only a limited number of physiological processes, such as the female reproductive cycle and wound healing. Therefore, it has been proposed that the expanding microvascular endothelial cell compartment in tumors might be an important specific target in cancer therapy [4].

Correspondence to: Dr K. Hoekman, Vrije Universiteit Medical Center, Department of Medical Oncology, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Tel: +31-20-4444319; Fax: +31-20-4444355; E-mail: [email protected]. Ó 2005 European Society for Medical Oncology

The purification and identification of angiostatin [5] and endostatin [6] as endogenous circulating protein fragments with anti-angiogenic activity provided a major conceptual and practical step forward for the translation of preclinical angiogenesis research to clinical trials [7]. Endostatin is a 20 kDa naturally occurring COOH-terminal fragment derived from collagen XVIII by proteolytic activities [8]. It was originally isolated from the conditioned medium of a murine hemangio-endothelioma cell line as an inhibitor of proliferation of endothelial cells in vitro and a strong inhibitor of angiogenesis in vivo [6]. Repeated administration of endostatin in several mouse-tumor models induced prolonged tumor regression [9]. Mechanistic studies have not firmly established a receptor for endostatin, but possible interactions with integrins [10, 11], tropomyosin [12], cell surface glypicans [13] and the VEGFR-2 receptor (KDR/flk-1) [14] have been described.

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Received 27 May 2005; accepted 10 June 2005

1696

Patients and methods Patients and eligibility criteria Eligibility criteria included patients with progressive advanced solid tumors, for whom no standard treatment was available, aged between 18 and 70 years. They had to have at least one measurable lesion of at least 1 cm. Aspartate aminotransferase (AST) and adenosine triphosphate (ALT) values were to be less than 2.5 times the upper limit of normal, a bilirubin level of less than 25 lmol/l, a creatinine value less than 130 lmol/l, a white blood count (WBC) greater than 3000/mm3, platelets greater than 100 000/mm3, and normal coagulation values. The patients had to have a life expectancy of at least 3 months and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients with child-producing potential had to agree to the use of effective contraceptive methods during the study. Informed consent was obtained from all patients. Excluded were patients having a brain tumor or brain metastases, a bleeding disorder, receiving anti-coagulant therapy, a history of myocardial infarction or angina pectoris in the last 6 months or uncontrolled congestive heart failure, having an active infection or receiving radio- or chemotherapy within 4 weeks before study. Also not eligible were patients with uncontrolled serious medical or psychiatric illness or having any other condition that was likely to interfere with regular follow-up.

Design of the study The patients were treated according to a dose escalation schedule. Originally, four cohorts of eight patients receiving 3.75, 7.5, 15 or 30 mg rhendostatin/m2/day were planned. Since no side-effects were encountered, and the lower dosages were shown to be safe in other studies running at the same time [17, 22], it was decided to limit the 7.5 and 30 mg cohorts to four patients and to expand the study with two more cohorts of four patients each (60 and 120 mg/m2/day). The patients received a 28-day continuous intravenous infusion (i.v.) of rh-endostatin via a Porth-a-cath system. After a 7-day observation period, subcutaneous (s.c.) injections were given daily every 12 h. The 7-day observation period was considered an adequate washout period for pharmacokinetic sampling. Assessment of tumor burden had to be completed at the end of the first s.c. treatment period and then every other treatment cycle of 4 weeks. rhendostatin administration was discontinued in case of progressive disease (PD), which was defined as more than a 100% increase in the sum of unidimensional tumor measurements or the sum of the products of the bidimensional tumor measurements or the appearance of a new lesion. This unusual definition of PD was based on the assumption that rh-endostatin was expected to induce delayed anti-tumor effects, where initial tumor progression was acceptable. In addition, rh-endostatin administration had to be discontinued in the case of grade 3 or greater non-hematological, or grade 4 hematological toxicity (using the Common Toxicity Criteria, version 2.0), missing of 14 continuous days of planned rh-endostatin administration, or the decision of the patient or the principal investigator.

Administration of rh-endostatin The rh-endostatin protein from the yeast strain, Pichia pastoris, purified to homogeneity was supplied by EntreMed (Rockville, MD) as a frozen liquid or as a lyophilized formulation. For the i.v. infusions we used endostatin as a frozen liquid formulation at a concentration of 8 mg/ml. It was stored at ÿ70°C until the day of administration. The buffer solution consisted of 17 mM citric acid, 66 mM sodium phosphate and 59 mM sodium chloride (pH = 6.2). Continuous i.v. rh-endostatin was delivered by a Deltec CADD-microÒ infusion pump via a central line. A disposable reservoir was connected with the Deltec pump. The reservoir was replaced every 48 h. For the s.c. injections, patients in cohorts 1–3 received the same formulation used for the i.v. administration prepared by the hospital pharmacy in syringes, which were stored at ÿ18°C for a maximum of 4 days. Patients in cohorts 4, 5 and 6 received a lyophilized formulation of 80 mg/vial for the s.c. administration. The endostatin was reconstituted with sterile water to a concentration of 110 mg/ml and transferred to syringes by the hospital pharmacy. Patients stored these syringes at 4°C for a maximum of 4 days.

Pharmacokinetics of rh-endostatin Blood samples for pharmacokinetic analysis were drawn in all patients before and at 2, 4, 8, 12 and 24 h after the start of the continuous i.v. infusion on days 9, 15 and 23, and on day 28, at 1, 2, 4, 8, 12 and 24 h after the infusion was stopped. During the s.c. administration, samples were drawn before and at 1, 2, 3, 4, 5, 6, 7, 8, 12 and 24 h after the first injection, and on days 9, 15, 23 and 28, followed by one sample every 28 days in case the treatment continued. The blood samples were allowed to clot for 4 h at room temperature and were then placed overnight at 4°C to allow the clot to retract. The clot was removed by centrifugation (10 min, 4000 rpm, 4°C) and the serum samples were stored at ÿ80°C. The samples were analyzed for human endostatin concentrations using a competitive enzyme immunoassay (Accucyte; Cytimmune Sciences, College Park, MD). Each sample was measured in duplicate. For calculations of the serum concentrations of rhendostatin, the patient’s basal endostatin level as measured on day 1 prior to dosing was subtracted from all other values. The non-linear regression

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Recently, extensive downstream changes in the expression of cell signaling molecules, known to be involved in angiogenic responses, have been identified in a study where human dermal microvascular endothelial cells where exposed to rh-endostatin [15, 16]. These effects were observed after only 4 h of treatment with concentrations of soluble rh-endostatin as low as 100 ng/ml, and involved 74834 clones tested by microarray, of which >12% had a greater than two-fold (up or down) change in expression. The circulating levels of endostatin in healthy individuals are approximately 20–40 ng/ml [17, 18]. Higher concentrations of at least 1 lg/ml rh-endostatin were necessary to inhibit migration of endothelial cells, probably as a result of binding to amb5 integrins and subsequent inhibition of the focal adhesion kinase/c-Raf and mitogen-activated protein kinase (MAPK) pathways [11]. A completely different mechanism of action for endostatin has recently been reported by Kranenburg et al. [19]. These authors suggested stimulation of proteolysis by tissue-type plasminogen activator (tPA) interactions, due to the occurrence of a cross-b structure of endostatin, as the essential anti-angiogenic activity. We obtained data in line with this hypothesis since tumor fluid from a patient with a gastrointestinal autonomic nerve tumor contained an excessive amount of endostatin, which was accompanied by high tPA and plasmin activity in this fluid [20]. In theory, continuous administration of anti-angiogenesis agents may have advantages over intermittent dosing. Interestingly, the continuous administration of endostatin to mice via an i.p. implanted mini-osmotic pump, leading to sustained endostatin blood levels, resulted in the same antitumor efficacy as a 8–10 times higher dose administered by daily i.p. bolus injections [21]. In the present trial we studied the feasibility, safety and pharmacokinetics of rh-endostatin, given in escalating doses, by continuous intravenous infusion and by twice daily subcutaneous injections in advanced cancer patients.

1697 program WinNonlin (Scientific Consulting Inc., Apex, NC) was used to determine pharmacokinetic values. The calculations were done using standard non-compartmental methods. The area under the curve (AUC) was calculated from the start until the end of the intravenous infusion (from 0 to 672 h) by linear trapezoid method. The steady-state concentration (Css) was calculated as the mean of the serum levels after apparent steadystate was achieved, i.e. on days 9, 15, 23 and 28 for the i.v. as well as the s.c. period. The serum half-life (t½a) of endostatin after discontinuation of the infusion was calculated from the data obtained from samples up to 8 h, including the Css as the starting point for the elimination curve. Total body clearance and apparent volume of distribution were also calculated using this part of the elimination curve.

Thirty-two patients with advanced solid tumors were enrolled in this study. They received, in total, 32 cycles of continuous i.v. infusion and 128 cycles of s.c. injections, with doses ranging from 3.75 to 120.0 mg/m2/day. The patient characteristics are listed in Tables 1 and 2. All patients completed the planned i.v. administration period. Of the 32 patients enrolled, four patients did not start s.c. administration because of disease progression. The longest s.c. administration was 20 months at a dose level of 60 mg/m2.

Toxicities No grade 3 or 4 toxicity was seen. Grade 1 and 2 adverse events occurred, but were generally not considered to be drug related. One patient (cohort 1) developed swelling and redness at the injection site after the first injection only, which resolved spontaneously within 24 h and did nor recur. One patient (cohort 4) experienced repeated redness at the injection site.

Table 1. Patient characteristics Total number of patients

32

Gender Female

16

Male

16

Data from all 32 patients were available for the analysis of intravenous infusion. A summary of the pharmacokinetic analysis is shown in Table 3. The level of endogenous serum endostatin, as measured before the start of the administration, was 33 ± 16 ng/ml (N = 32). At all dose levels an immediate rise over the endogenous levels was seen after administration of rh-endostatin. Examples of the time-course of exogenous endostatin serum levels are shown for two patients on the 60 mg/m2 cohort (Figure 1). In general, there was a larger variation in the concentrations in samples taken weekly during the s.c. cycles than during the i.v. cycles. The calculated steady-state concentration (Css), however, showed a linear correlation with dose (Figure 2). The mean t½ per cohort, estimated from the part of the curve where frequent sampling was done (time-points 0–8 h after the end of the i.v. administration) was 4.2–12.9 h with no apparent relation to dose. This t½ from our study appears to reflect the t½ reported in two of the phase I trials of bolus administration [17, 23]. In two patients in the 60 mg/m2/day cohort there was an increased serum endostatin level 1 week after the end of the i.v. administration compared with the baseline level. This increase was present in all patients in the 120 mg/m2/day cohort (’100–200 ng/ml). This suggests drug accumulation at the highest dose levels. The calculated values for total body clearance and apparent volume of distribution varied between patients to a similar extent as between the cohorts; thus, no evidence for dose dependency was seen (Table 3).

Response evaluation No objective responses were seen. Stabilization of disease, in this study defined as less than 100% progression after 4 months of treatment (on two subsequent radiographic assessments), was observed in eight patients, treated with 15–120 mg/m2 rhendostatin. Long stabilizations were observed in a patient with metastatic para-aortal lymph nodes originating from ovary cancer, treated for 17 months, and a patient with a locally advanced hemangiopericytoma, treated for 21 months. Three patients were still alive at the time of data analysis (32+, 38+ and 40+ months).

Age (years) Median

54

Range

40–70

Performance status 0

12 patients

1

20 patients

Type of tumor Colon cancer Sarcoma

13 4

Ovarian cancer

4

Esophageal cancer

3

Miscellaneous

8

Discussion Endostatin has been shown to inhibit potently angiogenesis in vitro and in vivo. Regression of several tumor types, without occurrence of drug resistance after repeated administration of mouse endostatin, has been reported [9]. Human recombinant endostatin has been generated in Pichia pastoris, and after dosefinding studies had been performed in mice and Cynomolgus monkeys, rh-endostatin entered clinical trials [17, 22, 23]. The present study was initiated to test the feasibility and safety of continuous rh-endostatin administration, based on the observation by Kisker et al. [21] that continuous administration compared with intraperitoneal injections had superior antitumor activity in mouse xenograft models. The subcutaneous route

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Results

Pharmacokinetics

1698 Table 2. Patient characteristics and treatment duration Patient no.

Age (years)

Sex

PS

Diagnosis

Prior (CT) regimens

Dose level (mg/m2)

N cycles

Overall survival (months)

1

53

F

1

Acup + lymph + lung

0

3.75

3

4

2

53

M

1

Oesophagus + lymph + lung + liver

1

3.75

1

2

3

61

F

1

Coecum + liver + lung

2

3.75

2

4

4

62

F

0

Colon + liver + lung

2

3.75

4

7

5

58

M

1

Osteosarcoma + lung

SU6668

3.75

2

2

6

51

M

1

Parotis + lung

RT

3.75

4

14

7

51

F

1

Mamma + lymphnodes + skin

Horm. + 4

3.75

1

2

8

52

M

1

Oesophagus + lymph + pleural + liver

3

3.75

2

3

70

M

1

Carcinoid + lung + skin

1

7.5

4

9

52

M

0

Melanoma + lymph + skin + liver + bone

2

7.5

2

5

11

56

F

1

Ovary + lymph

4

7.5

4

12

12

57

F

0

Oesophagus + liver + lung

RT

7.5

6

8

13

50

M

0

Colon + liver

5

15

8

27

14

50

F

0

Colon + liver + abdominal wall

2

15

2

27

15

47

F

0

Ovary + lymph

4

15/60

21

38+

16

58

M

0

Colon + liver + lung

1

15

4

30

17

54

F

0

Liver + lymph

2

15

2

10

18

54

M

0

Locally advanced sarcoma

1

15

17

36+

19

40

F

1

Ovary + lymph + pleural

4

15

2

4

20

43

F

1

Ovary + lymph + bone

2

15

6

10

21

51

M

1

Colon + liver + lung

4

30

2

3

22

49

M

0

Pancreas + liver

0

30

2

3

23

69

F

1

Thyroid + lymph + lung + bone

0

30

7

30+

24

49

M

1

Prostate + lymph + bone

Horm

30

8

12

25

40

F

1

Sarcoma + liver + lung

1

60

2

6

26

57

M

1

Colon + liver + lung

3

60

3

3

27

55

F

1

Colon + liver + lymph + abdominal wall

3

60

3

4

28

58

F

1

Locally advanced sarcoma + lung

3

60

1

4

29

61

M

0

Colon + liver

3

120

1

3

30

43

M

0

Colon + lymph + abdominal wall

3

120

2

6

31

66

F

1

Colon + lymph + abdominal wall + adrenals

4

120

6

15

32

56

F

1

Colon + lymph + lung

3

120

4

7

of administration was investigated to allow easier ambulatory treatment and the achievement of more steady blood levels of rh-endostatin. The data presented here show that by continuous infusion of rh-endostatin, a steady-state serum level of about 200 ng/ml immunoreactive endostatin was reached at a dose level of 60 mg/m2/day. In the 120 mg/m2/day cohort, three of four patients had a Css of >1000 ng/ml, which might indicate non-linear pharmacokinetics above 60 mg. In any case, the target serum level of 200–300 ng/ml, associated with evident biological activity in preclinical studies [21], was reached during this study. Importantly, this also holds for the s.c. twice daily administration, which produced a Css of 292 and 722 ng/ml for the 60 and 120 mg/m2/day cohort, respectively. In fact no

obvious differences in pharmacokinetic parameters between the two administration routes were observed. In order to be able to make a comparison of our AUC values with those obtained in other phase I trials, we recalculated the values from Table 3 by dividing them by the number of days from one cycle (28 days). We then obtained an apparent AUC24 h of ’320 and ’1280 lg/ml.min for the 60 mg/m2 and 120 mg/m2 dose, respectively, compared with an AUC24 h of 677 and 1545 lg/ml.min for the 300 mg/m2 and 600 mg/m2 dose found by Herbst et al. [17] and 688 lg/ml.min at a dose of 240 mg/m2 as reported by Eder et al. [22]. The value of AUC24 h after a 1-h infusion, as reported by Thomas et al. [23], was 864 lg/ml.min (14.4 lg/ml.h) at 225 mg/m2 and

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9 10

1699 Table 3. Serum pharmacokinetics of continuous intravenous administration and twice daily subcutaneous injections 3.75 (n = 8)

7.5 (n = 4)

15 (n = 8)

30 (n = 4)

60 (n = 4)

120 (n = 4)

Median (range) basal endostatin (ng/ml)

23 (17–31) n=8

37 (29–45) n=4

30 (15–55) n=8

28 (24–36) n=4

30 (21–50) n=4

64 (43–77) n=4

Mean (range) Css during i.v. period (ng/ml)

15 (5–42) n=8

49 (41–54) n=4

80 (45–146) n=8

135 (59–226) n=4

227 (189–264) n=4

1068 (651–1277) n=4

Mean (range) Css during s.c. period (ng/ml)

20 (14–35) n=5

58 (44–70) n=4

56 (35–97) n=7

146 (42–296) n=3

292 (275–313) n=3

722 (511–920) n=3

Mean (range) AUC during i.v. period (lg.h/ml)

10.3 (3.5–26.6) n=8

33.4 (24.2–35.0) n=4

52.7 (28.6–92.8) n=8

84.0 (40.3–132.7) n=4

149.5 (130.7–170.2) n=4

596.6 (396.0–737) n=4

Mean (range) t½ 0–8 h (h)

4.2 (2.3–6.8) n=7

7.6 (3.3–12.5) n=4

7.9 (3.3–13.1) n=8

12.9 (3.5–39.7) n=4

8.6 (4.4–12.1) n=4

4.4 (1.1–8.6) n=4

Mean (range) Vd (l)

195 (66–518) n=7

152 (72–268) n=4

205 (80–423) n=8

256 (142–493) n=4

262 (139–398) n=4

76 (15–164) n=4

Mean (range) Cl (l/h)

33.2 (7.0–53.0) n=7

13.9 (11.0–16.2) n=4

18.7 (8.5–31.0) n=8

25.7 (8.6–41.0) n=4

20.9 (19.0–22.7) n=4

11.3 (9.2–13.4) n=4

serum endostatin (ng/mL)

Css, steady-state concentration (basal value before endostatin administration subtracted) is mean of 4 weekly determinations; Vd, volume of distribution; AUC, area under the serum concentration curve until the end of i.v. (0–672 h); t½, elimination serum half-life; Cl, clearance.

450 400 stop iv

350 300 250 200 150 100

start sc

50 0 0

200

400

600

800

1000 1200 1400 1600

Time after start i.v. (Hours) Figure 1. Representative serum endostatin concentrations as a function of time after start of the intravenous infusion, including cycle 1 (i.v.) and cycle 2 (s.c.) of two patients receiving 60 mg/m2/day rh-endostatin.

concentration (ng/mL)

Endostatin Steady-state Concentration 1200 1000 I.V.

800 600 S.C.

400 200 0 0

50

100

150

dose (mg/m2/day) Figure 2. The steady-state serum endostatin concentration (pretreatment value subtracted). Correlation coefficient for i.v. = 0.96 and for s.c. = 0.99.

1470 lg/ml.min (24.5 lg/ml.h) at 300 mg/m2 dose. It can be concluded, therefore, that 60–90 mg/m2 continuous infusion gives an AUC of about 600–1000 lg/ml.min. This AUC meets the target AUC of 600–700 lg/ml.min set for antitumor activity by Sim et al. from mice studies [24]. Some pharmacokinetic parameters from four clinical studies of rh-endostatin are listed in Table 4. Taking into consideration

that routes of administration were different, that the groups of patients were small and heterogeneous, and that some values could not be calculated exactly in the same way, the selected pharmacokinetic values are similar in these four trials. Despite the fact that target serum concentrations of endostatin were reached in our study, no clear evidence for antitumor activity, defined as partial or complete responses, was observed. Three patients received rh-endostatin for an extended period of time (7, 17 and 21 months), and long-term survivors (>30 months) were present in this series. Interestingly, from the data of Kisker et al. [21] it has been argued that continuous rh-endostatin administration suppressed the slow-growing tumors more effectively than the fast-growing tumors. This observation was also reported for other anti-angiogenic agents [25]. Lack of antitumor activity may not be considered as a failure, because it is not the major endpoint of phase I studies. Most patients treated in our trial were heavily pretreated (see Table 2 for prior regimens in our study), comprising a population in which few responses may be expected. In the other three phase I trials of rh-endostatin [17, 22, 23] stabilization of disease was also observed in a few patients. Possible causes for lack of antitumor activity of anti-angiogenic agents may be due to the fact that the biology of advanced human cancer is more complex, redundant and less dependent on neo-vascularization compared with implanted human tumors in immunocompromised mice. The factors and processes, which may contribute to resistance against anti-angiogenic agents, have been discussed extensively [17, 26–32]. The interpretation of our trial and the other rh-endostatin phase I trials has been hampered by the lack of convenient and validated surrogate marker assays for anti-angiogenic activity. In general, the monitoring of serum markers, circulating endothelial cells (CECs), imaging of tumors and biopsies of tumors have been used for this purpose. These results, however, were not conclusive. rh-endostatin treatment of cancer patients induced no significant changes in the concentration of some well-known serum endothelial markers [17, 22, 23]. A decrease

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Dose (mg/m2/day)

1700 Table 4. Pharmacokinetic parameters from phase I clinical trials with soluble rh-endostatin Basal endostatin (ng/ml)

Vd (l/m2)

Cl (l/h) a

t½ (h)

Herbst [17]

20 6 10

’48

50 6 25

10.7 6 4.1 (‘t½b’)

Eder [22]

5–42 (mean 18)

43 6 20a

114–198 (3 highest doses)

10 (‘t½z’)

Thomas [23]

8–54 (28 6 13)

38 (not dose-dependent)

not reported

12.9 (‘t½b’)

This study

15–77 (33 6 16)

11–33 (not dose-dependent)

76–262

4–13b

a

Total body clearance was reported not to be dependent on body surface area by Herbst et al. [17] and Eder et al. [23]. t½ calculated from 0 to 8 h after i.v. stopped.

b

Acknowledgements Endostatin was provided by Entremed Inc, who also supported the study.

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in the number of CECs due to endostatin treatment has only been shown in a preclinical tumor model [33]. Herbst et al. [34], using PET (labeled water), observed a decrease in tumor blood flow that was maximal at about 250 mg/m2 rh-endostatin administered, but this was not evident at similar doses in the studies by Eder [22] and Thomas [23], using MRI or dynamic CT scans, respectively. In tumor biopsies, Herbst et al. [17, 34] observed a significant increase in endothelial cell death and a decreased microvessel density at 250 mg/m2 of rh-endostatin; this could, however, not be reproduced by Thomas et al. [23] and Mundheke et al. [35]. The latter group also used a skin wound assay to monitor possible rh-endostatin effects on angiogenesis, but wound healing was not affected at 8 weeks after treatment with doses up to 300 mg/m2/day [35]. Recently, Macpherson et al. [36] reported that P. pastoris rhendostatin from Entremed Inc. was inactive in an endothelial tube forming assay in concentrations of 250 ng/ml or higher, while the same product purchased by Calbiochem inhibited tube formation. These authors concluded that differences in storage or handling conditions, such as concentration, temperature or pH, rather than breakdown of the primary product might be responsible for this finding. Since lack of activity has also been reported from endostatin produced by retroviral gene approach in mouse tumor models in which serum concentrations of 746 ng/ml [37] were reached, the proper folding of endostatin is an issue of intensive debate [38]. Gebbink et al. [39] found differences in folding between endostatin produced in bacteria used in the original studies and endostatin from Entremed. They postulated that active endostatin adopts a conformation with cross-b structure, similar to structures found in amyloid. This might explain the discrepancies in the results obtained with endostatin from different sources. Finally, the dependence on E-selectin expression by endothelial cells [40] and the requirement of circulating adhesion proteins for anti-angiogenic activity of endostatin [41] suggests possible differences in patients with cancer. These data suggest that co-administration of an activator of endothelial cells may modulate endostatin activity. In conclusion, it is questionable if additional clinical work with rh-endostatin is indicated at this moment, despite the lack of toxicity and predictable pK. The development of a simple ex vivo activity test and/or appropriate surrogate markers for anti-angiogenic activity in patients, in analogy to the recent experience with bevacizumab [42], an antibody against vascular endothelial growth factor, would be highly recommended in order to improve our understanding of the results of any new trial.

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