Purpose: Squalamine is an antitumor agent that has been shown to have antiangiogenic activity in animal models. This Phase I/IIA study was designed to assess the safety, clinical response, and pharmacokinetics of squalamine when administered as a 5-day continuous infusion in conjunction with standard chemotherapy every 3 weeks in patients with stage IIIB (pleural effusion) or stage IV non-small cell lung cancer.

Experimental Design: Patients with chemotherapy-naive non-small cell lung cancer were treated with escalating doses of squalamine in combination with standard doses of paclitaxel and carboplatin. Paclitaxel and carboplatin were administered on day 1, followed by squalamine as a continuous infusion on days 1–5, every 21 days.

Results: A total of 45 patients were enrolled (18 patients in the Phase I dose escalation arm and 27 in the Phase IIA arm). The starting dose of squalamine was 100 mg/m2/day and escalated to 400 mg/m2/day; two of three patients at 400 mg/m2/day had dose-limiting toxicity that included grade 3/4 arthralgia, myalgia, and neutropenia. On the basis of safety and toxicity, 300 mg/m2/day was selected as the Phase II dose of squalamine in this combination regimen. An additional 27 patients (a total of 33) were enrolled according to the protocol treatment schema at 300 mg/m2/day. There was no pharmacokinetic evidence of drug interactions for the combination of squalamine, carboplatin, and paclitaxel. Forty-three patients were evaluable for response. Partial tumor responses were observed in 12 (28%) of these patients; an additional 8 evaluable patients (19%) were reported to have stable disease. For all of the patients treated, the median survival was 10.0 months; and 1-year survival was 40%.

Conclusions: The combination of squalamine given continuously daily for 5 days, with paclitaxel and carboplatin given on day 1, is well tolerated. Patient survival data and the safety profile of this drug combination suggests that the use of squalamine given at its maximum tolerated dose with cytotoxic chemotherapy should be explored further as a potentially effective therapeutic strategy for patients with stage IIIB or IV non-small cell lung cancer.

Squalamine is a chemically synthesized aminosterol, originally isolated from the liver of the dogfish shark and identified in a search for antimicrobial agents. Squalamine selectively inhibits new blood vessel formation (1, 2); this activity is thought to be mediated through inhibition of the sodium-hydrogen antiporter sodium-proton exchangers (specifically the NHE3 isoform) causing inhibition of hydrogen ion efflux from endothelial cells, with subsequent reduction of cellular proliferation (3, 4). Studies in tumor-bearing mice have shown that squalamine inhibits angiogenesis and tumor growth in xenograft models of lung (5, 6), breast (7), ovarian (8), and prostate (9) cancer and in brain (2) and breast (5) allograft tumor models in rats. Squalamine also has been shown to prevent lung metastases in the murine Lewis lung carcinoma model, both as a single agent and in combination with various other chemotherapeutics (5). The antitumor effects of squalamine appear to be the result of blocking mitogen-induced proliferation and migration of endothelial cells, because squalamine does not affect the growth of tumor cells in culture (2, 5). It also has been noted that squalamine seems to have the greatest effect on newly emerging vessels with no appreciable effect on unstimulated endothelial cells (4).

Squalamine does not appear to have substantial direct effects on primary tumor growth in animal models when administered as a single agent. However, enhanced antitumor responses are observed when squalamine is administered in combination with cytotoxic chemotherapeutic agents when compared with cytotoxic agents used alone (5, 6, 10). We have previously shown that squalamine inhibits the establishment of H460 human tumors in nude mice but has no effect on the growth of H460, CALU-6, and NL20T-A human NSCLC3 xenografts when administered after the tumors are established (6). However, when squalamine was combined with cisplatin or carboplatin during treatment of these xenografts, there was a significant increase in tumor growth delay and a decrease in blood vessel formation. Neither the effects on tumor growth delay nor the effects on blood vessel formation were observed when squalamine was administered with paclitaxel, vinorelbine, gemcitabine, or docetaxel in these NSCLC models, suggesting a specificity for platin agents (6). Squalamine induces disorganization of F-actin stress and causes a concomitant reduction in the endothelial cell surface molecule cadherin that disrupts cell-cell adhesion (10), two events necessary for angiogenesis. We have hypothesized, on the basis of these observations, that squalamine acts to augment tumor cell cytotoxicity from platinum exposure by interfering with the promotion of endothelial cell movement and cell-cell communication necessary for the growth of new blood vessels after platin-induced tumor damage.

The combination of carboplatin and paclitaxel is a commonly administered chemotherapy regimen for NSCLC in the United States. However, NSCLC response rates remain poor and median survival has not been extended beyond 8–10 months (11, 12, 13). On the basis of its unique mechanism and animal model data, it is speculated that squalamine may demonstrate clinical activity in vascular tumors and may prevent metastasis when administered in combination with platin-containing chemotherapy regimens. Given the enhancement in antitumor activity observed when squalamine is administered with cisplatin and carboplatin in human tumor xenograft or allograft models, and the widespread use of carboplatin and paclitaxel in advanced NSCLC, we initiated a Phase I/IIA study to evaluate the safety and antitumor activity of squalamine administered in combination with carboplatin and paclitaxel in patients with NSCLC. The primary objectives of this study were to evaluate the objective response rate and safety of squalamine administered as a 5-day continuous infusion in combination with carboplatin and paclitaxel for patients with stage IIIB or IV NSCLC. An additional objective of this study was to determine the pharmacokinetic characteristics of squalamine, carboplatin, and paclitaxel when administered in combination and to assess any pharmacokinetic evidence of drug interaction.

Patient Selection.

Patients with histologically confirmed stage IIIB (pleural effusion) or stage IV non-small cell bronchogenic carcinoma were eligible for this study. All of the patients were required to have bidimensional measurable or evaluable disease; patients with clinically stable brain metastases were allowed to participate. Other eligibility criteria included: age ≥18 years with ECOG performance status ≤1, able and willing to provide written informed consent for participation, absence of other serious medical disorders or neuropathy, and adequate bone marrow function (WBC count ≥4000/mm3 or absolute neutrophil count ≥1500/mm3 and platelet count ≥100,000/mm3 ), liver function (total bilirubin ≤2 times upper limit of normal and AST/SGOT <5 times upper limit of normal), and renal function (serum creatinine ≤1.5 mg/dl or calculated creatinine clearance ≥60 ml/min). Sexually active male and female patients were required to practice medically acceptable birth control, and female patients who were either pregnant, lactating, or attempting to become pregnant were not eligible for participation. Other exclusion criteria included prior treatment with chemotherapeutic agents, biological response modifiers, or antiangiogenesis agents (prior chemotherapy or radiation therapy for other malignancies was allowed if at least 5 years had elapsed since completion of treatment), uncontrolled diabetes mellitus, or significant cardiac disease. In patients with only one site of measurable disease, previous irradiation to that site was not allowed unless the patient had experienced disease progression.

Drug Administration and Study Design.

Squalamine was supplied by Genaera Corporation in 10-ml stoppered glass vials containing 50 mg of lyophilized active drug substance; the lyophilized powder was reconstituted with water (5 ml). The appropriate dose of the drug was then drawn from the vial(s) and transferred aseptically to continuous infusion bags containing 5% dextrose such that the final squalamine concentration was 3 mg/ml. Commercially available preparations of carboplatin and paclitaxel were used according to the manufacturer’s instructions.

On day 1 of each cycle, paclitaxel at a dose of 225 mg/m2 was administered as an i.v. infusion over 3 h with an in-line 0.22 μm filter. Immediately after the paclitaxel administration, carboplatin was administered at a dose of AUC = 6 in normal saline as an i.v. infusion over 15–30 min through a vein or venous access device. Squalamine was administered immediately after carboplatin administration by continuous infusion via a centrally placed infusion line using an ambulatory infusion pump system (SIMS Deltec, Inc., St. Paul, MN). The squalamine dose was escalated in sequential cohorts from 100 to 400 mg/m2/day and was administered as a 5-day (120 h) continuous infusion. The dose was varied by controlling the infusion pump rate. This 5-day dosing regimen was repeated every 21 days unless the clinical condition of the patient required a delay in further drug administration.

Four cohorts were studied. In each cohort, the paclitaxel dose (225 mg/m2) and carboplatin dose (AUC = 6) were held constant and the squalamine dose was varied. The first cohort of patients was treated with squalamine at a dose of 100 mg/m2/day; subsequent cohorts received 200, 300, or 400 mg/m2/day squalamine. Initially, three patients were enrolled in a cohort, and all of the patients in a cohort were required to complete the first 21-day cycle before evaluation of DLT could be made; therefore, decisions regarding dose escalation were deferred until after the third patient in a cohort completed the first 21-day cycle. In the absence of DLT in the first three patients, dose escalation to the next dose was permitted. If one of the three patients experienced DLT, up to three additional patients were entered into that cohort. If at least one additional patient experienced DLT, the next lowest squalamine dose was determined to be the MTD, or Phase II dose. DLT was defined as any of the following: grade IV neutropenia (absolute neutrophil count <500/mm3 ) for more than 7 days; grade IV neutropenia accompanied by a fever (single elevation in oral temperature to >38.5°C or three elevations to >38°C during a 24-h period) that requires parenteral antibiotics; grade IV thrombocytopenia; delay in carboplatin/paclitaxel administration of more than 2 weeks; or other hematological grade III or IV treatment-related toxicity. Once the squalamine MTD was established, additional patients were enrolled as described in the “Statistical Considerations” section to evaluate clinical efficacy. In the absence of disease progression or unacceptable toxicity, patients were allowed to receive a maximum of six cycles of treatment.

Toxicity Assessments.

Any patient who received a single dose of squalamine was considered evaluable for safety analysis. Before treatment and at least weekly during the study, routine laboratory studies, including complete blood count and evaluation of liver and renal function, were performed. Serum pregnancy tests were performed as applicable. All adverse clinical events were categorized according to the NCI common toxicity criteria (CTC) or, if the NCI CTC were not applicable, as mild, moderate, or severe by the investigator.

Determination of Objective Response.

Objective clinical response was determined by quantitative assessment of measurable lesions. At baseline and before initiation of treatment cycles 2, 4, and 6, tumor size assessment was made by X ray, computed tomography, and/or magnetic resonance imaging evaluation. For this study, complete clinical response was defined as complete disappearance of all clinically detectable malignant disease for at least 4 weeks. A partial clinical response was defined as a ≥50% decrease in tumor size for at least 4 weeks without increase in size of any area of known malignant disease of >25% or the appearance of new areas of malignant disease. Stable disease was defined as no significant change in measurable or evaluable disease for at least 4 weeks and no appearance of new areas of malignant disease; stable disease also included a decrease of <50% in malignant disease, a decrease in unidimensional measurable disease of <30%, or an increase in malignant disease of <25% at any site. Progressive disease was defined as a significant increase (i.e., >25%) in the size of lesions present at the start of treatment or after a response, or the appearance of new metastatic lesions not present at the start of treatment.

Patients were considered evaluable for response if they had completed one cycle of chemotherapy, although they were considered evaluable for toxicity assessment and survival if they had received any treatment therapy.

Pharmacokinetic Assessment.

For all of the patients, blood samples (10 ml in heparinized collection tubes) were obtained before treatment, during the first 27.5 h after treatment was initiated, and during 48 h on days 6–7 of the first cycle. Blood samples were specifically collected 1.5 h after initiation and at completion of the 3-h paclitaxel infusion and at the end of the 30-min carboplatin infusion that immediately followed paclitaxel infusion. Additional samples were collected 2, 4, 12, 24, and 120 h after the initiation of squalamine administration and 15 and 30 min and 1, 2, 4, 12, 24, and 48 h after the 5-day squalamine infusion was completed. All of the plasma samples were centrifuged at 3000 rpm for 15 min at 4°C immediately after collection, and the resulting plasmas were stored at −20°C until analysis.

Squalamine concentrations in plasma samples were determined by a contract laboratory (Taylor Technology, Princeton, NJ) with a validated high performance liquid chromatography-electrospray mass spectrometry (LC/ESI/MS) method after sample extraction on OASIS HLB Solid Phase Extraction cartridges (Waters Company, Milford, MA). Deuterated (d6)-squalamine was used as an internal reference standard with all of the samples. Extracted samples were subsequently analyzed on a Hewlett-Packard Series II 1090L liquid chromatography system (Agilent Technologies, Palo Alto, CA) coupled with a Finnigan TSQ mass spectrometer (Finnigan Corporation, San Jose, CA) by an electrospray interface. The mass spectrometer was operated in a selected ion scanning (SIM) mode with windows at 626.5 and 632.5 ± 0.4 atomic mass unit and exhibited a linear detection range of 10–1000 ng/ml squalamine.

Paclitaxel levels in duplicate human plasma samples were quantified by a second contract laboratory (MDS Pharma, Montreal, Canada) using high-performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). A protein precipitation was performed before analysis on a Waters Alliance 2690 liquid chromatograph (Waters Corporation, Milford, MA) coupled to an Applied Biosystems/PE Sciex API300 mass spectrometer (Applied Biosystems Corporation, Foster City, CA) fitted with a turbo ion spray source operating in the positive ion mode. N-octylbenzamide was added as an internal standard before the protein precipitation. The range of reliable paclitaxel quantitation on this instrument was determined to be 5–5000 ng/ml.

Free platinum (Pt) levels from carboplatin in human plasma were also determined at MDS Pharma by a validated, flameless atomic absorption method. Quantitation over a linear dynamic concentration range of 50–2220 ng Pt/ml was conducted on a Varian SpectrAA 220 Zeeman atomic absorption system (Varian Inc., Palo Alto, CA).

Pharmacokinetic modeling was performed using the WinNonLin data package (version 3.l; Pharsight Corporation, Mountain View, CA). Maximum concentration (Cmax) and time to Cmax (tmax) were actual observed values, and area under the concentration-time curve (AUC) was estimated using a trapezoidal rule. For squalamine, both a noncompartmental model and a two-compartment model with constant i.v. input and first order output were used to evaluate each individual patient’s set of concentration-time data. Squalamine pharmacokinetic data for a patient were used in population pharmacokinetic analyses as long as the data were missing from no more than five time points, including no more than two consecutive time points. In addition, pharmacokinetic analyses were performed for an individual patient if quantifiable concentration data for at least three of the last four time points could be joined by a straight line when graphed on a linear-linear or log-linear scale (noncompartmental analysis) or if the data were not rejected by the WinNonLin software for two-compartment analysis. Pharmacokinetic parameters of paclitaxel and carboplatin were evaluated using a noncompartmental model of plasma concentration-time data for each patient for whom data were available.

Statistical Considerations for Patient Cohort Size.

The primary study objective was to determine the squalamine MTD in the context of any DLTs of squalamine when administered in combination with carboplatin and paclitaxel therapy. The sample size of the study was increased at the MTD to test a null hypothesis; the number of patients to be enrolled was selected by statistical consideration of whether or not the efficacy of this treatment regimen was quantitatively worse than the combination of carboplatin and paclitaxel alone. Once the squalamine MTD was identified, the number of additional patients treated at the MTD dose was fixed at 16, and the incidence of objective response (i.e., complete response and partial response) was examined. If two or more objective responses were observed among the 16 patients at the squalamine MTD, an additional 9 patients were enrolled to further explore the therapeutic benefit of the three-drug combination. A 33% or greater objective response rate was considered clinically meaningful; representing a 50% improvement in response over the rate for carboplatin/paclitaxel reported in the ECOG 1594 trial (11).

Demographic and Baseline Patient Profile.

This Phase IIA study was conducted at four sites: The University of Texas M. D. Anderson Cancer Center, the University of Wisconsin, the Cancer Therapy and Research Center (CTRC; San Antonio, TX), and Vanderbilt University. Forty-five patients, whose demographics are presented in Table 1, were enrolled in this study during the period from June 1999 to March 2001 and received a total of 172 courses of squalamine plus carboplatin and paclitaxel (number of patients: M. D. Anderson, 23; Wisconsin, 16; CTRC, 4; Vanderbilt, 2). Using the ECOG Performance Status Scale, we determined that the median performance status for all of the enrolled patients was 1, with 36 patients having a performance status equal to 1, and 9 patients with performance status equal to 0. Although patients with prior chemotherapy were excluded from the study, there were eight patients who had received prior radiotherapy.

Patient Disposition.

Eighteen (40%) of the 45 patients in this study completed the full six-cycle chemotherapy regimen as required by the clinical protocol. Reasons for early discontinuation of treatment for the remaining 27 patients are presented in Table 2. Two patients did not complete 5 days of squalamine therapy in cycle one, one because of the development of pneumonia on day 3, and the second because of a pulmonary embolism on day 2.

In the first three patients in cohort 1 (100 mg/m2/day), one patient developed DLT (grade 3 sodium increase); therefore, in accordance with the study design, an additional three patients were enrolled at this initial squalamine dose. There were no further occasions of DLT in cohorts 1, 2, or 3. One patient in cohort 4 receiving 400 mg/m2/day squalamine experienced dose-limiting grade 3 increases in sodium, potassium, AST, ALT, and total bilirubin levels, and a second patient had Grade 3 arthralgias and myalgias that were considered DLT. On the basis of these data and after consultation among all of the investigators at the four study sites, it was decided that the fourth dose level was not the dose level that we wanted to take forward into Phase II trials. Therefore, an additional three patients were entered at dose level 3 to confirm this as the Phase II dose. None of the patients at this dose level had DLT. Therefore, the cohort 3 squalamine dose of 300 mg/m2/day was determined to be the MTD when given in combination with paclitaxel (225 mg/m2) and carboplatin (AUC = 6).

Safety Assessments.

Table 3 summarizes grade 3/4 and total adverse events that were reported during the study. The majority of patients tolerated the treatment regimen well. The clinically significant adverse events noted were alopecia, arthralgias and myalgias, anorexia, nausea/vomiting, dyspnea, and leukopenia/neutropenia, of which neutropenia was the most frequent grade 3/4 serious toxicity. Other frequent grade 3/4 toxicities included dyspnea, elevated AST, and nausea/vomiting episodes. Accrual was stopped at dose level 4 when two of three patients experienced numerous grade III toxicities, even though none were previously defined as dose limiting. No cumulative toxicities were observed.

Squalamine, Carboplatin, and Paclitaxel Pharmacokinetics.

A total of 42 patients had a full or nearly full complement of blood samples collected for analysis and pharmacokinetic modeling, of whom samples from 41 patients were potentially available for determination of squalamine content. Noncompartmental pharmacokinetic analysis of physiologically relevant parameters is detailed for squalamine, paclitaxel, and carboplatin in Table 4.

Squalamine was administered as a continuous infusion over 5 days. The initial phase was quite short, as reflected in the distribution half-life ranging from 0.94 to 1.74 h. Squalamine plasma clearance follows a second order of elimination and ranged from 4.67 to 7.55 liter/hr. The terminal half-life ranged from 16.7 to 203.6 h. The long terminal half-life in one patient at the 100-mg/m2/day dose level led to this skewed data. This prolonged clearance was attributable to a very high concentration of squalamine observed during the terminal portion of the curve. This may have been caused by inadequate flushing of squalamine in the central line after the completion of squalamine infusion and before the collection of blood specimen. As expected, the linear pharmacokinetic, observed over the four dose levels in the Cmax and AUC, ranged from 2.7 to 10.7 μg/ml and 348 to 1301 μg/ml∗h, respectively. There was no evidence of any relationship between squalamine pharmacokinetic parameters other than AUC and Cmax with the daily dose of squalamine.

Across the four squalamine dose levels, the mean paclitaxel clearance ranged from 12.1 to 16.1 liter/h. With paclitaxel dose of 225 mg/m2, the resulting mean AUC ranged from 32 to 40.3 μg/ml∗h. For carboplatin, ultrafiltrate-plasma (free carboplatin) clearance ranged from 3.3 to 5.8 liter/h. On the basis of the individual’s estimated glomerular filtration rate (creatinine clearance), the dose of carboplatin administered to target a carboplatin AUC of 6 mg/ml × min resulted in an AUC ranging from 4.6 to 8.3 mg/ml × min. The mean carboplatin AUC was 5.67 + 1.64 mg/ml × min for the group with the most number of patients (n = 25) and was at the MTD dose level of squalamine at 300 mg/m2/day. This result nearly reached the targeted carboplatin AUC of 6 mg/ml × min.

The plasma clearances for both paclitaxel and carboplatin, analyzed using ANOVA parametric testing, did not reveal any significant effects from the coadministration with squalamine. However, using the same ANOVA testing, the AUC for carboplatin slightly correlated with the squalamine dose (P = 0.041), suggesting a possible change in the disposition of carboplatin in the presence of squalamine.

Clinical Response.

Antitumor activity was assessed in any patient who completed at least two cycles of treatment. The intent-to-treat population was defined as anyone who received a single daily infusion of squalamine. There were 45 patients in the intent-to-treat population; however, 10 patients withdrew during, or just subsequent to, the first cycle of chemotherapy and were not evaluated for response, leading to an evaluable population of 35 patients (Table 2). The objective tumor response, defined as the tumor assessment made after completion of at least two cycles, is shown in Table 5. There were no observations of complete response, but there were 12 documented partial responses among the 35 evaluable patients, leading to an objective clinical response rate of 34%. There also were an additional 8 patients with stable disease and 15 patients with progressive disease that had no degree of response to the triple chemotherapy combination.

Survival.

Kaplan-Meier survival analysis was performed for all of the 45 enrolled patients in the intent-to-treat group. In the intent-to-treat group, the median survival was 10.0 months and the 1-year survival was 40% (Fig. 1). A similar analysis was also performed on the 33 patients treated at the squalamine MTD of 300 mg/m2/day. In the patient population who received squalamine at 300 mg/m2/day, median survival was 8.5 months and 1-year survival was 33%.

Therapy for advanced lung cancer has improved only moderately in the last 20 years. Despite the most aggressive approaches of single-agent and combination chemotherapy with these cytotoxic agents, response rates have improved only modestly, ∼20–25%, and median survival remains at 8–10 months (11, 12, 13). Clearly, there is a need for development of novel and rationally designed therapeutic agents that can be used either as single agents or in combination with conventional cytotoxic drug regimens.

Angiogenesis inhibitors represent a very exciting approach for NSCLC patients. There is a wealth of data in the literature now documenting the requirement for angiogenesis in human tumor growth and metastasis. The inhibition of angiogenesis has proved to be an important concept and a potentially effective strategy for treatment of existing tumors and prevention of metastasis both in animals and humans. In addition to eliminating the tumor’s “supply line,” other theoretical advantages of targeting the endothelial cells include the possibility of overcoming drug resistance, because endothelial cells are genetically stable and, thus, less likely to evolve mechanisms of resistance. In addition, theoretically, death on one blood vessel may result in death of thousands of tumor cells downstream and, when combined with cytotoxic agents, may result in enhanced toxicity. Antiangiogenic treatment strategies, including the use of endostatin, angiostatin, TNP-470, metalloprotease, or tyrosine kinase inhibitors, or the employment of various anti-VEGF approaches have been enormously successful in preclinical animal models (in some cases producing complete tumor eradication) and are now being evaluated in clinical trials (14, 15, 16). Because of their specificity for endothelial cells, these agents are generally well tolerated.

On this basis, we have combined the antiangiogenic aminosterol squalamine with a conventional cytotoxic regimen of carboplatin plus paclitaxel for treatment of patients with advanced NSCLC. The goal of this study was to determine, as a preface for larger studies, whether the safety and response rates with the combination regimen were at least equivalent to the rates with the cytotoxic regimen alone.

In this study, we chose to combine squalamine with carboplatin because of preclinical models suggesting a positive interaction with platin (6). We chose the combination of carboplatin and paclitaxel rather than carboplatin alone because of clinical data demonstrating doublets are better than a single agent (17, 18). In this study, we found that aminosterol squalamine can be safely combined with carboplatin and paclitaxel; the addition of squalamine to the chemotherapy regimen produced only minimal additional side effects. Under the schedule evaluated in this study (5-day continuous infusion of squalamine at doses up to 400 mg/m2/day in combination with paclitaxel at 225 mg/m2 and carboplatin at AUC = 6), the response rate and time to tumor progression as well as median and one-year survival all are consistent with the historical studies of carboplatin and paclitaxel.

At the squalamine doses evaluated (100–400 mg/m2/day) in combination with chemotherapy, significant toxicities were limited to mild myelosuppression. The maximum squalamine dose that could be safely combined with carboplatin and paclitaxel was determined to be 300 mg/m2/day; this dose was safely administered for up to six 21-day cycles in this patient population. Under this treatment regimen and at this dosage, a 10-month median survival was observed in patients with stage IIIB (pleural effusion) and IV NSCLC on an intent-to-treat basis; at the MTD, an 8.5-month survival was observed. The difference in outcome between the intent-to-treat group and the patients treated at the MTD probably reflects the small patient numbers; however, it also suggests that the benefit of squalamine may be independent of dose.

On the basis of small-animal data, a dose of 20 mg/kg squalamine injected i.v. was shown to inhibit tumor growth with a resultant AUC of 215 μg/ml∗h (Ref. 5, and unpublished data).4 In another preclinical paper, an additive or synergistic effect with squalamine and cisplatin was observed at 6 mg/m2 (2 mg/kg) in mice. Squalamine at a concentration of 1.6 μm inhibited the VEGF signaling pathway in human umbilical vascular endothelial cells (19). In this clinical study, all of the dose levels of squalamine achieved or exceeded this exposure (AUC) required to have biological activity based on the preclinical data.

The pharmacokinetic parameters of squalamine in this combination study with two standard cytotoxic agents, paclitaxel and carboplatin, displayed similar disposition to squalamine administered as a single agent in the Phase I study as described by Bhargava et al.(20). With the four dose levels of squalamine used in this combination study, squalamine displayed linear pharmacokinetics. Plasma clearance of squalamine did not differ significantly among the four dose levels. Paclitaxel pharmacokinetics was not altered in this combination therapy. Carboplatin clearance was not significantly altered by the concomitant administration of squalamine. When carboplatin AUC was analyzed, there was a trend for an effect of increasing dosage of squalamine on the disposition of carboplatin. This correlation was not significant if the three data points from the 400 mg/m2/day squalamine group were omitted from the analysis. This suggests a possible drug interaction with squalamine but only at the higher dosage level. Additional data at this higher squalamine dosage level would be needed to support this higher-dose effect.

The pharmacokinetic analysis of squalamine clearance suggests that by this route and schedule of administration, this agent reaches serum concentrations consistent with preclinical activity without unacceptable toxicity. Studies involving other antiangiogenic agents, notably bevacizumab have observed increased rates of hemoptysis, including fatal events (21). No grade 3–5 episodes of hemoptysis were observed in our study.

Future studies will delineate the optimal method of combining the potent antiangiogenic agent squalamine with standard chemotherapy regimens. Although the results reported here are promising, additional effort is need to capitalize even further on the interaction of the antiangiogenic and cytotoxic agents. Although it is possible that squalamine has a negative interaction with paclitaxel, this was observed in preclinical models (22, 23). Preclinical studies had demonstrated that the antitumor effect is greatest when squalamine is administered with a platin on a frequent basis (17). To maximize this approach, a Phase II study in NSCLC patients involving weekly squalamine with weekly carboplatin and paclitaxel is ongoing.

Although angiogenesis inhibitors represent a very exciting approach for additional clinical trials, they pose a number of clinical dilemmas. Because this class of agents works indirectly against the tumor vasculature, they will most likely be cytostatic agents with no direct antitumor activity. Not unexpectedly, preclinical studies of squalamine given as single agent have not shown any significant antitumor activity. The best use of such compounds ultimately may be to prevent disease progression and/or to maintain stable disease, the induced dormancy theory of Folkman (24). However, this type of activity will be difficult to evaluate in classic Phase I and II trials, and will likely require a combination with more classic cytotoxic agents in the setting of patients with advanced disease, as we have undertaken in this study. Incorporation of surrogate biomarkers, or application of new genetic microarray technology, may be useful for the demonstration of benefit from the added, nonclassic agent. For antiangiogenic agents like squalamine, it is anticipated that noninvasive imaging studies will show a beneficial clinical effect on blood vessel density, blood flow, or tissue metabolism in the target tissue. With those data in hand, it will be possible to optimize the dose, the schedule, and the route of administration of squalamine for maximal clinical benefit.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by an American Society of Clinical Oncologists Career Development Award and an M. D. Anderson Cancer Center Physician-Scientist Award. J. H. S. is supported by a NCI K24 Mid-career Development Award.

3

The abbreviations used are: NSCLC, non-small cell lung cancer; ECOG, Eastern Cooperative Oncology Group; AST, aspartate aminotransferase; SGOT, serum glutamic oxaloacetic transaminase; AUC, area under the concentration curve; MTD, maximum tolerated dose; DLT, dose-limiting toxicity; NCI, National Cancer Institute; ALT, alanine aminotransferase.

4

R. S. Herbst, unpublished observations

Fig. 1.

Survival curves for intent-to-treat (ITT) population. A, survival. B, time to progression.

Fig. 1.

Survival curves for intent-to-treat (ITT) population. A, survival. B, time to progression.

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Table 1

Patient characteristics

CharacteristicData
No. of patients  
 Total 45 
 Evaluable 43 
No. of courses  
 Total 172 
 Median, per patient 
 Range, per patient 1–6 
Age, yr  
 Median 60 
 Range 41–84 
Sex, no. of patients  
 Male 22 
 Female 23 
Performance status at study entry, no. of patients  
 0 9 (20%) 
 1 36 (80%) 
Previous therapy, no. of patients  
 Chemotherapy 0 (0%) 
 Radiation therapy 8 (9%) 
Disease stage at study entry, no. of patients  
 Stage IIIB with pleural effusion 5 (11%) 
 Stage IV 40 (89%) 
CharacteristicData
No. of patients  
 Total 45 
 Evaluable 43 
No. of courses  
 Total 172 
 Median, per patient 
 Range, per patient 1–6 
Age, yr  
 Median 60 
 Range 41–84 
Sex, no. of patients  
 Male 22 
 Female 23 
Performance status at study entry, no. of patients  
 0 9 (20%) 
 1 36 (80%) 
Previous therapy, no. of patients  
 Chemotherapy 0 (0%) 
 Radiation therapy 8 (9%) 
Disease stage at study entry, no. of patients  
 Stage IIIB with pleural effusion 5 (11%) 
 Stage IV 40 (89%) 
Table 2

Dosing data for patients receiving chemotherapy and reasons for discontinuation of treatment

No. of patients at various squalamine dose levels, mg/m2/dayTotal
100200300400
Cycles completed      
 Less than one 
 1 
 2 
 3 
 4 
 5 
 6 13 18 
 Total 33 45 
Reasons for discontinuation before cycle 6 (n = 27)      
 Disease progression 12 18 
 Unacceptable toxicity 
 Withdrawal of consent 
 Intercurrent illness (non-cancer) 
 Death 
 Other 1a 
No. of patients at various squalamine dose levels, mg/m2/dayTotal
100200300400
Cycles completed      
 Less than one 
 1 
 2 
 3 
 4 
 5 
 6 13 18 
 Total 33 45 
Reasons for discontinuation before cycle 6 (n = 27)      
 Disease progression 12 18 
 Unacceptable toxicity 
 Withdrawal of consent 
 Intercurrent illness (non-cancer) 
 Death 
 Other 1a 
a

Pathological fracture.

Table 3

Presentation of adverse events data

ToxicityNo. of patients (%)
NCI toxicity gradeTotal
Grade 1–2Grade 3–4
Nonhematological    
 Nausea 29 (85%) 5 (15%) 34 
 Myalgia 28 (90%) 3 (10%) 31 
 Anorexia 26 (93%) 2 (7%) 28 
 Vomiting 21 (84%) 4 (16%) 25 
 Dyspnea 18 (75%) 6 (25%) 24 
 Arthralgia 22 (100%) 0 (0%) 22 
 Constipation 20 (91%) 2 (9%) 22 
 Diarrhea 20 (95%) 1 (5%) 21 
 Peripheral neuritis 14 (87.5%) 2 (12.5%) 16 
 Paresthesia 14 (100%) 0 (0%) 14 
 Increased SGOT 10 (77%) 3 (23%) 13 
 Increased SGPT 8 (62%) 5 (38%) 13 
 Neuropathy 9 (69%) 4 (31%) 13 
 Dehydration 7 (70%) 3 (30%) 10 
 Lung disorder 6 (75%) 2 (25%) 
 Peripheral edema 7 (87.5%) 1 (12.5%) 
 Hemoptysis 8 (100%) 0 (0%) 
 Asthma 7 (100%) 0 (0%) 
 Hypokalemia 3 (50%) 3 (50%) 
 Hyponatremia 2 (40%) 3 (60%) 
 Pleural effusion 1 (20%) 4 (80%) 
 Pneumonia 1 (20%) 4 (80%) 
 Deep venous thrombophlebitis 1 (25%) 3 (75%) 
Hematological    
 Leukopenia/neutropenia 4 (18%) 18 (82%) 22 
 Anemia 11 (73%) 4 (27%) 15 
 Thrombocytopenia 8 (89%) 1 (11%) 
ToxicityNo. of patients (%)
NCI toxicity gradeTotal
Grade 1–2Grade 3–4
Nonhematological    
 Nausea 29 (85%) 5 (15%) 34 
 Myalgia 28 (90%) 3 (10%) 31 
 Anorexia 26 (93%) 2 (7%) 28 
 Vomiting 21 (84%) 4 (16%) 25 
 Dyspnea 18 (75%) 6 (25%) 24 
 Arthralgia 22 (100%) 0 (0%) 22 
 Constipation 20 (91%) 2 (9%) 22 
 Diarrhea 20 (95%) 1 (5%) 21 
 Peripheral neuritis 14 (87.5%) 2 (12.5%) 16 
 Paresthesia 14 (100%) 0 (0%) 14 
 Increased SGOT 10 (77%) 3 (23%) 13 
 Increased SGPT 8 (62%) 5 (38%) 13 
 Neuropathy 9 (69%) 4 (31%) 13 
 Dehydration 7 (70%) 3 (30%) 10 
 Lung disorder 6 (75%) 2 (25%) 
 Peripheral edema 7 (87.5%) 1 (12.5%) 
 Hemoptysis 8 (100%) 0 (0%) 
 Asthma 7 (100%) 0 (0%) 
 Hypokalemia 3 (50%) 3 (50%) 
 Hyponatremia 2 (40%) 3 (60%) 
 Pleural effusion 1 (20%) 4 (80%) 
 Pneumonia 1 (20%) 4 (80%) 
 Deep venous thrombophlebitis 1 (25%) 3 (75%) 
Hematological    
 Leukopenia/neutropenia 4 (18%) 18 (82%) 22 
 Anemia 11 (73%) 4 (27%) 15 
 Thrombocytopenia 8 (89%) 1 (11%) 
Table 4

Pharmacokinetic parameters by squalamine dose levelsa

Parameters100 mg/m2/day (n = 5)200 mg/m2/day (n = 3)300 mg/m2/day (n = 19)400 mg/m2/day (n = 2)
Squalamine     
Cmax (μg/ml)b 2.7 ± 0.5 7.3 ± 1.1 9.1 ± 4.9 10.7 ± 0.9 
AUC (μg/ml*h) 347.98 ± 38.87 861.19 ± 140.44 1076.41 ± 592.21 1300.90 ± 79.76 
CL (liter/h) 6.54 ± 3.60 4.72 ± 3.46 4.67 ± 2.62 7.55 ± 3.79 
 Volume of distribution (Vss250 ± 493 14.4 ± 11.6 21.4 ± 38.4 17.6 ± 8.8 
t1/2-α (h) 1.31 ± 0.73 1.22 ± 1.0 0.94 ± 0.47 1.74 ± 0.70 
t1/2-β (h) 203.6 ± 335.5 16.7 ± 11.5 37.8 ± 73.8 24.8 ± 28.4 
Paclitaxel     
CL (liter/h) 16.0 ± 6.4 (n = 5) 14.9 ± 6.7 (n = 3) 16.1 ± 7.4 (n = 25) 12.1 ± 6.2 (n = 2) 
AUC (μg/ml*hr) 32.3 ± 6.4 (n = 5) 33.3 ± 16.8 (n = 3) 32.0 ± 15.8 (n = 25) 40.3 ± 21.2 (n = 2) 
Carboplatin     
CL (liter/h) 5.8 ± 2.9 (n = 4) 4.9 ± 3.6 (n = 3) 4.2 ± 1.5 (n = 25) 3.3 ± 0.4 (n = 3) 
AUC (μg/ml*h) 144.7 ± 37.2 (n = 4) 169.7 ± 31.1 (n = 3) 178.5 ± 51.6 (n = 25) 261 ± 82.5 (n = 3) 
Parameters100 mg/m2/day (n = 5)200 mg/m2/day (n = 3)300 mg/m2/day (n = 19)400 mg/m2/day (n = 2)
Squalamine     
Cmax (μg/ml)b 2.7 ± 0.5 7.3 ± 1.1 9.1 ± 4.9 10.7 ± 0.9 
AUC (μg/ml*h) 347.98 ± 38.87 861.19 ± 140.44 1076.41 ± 592.21 1300.90 ± 79.76 
CL (liter/h) 6.54 ± 3.60 4.72 ± 3.46 4.67 ± 2.62 7.55 ± 3.79 
 Volume of distribution (Vss250 ± 493 14.4 ± 11.6 21.4 ± 38.4 17.6 ± 8.8 
t1/2-α (h) 1.31 ± 0.73 1.22 ± 1.0 0.94 ± 0.47 1.74 ± 0.70 
t1/2-β (h) 203.6 ± 335.5 16.7 ± 11.5 37.8 ± 73.8 24.8 ± 28.4 
Paclitaxel     
CL (liter/h) 16.0 ± 6.4 (n = 5) 14.9 ± 6.7 (n = 3) 16.1 ± 7.4 (n = 25) 12.1 ± 6.2 (n = 2) 
AUC (μg/ml*hr) 32.3 ± 6.4 (n = 5) 33.3 ± 16.8 (n = 3) 32.0 ± 15.8 (n = 25) 40.3 ± 21.2 (n = 2) 
Carboplatin     
CL (liter/h) 5.8 ± 2.9 (n = 4) 4.9 ± 3.6 (n = 3) 4.2 ± 1.5 (n = 25) 3.3 ± 0.4 (n = 3) 
AUC (μg/ml*h) 144.7 ± 37.2 (n = 4) 169.7 ± 31.1 (n = 3) 178.5 ± 51.6 (n = 25) 261 ± 82.5 (n = 3) 
a

All values are expressed as mean ± SD.

b

Cmax, maximum plasma drug concentration; CL, plasma clearance; Vss, volume of distribution at steady state; t1/2-α, distribution half-life; t1/2-β, terminal (elimination) half-life.

Table 5

Response rate (best tumor response)

Best tumor responseNo. of patientsResponse rate
ITTa patients n = 45Evaluable patients n = 43
Complete response 0% 0% 
Partial response 12 27% 28% 
Stable disease 18% 19% 
Disease progression 23 51% 53% 
Not evaluable 4%  
Best tumor responseNo. of patientsResponse rate
ITTa patients n = 45Evaluable patients n = 43
Complete response 0% 0% 
Partial response 12 27% 28% 
Stable disease 18% 19% 
Disease progression 23 51% 53% 
Not evaluable 4%  
a

ITT, intent to treat.

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