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Clinical Trials |
1 The University of New Mexico, Albuquerque, New Mexico; 2 Baylor College of Medicine, Houston, Texas; and 3 The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Patients with primary or metastatic lung cancer received aerosolized liposomal 9-nitrocamptothecin for 5 consecutive days/week for 1, 2, 4, or 6 weeks followed by 2 weeks of rest to determine feasibility. For the Phase I part, the dose was increased stepwise from 6.7 up to 26.6 µg/kg/day Monday to Friday for 8 weeks followed by 2 weeks of rest.
Results: Twenty-five patients received treatment. The mean baseline forced expiratory volume in 1 second for all patients was 85% of predicted. A dose-limiting toxicity was chemical pharyngitis seen after 1 week in 2 of 2 patients at 26.6 µg/kg/day. At 20.0 µg/kg/day, grade 2 and 3 fatigue prompting a dose reduction was seen after 4 weeks in 2 of 4 patients. Grade 2 toxic effects included nausea/vomiting (9 patients), cough and bronchial irritation (6 patients), fatigue (5 patients), anemia (4 patients), neutropenia (2 patients), anorexia (1 patient), and skin rash around the face mask (1 patient). 9-Nitro-20(S)-camptothecin (9NC) was absorbed systemically. Partial remissions were observed in 2 patients with uterine cancer, and stabilization occurred in 3 patients with primary lung cancer.
Conclusions: Aerosol administration of liposomal 9NC was found to be feasible and safe. 9NC delivered as an aerosol was detected in patient’s plasma shortly after the start of treatment. The recommended dose for Phase II studies is 13.3 µg/kg/day (equivalent to 0.5 mg/m2/day), which constitutes two consecutive 30-min nebulizations/day from a nebulizer reservoir with 4 mg of 9NC in 10 ml of sterile water, Monday to Friday for 8 weeks every 10 weeks.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first objective of this study was to determine the safety and feasibility of aerosol administration of DLPC-9NC to human patients for 5 consecutive days/week for increasing periods of time. Secondary study objectives included determinations of the recommended Phase II dose for an 8-week course of treatment and the dose-limiting toxicity (DLT) profile for this length of administration. Concentrations of 9NC in plasma of volunteering patients were measured to determine preliminary pharmacokinetics of the aerosolized preparation.
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PATIENTS AND METHODS |
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50% of the predicted values. All patients signed an informed consent form approved by the human-subject institutional review board.
Treatment.
9-Nitrocamptothecin (ChemWerth, Woodbridge, CN) and dilauroylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) were formulated by S.P. Pharmaceuticals LLC (Albuquerque, NM) and lyophilized to a dry powder as described previously (4)
. Sterile vials of 2 mg of 9NC and 100 mg of DLPC were stored at –20°C until ready for patient use. The drug was mixed with 10 ml of sterile water that provided an approximate 30-min nebulization treatment.
A feasibility evaluation of DLPC-9NC administered 5 consecutive days/week by aerosol was done first, followed by a Phase I trial. Six patients (cohort 1) were entered in the feasibility cohort and treated twice for 30 min every day for 5 consecutive days, followed by an observation period of 2 weeks. The daily dose was 6.7 µg/kg 9NC. Once the feasibility of the aerosol treatment was established, the Phase I study began, first by increasing the number of weeks of delivery with 1 patient/each test period followed by 2 weeks of observation. The test periods were 2, 4, and 6 weeks (cohorts 1A, 1B, and 1C). Dose escalation started with 8 weeks of administration with cohorts of 3–6 patients (cohorts 2–5). Increasing drug dosage from 6.7 µg/kg/day was done first by doubling the concentration of drug in the nebulizer, then by increasing the amount of time/daily administration. Estimated daily doses tested were 6.7, 13.3, 26.6, and 20.0 µg/kg/day (Table 1
; Ref. 9
). The drug was administered through an AeroMist nebulizer (CIS-US, Bedford, MA) flowing at 10 liters of air/min with a mouth breathing-only face mask (Hans Rudolph, Inc., Kansas City, MO). Patients and the aerosol nebulizer were enclosed in a HEPA-filtered airborne scavenging tent (Peace Medical, Inc., Orange, NJ). Premedication was introduced after 1 patient (cohort 2) developed a grade 2 bronchial irritation after a treatment. Patients in later cohorts were premedicated for 3 days before starting chemotherapy with Albuterol and Fluticasone propionate inhalers, which were then given on an as needed basis throughout the aerosol treatment period. Before chemotherapy administration, patients simulated a treatment to familiarize themselves with the tent environment, face mask, and respiratory techniques. The first 8-week course and the first week of the second course of treatment were administered at M. D. Anderson Cancer Center under clinical supervision to observe and train patients. Afterward, if no side effects greater than grade 2 were observed, patients were allowed to self administer treatment at home with a portable air compressor (Easy Air 15; Precision Medical, Northampton, PA) and an Enviracaire HEPA-filtering system (Honeywell, Golden Valley, MN). Home treatment was supervised weekly by telephone contact and once by a home visit by a member of the research team. Repeated courses at the same or the previous dose level were administered to the patients who benefited from the treatment as determined by either remission or stabilization of disease.
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Pharmacological Procedures.
Because this was the first use of 9NC as an aerosol, it was important to determine systemic absorption after inhalation. For subjects who volunteered for pharmacokinetic studies, blood was obtained at various time points from the start of the aerosol treatment depending on the cohort. For cohort 1 (feasibility study), blood was obtained on either day 5 of the treatment week. Samples were taken at 0, 0.5, 1, 1.25, 1.5, 2, 3, 5, 7, and 24 h from the start of the 1-h aerosol exposure. For cohorts 1A, 1B, 2, and 3, samples were obtained at 0, 2, and 5 h on days 1 and 5 of weeks 1, 4, and 8 where appropriate. For cohort 5, samples were obtained at 0, 2, 5, 8, and 24 h on day 1 of weeks 1, 4, and 8 and at 0, 2, 5, and 8 h on day 5 of weeks 1, 4, and 8. For cohort 4 (twice daily treatment), blood was obtained at 0, 2, 5, 10 (before start of second treatment), 12, 15, and 24 h from the start of the first treatment.
Extraction of total 9NC and its carboxylate and lactone forms were performed using Waters C18 Sep-Pak Light cartridges immediately after separating the plasma. The cartridges were activated with 1 ml of 100% methanol, followed by 1 ml of water. Fifty µl of the lactone form of CPT in 100% methanol (CPT; final concentration, 5 or 10 ng CPT/ml) were added as internal standard to 1 ml of plasma in a 3-ml syringe. This sample was immediately loaded onto the cartridge. The fraction that did not bind to the cartridge was collected as the carboxylate form of 9NC. Then the C18 cartridge was washed with 1 ml of water, followed by 1 ml of 20% methanol in water, and the lactone form (containing the internal standard) was collected by eluting the cartridge with 1 ml of 100% methanol. Another 1 ml of plasma was used to extract the total 9NC.
For cohorts 1, 1A, 1B, and 2, liquid chromatography/mass spectrometry (MS) analysis (see below) was used to detect all 9NC fractions and done at M. D. Anderson Analytical Core Laboratory. The following extraction technique was used. Fifty µl of CPT and 2 ml of acetonitrile were added to 1 ml of the carboxylate eluate, and to 1 ml of plasma (total 9NC fraction), the mixtures vortexed for 5 min and then centrifuged for at 2500 rpm for 5 min in a refrigerated (4°C) centrifuge. Five ml of a 95:5 mixture of chloroform:2-propanol (v/v) were added to the supernatants, vortexed for 5 min, and then centrifuged for at 2500 rpm for 5 min. The lower layer was placed into glass tubes and placed in a heating block at 40°C and evaporated to dryness with air. The lactone eluate was also dried. All three 9NC fractions were reconstituted with 50 µl of 100% methanol, vortexed briefly, and then 50 µl of 0.02% formic acid were added and vortexed. After centrifugation at 2500 rpm for 5 min, the supernatants were centrifuged again in a microfuge at 15,000 rpm for 5 min. The resulting supernatant fractions were used for LC/MS analysis.
For cohorts 3, 4 and 6, a high-performance liquid chromatography detection method was used at Baylor College of Medicine. Fifty µl of CPT was added to 1 ml of plasma and to 1 ml of carboxylate eluate as described above and the mixture vortexed for 10 s. To precipitate protein and acidify the fraction to convert all 9NC to the lactone form, 1 ml of 8.5% H3PO4 was added, vortexed, and allowed to incubate for 15 min at room temperature. After centrifugation (International Electrotechnical Commission centrifuge; 2000 rpm for 10 min), the supernatants were loaded onto C18 Sep-Pak cartridges and processed as described above. The 9NC was collected as the lactone form in the 100% methanol fraction. An aliquot (0.5 ml) of all three 9NC fractions was added to 15-ml plastic centrifuge tubes, placed in a heating block at 50°C, and evaporated to dryness with air. The samples were reconstituted 5-fold by adding 100 µl of high-performance liquid chromatography mobile phase as described below. The reconstituted samples were centrifuged in a microfuge at 13,000 rpm for 10 min and the supernatants used.
The LC/MS instrument (MicroMass, Beverly, MA) was operated in the electrospray-positive ionization mode. 9NC and CPT (internal standard) were detected in selected ion recording mode at m/z 394 for 9NC and m/z 349 for CPT. Analytes were separated from background plasma components by running extracted samples through a microbore YMCbasic 5-µm 1 x 100-mm analytical column (Waters, Milford, MA). Column temperature was maintained at 30°C. The isocratic mobile phase consisted of 40% acetonitrile and 60% 0.1% formic acid (aqueous; pH 3.0) at a flow rate of 100 µl/min. The lowest level of detection for 9NC was 0.5 ng/ml plasma.
The Waters HPLC system used a Waters Xterra MS C-18 guard column with a Waters Xterra MS C-18 column (2.1 x 150 mm) heated to 50°C. The isocratic mobile phase consisted of 40% methanol and 60% water containing 0.5% acetic acid (pH 1.5) flowing at 0.25 ml/min. 9NC was detected using a Waters 2487 absorbance detector set at 228 nm, whereas CPT was detected using a Waters 470 scanning fluorescence detector (excitation wavelength, 360 nm; emission wavelength, 455 nm). Minimal level of detection was 2.5 ng 9NC/ml plasma. Selected samples were compared with the LC/MS method and found to be equivalent.
Standard curves for 9NC were prepared in human serum and processed as described above for each cohort. Concentrations of 0, 0.5, 2.5, 10, 50, 100, and 250 ng 9NC/ml of plasma each containing 10 ng CPT/ml as the internal standard were used. 9NC plasma concentrations were calculated from the ratio of 9NC to CPT.
Bronchoscopsy was performed by a pulmonologist (A. Huaringa) to obtain bronchoalveolar lavage (BAL) fluid from the right middle lobe of volunteering patients at various times after the completion of treatment. Concentration of total 9NC in the BAL fluid was corrected for dilution by determining the ratio of urea in the lavage and in a plasma sample taken at the same time (urea diffuses freely).
Criteria for Response and Toxicity.
Adverse events and DLTs occurring in the first course of therapy were based on the National Cancer Institute Common Toxicity Criteria version 2 to determine the appropriate starting dose for Phase II studies. DLTs were defined as the dose that produced a reversible grade 3 or 4 hematological toxicity lasting >7 days or a reversible grade 3 (grade 2 for neurotoxicity) nonmyelosuppressive toxicity in >33% of patients treated at a given dose level. Clinical responses were determined according to the WHO criteria (11)
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Statistical Analysis.
A standard 3 + 3 design was used. The recommended dose for Phase II study was defined to be the highest dose for which no patient of 3 or <2 patients of 6 developed a grade 3 toxicity.
Pulmonary function was compared by the two-tailed, paired t test to evaluate changes during and after treatment from baseline values. Total 9NC concentrations in plasma between cohorts were compared by two-tailed t test with unequal variances.
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RESULTS |
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and 3
. Mean FEV1 and other pulmonary functions before treatment were at least 85% of predicted values.
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All other side effects were grades 1 or 2. Table 4
shows the main side effects, with cough in 67% of patients, bronchial irritation in 46%, sore throat in 33%, nausea in 62%, vomiting in 33%, anorexia in 33%, dysgeusia in 33%, fatigue in 50%, anemia and neutropenia in 29%, and skin rash around the face mask in 21%. Grade 1 epistaxis, chills, dysuria, and thrombocytopenia were seen in 
4 patients. No grade 3 neutropenia was observed. No cumulative toxic effects were observed in 7 patients who received more than one course.
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and O2 saturation (data not shown) remained within an acceptable clinical range (>50% predicted value). Mean percent predicted FEV1 function dropped by 20% during aerosol therapy (P < 0.0001) and improved to 83% of predicted value upon cessation of therapy (Fig. 1)
. The mean end-of-treatment value was not significantly different from the baseline (P = 0.107). The 3 patients with a FVC and/or FEV1 < 50% of predicted value had extensive pulmonary tumor involvement at the start of treatment that was progressive despite therapy. TLC values were virtually unchanged and DLCO values were closely comparable with FVC results.
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. The first patient was treated at 13.3 µg/kg/day. The second at 20 µg/kg/day for 4 weeks, then at 13 µg/kg/day for the last 4 weeks. This reduction was prompted by a grade 2 fatigue, at the request of the patient. They both successfully underwent resection of the residual disease after two courses and remain free of cancer 18 months later. A partial remission of a liver metastasis was also observed in a patient with endometrial cancer treated at 13.3 µg/kg/day, demonstrating the systemic potential of aerosol delivery of this drug (Fig. 2B)
. In addition, 3 of the 6 patients with primary lung cancer had stabilization of their tumors. Two of these patients received 6.7 µg/kg/day dose for either multiple 1-week courses or a 6-week course of treatment. The third patient received 13.3 µg/kg/day dose for an 8-week course. Clinical evaluation of tumor mass was made during the follow up period from a computer-assisted tomography scan after either two 1-week courses or after a single 6- or 8-week course of treatment.
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. Total 9NC plasma concentrations continued to increase for 2–3 h from the start of treatment reaching a mean (±SD) peak concentration of 37.7 ± 20.2 ng/ml at 2 h (range, 13.6–58.0 ng/ml). Mean (±SD) clearance of 9NC was biphasic with a T1/2
of 1.9 ± 1.4 h and a T1/2ß of 16.4 ± 10.5 h. The area-under-the-curve (AUC) of the lactone form measured in the last 2 patients comprised only 3.2 and 3.5% of the total 9NC. Four of these 5 patients also volunteered for a bronchoscopy. The time of sampling was spread out among the patients from 1.2 to 4.8 h from the start of aerosol treatment. BAL 9NC concentrations varied from 51 to >361 ng/ml. Because of the large dilutions, urea levels in the BAL fluids were below detection (i.e., <0.5 mg/dl). Therefore, 9NC concentrations in BAL fluids were >4.2 to >10.6 times higher that those measured concurrently in the plasma.
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. Similar changes were observed in both CMAX and AUC results. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 fatigue was seen at all dose levels. Although the most dramatic dose-limiting toxicity was chemical pharyngitis, the mucosa of the bronchus was spared significant toxicity despite a reversible decrease in FEV1. Cough and bronchial irritation were clinically ameliorated by bronchodilators and steroids. Bronchial irritation was also more pronounced in patients with extensive pulmonary involvement with cancer. Changes in oxygen saturation and lung volumes were not clinically relevant. This study was limited in time because of the nature of the diseases treated; therefore, we do not have data on long-term pulmonary toxicity. It is encouraging that FEV1 values were reversible after cessation of treatment (Fig. 1)
. Nausea and vomiting were observed in a few patients, but these complaints did not reach the intensity seen with oral administration (14
, 16)
, and weight loss was not observed after aerosol delivery. Although 2 patients complained of dysuria, blood was not seen on the urine microscopic examination. Our pharmacology results demonstrated that there was a dose-dependent increase in both CMAX and AUC values at the two lower doses but not at the highest dose. This may have been due to a saturation effect on the lungs’ ability to transport drug from the alveolar side to the blood. Such an effect would be detected best in AUC values. Although the 9NC plasma AUC value at the highest dose was greater than the increase in the 9NC aerosol dose, the limited number of patients and time points prevented an accurate analysis. Future pharmacology studies will follow patients for longer periods of time. Our results also show that high levels of 9NC were found in the lungs at the end of treatment and that higher BAL to plasma ratios were noted at later time points. One weakness of our study is the commercial urea measurement in the BAL, which fell below the detection level of common laboratory techniques. Absorption of drug through the lung parenchyma into the systemic circulation after its inhalation is rapid and sustained. Alveolar-capillary exchange of drug is through the pulmonary vein, making inhalation therapy a method of sustained arterial delivery. This would allow first pass presentation of drug to cancer sites. It is also possible that 9NC deposited on lung surfaces is metabolized differently than after other routes of administration. The aerosol droplet size of the 9NC liposomal complex is in the range that optimizes alveolar deposition (1–3 µm).
Responses were observed in patients with endometrial cancer at the recommended Phase II dose, not only in the lungs, but also systemically. Responses occurred early after initiation of chemotherapy (within the first 4 weeks by chest X-ray, confirmed at 8 weeks by computerized tomography scanning) and led to resection of pulmonary disease with a possibility of cure in 2 patients as recommended (17) .
The recommended dose for Phase II studies is 13.3 µg/kg/day on a daily 60-min exposure, 5 consecutive days/week for 8 weeks, with a concentration of 9NC of 0.4 mg/ml in the nebulizer. The advantages of inhaled 9NC are rapid and efficient systemic absorption and the good side effect profile. Because of the promising anticancer activity, a Phase II study in patients with recurrent endometrial cancer is planned. Additional pharmacokinetics will also be performed to better define the disposition of 9NC after inhalation in plasma, tumor, and bronchoalveolar fluids.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Note: Presented in part at the New York Academy of Science Symposium on Camptothecin 2000, annual meetings of American Society Clinical Oncology 2000 and 2002, AACR 2002, and International Gynecologic Cancer Society 2002.
Requests for reprints: Claire F. Verschraegen, University of New Mexico, Cancer Research and Treatment Center, 900 Camino de Salud Northeast, Albuquerque, NM 87131. Phone: (505) 272-6760; Fax: (505) 272-2841; E-mail: cverschraegen{at}salud.unm.edu
Received 6/23/03; revised 12/15/03; accepted 1/ 4/04.
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REFERENCES |
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