Case Study Aerosol Delivery Of Camptothecin Analogues

During the replication of the genome, the double-helix must be unwound prior to the arrival of the DNA polymerase. As DNA helicase proceeds down the strand, unwinding the DNA ahead of the replication machinery, the double-stranded DNA in front of the replication fork becomes increasingly super-coiled. This increased coiling creates a stress that must be relieved or else the entire replication process would abruptly and prematurely terminate. The enzymes responsible for alleviating this super-helical tension are the topoisomerase family, which in humans consist of topo-I and topo-II. Topo-I relaxes DNA by cleaving a single strand of a duplex DNA, allowing passage of the other strand through the nick before religation, and it is this enzyme that is targeted by camptothecin (CPT) and its analogs. CPT destructively interferes with the replication process by stabilizing the normally transient covalent linkage between the topo-I and the DNA strand, exploiting the fact that malignant cells contain greater amounts of topo-I than normal cells, making them more sensitive to this cytotoxic effect (100,101).

CPT and its analogs exist in a pH-dependent equilibrium between two distinct chemical conformations: a closed lactone E ring, and an open carboxylate form. While the lactone ring is the active compound required for binding with the topo-I-DNA complex, at physiologic pH in human serum it is the carboxylate form that dominates (102,103). In human serum, the AUC of the lactone form is between 0 and 16% of the AUC of both forms combined. This observation may be the reason for the low therapeutic index observed with these compounds in humans (104). Preclinical and clinical data indicate that the cytotoxic activity of CPT resides in the E-ring lactone. Futhermore, correlations between the degree of neutropenia and plasma concentrations of lactone and open-ring forms have suggested that the open-ring carboxylate may contribute to myelosuppression (105,106).

When tested in vivo against human tumors (in nude mice models), CPT and its analogs have demonstrated the remarkable capacity to cure the affected animals, exhibiting potency at least one hundred times greater over their cytotoxic counterparts. Moreover, once tumors have been eradicated they usually do not recur (107). In these mice models, 50% of the drug present in serum is in the lactone form compared to only about 10% in the human serum, presumably attributing to the dramatically reduced efficacy in humans, where anticancer responses are observed in fewer than 20% of patients.

While these results are somewhat discouraging, it is surprising, considering the low amount of lactone that reaches the tumor in humans, that any significant responses are even observed (100). The clinical activity of topotecan and other analogues may be attributed to the higher concentration of lactone observed at physiologic pH (around 30% for topotecan), though their therapeutic actions are hindered by their rapid elimination from the plasma (108).

As attested by the preceding paragraphs, CPT and its analogs appear to possess significant potential for effective anticancer activity, though their efficacy is limited by pharmacokinetic and delivery issues. Importantly, within the human blood stream the active a-hydroxy-S-lactone ring moiety is significantly hydrolyzed to its inactive charged carboxylate form. Moreover, when administered systemically, CPTs exhibit significant toxicity, especially involving the bone marrow and gastrointestinal tract, prompting the investigation of alternative administration routes for this set of compounds (100). For example, 9-nitrocamptothecin (9-NC) has been orally administered at doses of 1 mg/m2/day safely for extended periods. However, similar to IV systemic administration of other analogues, dose-limiting toxicities, most notably myelosuppression, have been observed (109). Pulmonary delivery of 9-NC was then investigated as a possible method of mitigating the delivery and pharmacokinetic limitations.

9-NC, like many other chemotherapy agents, is water insoluble. Preclinical investigations of aerosol formulations therefore required the development of a dispersed liquid system to facilitate aerosol generation. Liposomal formulations of 9-NC were developed and studied in animal models (53). Dilauroylphosphatidylcholine (DLPC) liposome systems had been well characterized in terms of synthesis and safety in animal models (110,111). Rats exposed to 1 h of continuous aerosol for 28 consecutive days exhibited no effect of the phospholipid (112). Phase I/II studies in humans with DLPC aerosol have also demonstrated its safety and tolerability (113). Liposomes were prepared using DMSO to dissolve the lipophilic drug before addition to the phospholipids dissolved in butanol. The mixtures are snap frozen and then lyophilized. Reconstitution of the liposomes was a simple procedure, involving addition of water for injection and after which, aerosol formation by nebulization was readily achieved.

The pharmacokinetic studies of aerosolized CPT liposomes in mice showed that high concentrations of CPT in the lungs could be attained. However, the drug was rapidly distributed to the liver, brain, and other organs (59). As discussed below, the absorption and distribution from the lung may have significant effects on local and systemic exposures and may be interpreted from two points of view. First, treatment of tumors within the lung may require sufficient residence time in local tissues for clinical efficacy. Additionally, rapid absorption from the lung following inhalation results in significant systemic exposure that may give rise to unfavorable side effects. Alternatively, pulmonary delivery of chemotherapy agents that are rapidly absorbed may possess distinct advantages for treatment of tumors that do not reside within the respiratory tract.

In terms of pharmacokinetic advantage, 9-NC-DLPC liposome aerosols have demonstrated a favorable profile (significantly reduced tumor growth rate and shrinkage without serious side effects) in treating three different human cancer xenografts (breast, colon, and lung) in a nude mouse model (53). It is believed that the liposomal formulation protects the lactone-ring, increasing the concentration of this active conformation, thus allowing for enhanced penetration in tumor cells. In animal models, equivalent antitumor activity of liposomal 9-NC as compared to the non-liposomated drug is achieved at less than 20% of the dose. Toxicity profiles in mice indicate a maximum tolerated dose of approximately 4 mg/kg when administered orally. Accordingly, in these studies the maximum dose delivered via aerosol of 307 mg/kg/day was also non-toxic. Antitumor activity was noted in the absence of weight loss or other evident toxicity (54).

This same liposomal 9-NC-DLPC aerosol system was also studied in a phase I trial in patients with advanced pulmonary malignancies (60,61). Patients were eligible if they had primary or metastatic cancer in the lungs, had failed standard chemotherapy regimens for their disease, exhibited normal bone marrow function along with normal hepatic and renal functions, had no known respiratory disease other than cancer, and possessed acceptable pulmonary function. Treatment in the feasibility cohort consisted of 6.7 mg/ kg/day 9-NC in aerosol reservoir (nebulizer) for 60min per day for 5 consecutive days/week for 1,2,4, or 6 weeks, followed by observation for 2 weeks. For the phase I portion of the study, doses were increased stepwise from 6.7 up to 26.6 mg/kg/day for 5 consecutive days for 8 weeks followed by 2 weeks rest. Twenty five patients received treatment. Does-limiting toxicity was chemical induced pharyngitis seen in two of the patients at the highest dosage (26.6 mg/kg/day). At 20.0 mg/kg/day, a grade 2 and 3 fatigue required dose reduction in 2 of the 4 patients. A reversible decrease in forced expiratory volume was also noted in patients treated with the aerosolized drug. Significantly, there was no notable hematologic toxicity, while 9-NC plasma levels were similar to those observed after oral ingestion, though the mechanisms behind this different toxicity response are unknown. Based on these studies, the recommended dose for phase II studies was 13.3 mg/kg/day delivered as two consecutive 30 min nebulizations/day from a nebulizer reservoir with 4 mg 9-NC in 10 ml water, for 5 consecutive days for 8 weeks every 10 weeks. Partial remissions were observed in two patients with uterine cancer and stabilization occurred in three patients with primary lung cancer (Fig. 2). A partial remission of a liver metastasis was also observed in a patient with endometrial cancer, demonstrating the systemic potential of aerosol delivery (Fig. 2).

Pharmacokinetic studies in patients demonstrated that total 9-NC plasma concentration continued to increase for 2-3 h from the start of treatment reaching a mean (+SD) peak concentration of 37.7 ± 20.2ng/ml at 2 h (Fig. 3). Mean (+SD) clearance was biphasic with a T1/2a of 1.9 ± 1.4 h and a T1/2p of 16.4 ± 10.5 h. The AUC of the lactone form measured in two patients comprised 3.2% and 3.5% of the total 9-NC. 9-NC concentrations in bronchoalveolar lavage fluid were 4.2 to 10.6 times higher than those measured concurrently in plasma.

Several recent animal studies have focused on aerosol delivery of 9-NC in combination with other chemotherapeutic agents. A recent investigation in mice examined the anticancer properties of a vitamin E analogue and 9-NC delivered as an aerosol against mouse mammary tumor cells (88). This combination treatment significantly enhanced antiproliferative and

Patient Aerool Coverings
Figure 2 Response in lung and liver tumors after aerosol treatment (arrows). (A) partial response in a lung metastasis of endometrial cancer (arrow) and (B) partial response in a liver metastasis of endometrial cancer in another patient (arrow). Source: From Ref. 61.

proapoptotic activities both in cell culture, and when formulated in liposomes and delivered via aerosolization to treat metastatic murine mammary tumor. Administration of these agents in tandem exhibited a significant reduction in tumor volume when compared to either treatment alone.

Continue reading here: Pharmacokinetics Of Inhalation Aerosols Implications For Cancer Treatment

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