Liposomal Annamycin Research Articles

Preliminary Results from a Phase 1/2 Study of Liposomal Annamycin (L-ANN) in Combination with Cytarabin for the Treatment of Patients with Acute Myeloid Leukemia (AML) That Is Refractory to or Relapsed (r/r) after Induction Therapy

Background: Anthracylines still remain the backbone for the treatment of patients with acute myeloid leukemias (e.g., “7 + 3”), however, the dose escalation is impeded by a significant cardiotoxicity of these compounds. The design of the novel liposomal L-annamycin (L-ANN) was the replacement of a basic amine at the C-3' position with a hydroxy group, which was shown to significantly reduce cardiotoxicity when compared with doxorubicin. Moreover, this modification not only decreased cardiotoxicity, but also led to increased activity against mdr-1 tumors. In addition, L-ANN was found to be a TOPO-II poison, and it has been hypothesized that the drug may not alter TOPO-IIβ, an isoform which is known to be a key pathway involved in anthracycline-induced cardiotoxicity. However, this hypothesis has not yet been confirmed experimentally. L-ANN as a monotherapy has been evaluated in r/r AML patients and demonstrated an ORR of 80% (240 mg, administered on three consecutive days, MB-105, NCT03388749, Gil et al. 2023) with no sign of cardiotoxicity noted in any patient. L-ANN is currently being evaluated in a phase 1b/2 study in combination with cytarabine for the treatment of AML (MB-106; NCT05319587). Study Design: MB-106 (NCT05319587) is aphase 1/2, multi-center, open-label, dose-escalation study to evaluate safety, tolerability, pharmacokinetics (PK) and efficacy of L-ANN in combination with cytarabine in adult r/r AML patients after induction therapy. Approximately 63 subjects will be enrolled, including up to 42 in the dose escalation phase, and up to 21 in the phase 2 expansion at the established recommended phase 2 dose (RP2D). The dose escalation phase of the study has explored 2 dose levels of L-ANN with a starting dose of 190 mg/m 2/day and 230 mg/m 2/day for 3 consecutive days. All patients will also receive cytarabine 2.0 g/m 2/day for 5 consecutive days starting on the first day of L-ANN treatment. The primary objectives of the phase 1b portion of the study are to evaluate the safety and identify the RP2D of L-ANN in combination with a standard regimen of cytarabine. Routine safety monitoring including ECHOs, ECGs, GLS measurements, cardiac biomarkers, laboratory evaluations, and bone marrow assessments (BMA) will be performed. Secondary objectives include CR/CRi and mOS. Dose escalation was halted after the 230 mg/m 2/day cohort due to the safety and efficacy data identifying this as the RP2D. Results: To date, 6 patients have completed therapy cohort 1 (n=3) and cohort 2 (230 mg/m 2/day, n=3) with no dose limiting toxicities (DLTs) noted. Prescheduled interim reviews of the data took place after completion of each dosing cohort. In the first cohort one patient achieved a CR following the first cycle of therapy which continued to exhibit durability at 5 months. In the second cohort one patient achieved a CRi and two patients showed progression. In light of the safety and efficacy data from this trial and the prior MB-105 trial, the current study has begun to expand enrollment of up to 21 additional patients at the 230 mg/m 2 L-ANN level to quantify efficacy. Conclusion: Initial data suggested that L-ANN is safe and active in heavily pretreated r/r AML patients with no signs of cardiotoxicity. This study is currently being conducted in Europe across 5 sites in Poland and 3 in Italy. Results from the Phase 2 portion of this study will be presented at the meeting.

Results of a Phase I Study of Liposomal Annamycin for Treatment of R/R AML Patients after Induction Therapy

Results of a Phase I Study of Liposomal Annamycin for Treatment of R/R AML Patients after Induction Therapy

High Efficacy of Liposomal Annamycin (L-ANN) in Combination with Cytarabine in Syngeneic p53-Null AML Mouse Model

High Efficacy of Liposomal Annamycin (L-ANN) in Combination with Cytarabine in Syngeneic p53-Null AML Mouse Model

AML-221: Liposomal Annamycin Overcomes MDR and Has No Cardiac Toxicity in Two Clinical Trials for the Second-Line Treatment of R/R AML

Context This study is determining the lack of any cardiotoxicity and efficacy of L-Annamycin for the second-line treatment of R/R AML. Objective To demonstrate the safety and efficacy of L-Annamycin leading to its approval as the only approved therapy for most patients with R/R AML agnostic of mutational status. Design Two new phase Ib/IIa clinical trials were started in both the U.S. and Europe in 2018 using L-Annamycin for the second-line treatment of R/R AML. These are ongoing proof-of-concept studies that will hopefully lead to accelerated approval for this indication. All patients have received 3 days of intravenous L-Annamycin over 2 hours. Patients are being evaluated for efficacy using the International Working Group response criteria. For the efficacy analysis, 21 subjects will be evaluated and compared to historical controls. All patients will be followed, and OS, as well as subsequent therapies if any (including SCT) will be captured. An update to these results will be presented. Setting All patients are being treated in tertiary care academic medical centers specializing in hematological malignancies and hematopoietic SCT in both Poland and the U.S. Patients Patients who have primarily refractory AML or who have relapsed with AML are eligible for the study. The EF must be >50% to receive therapy. The study is a 3+3 dose escalation followed by an expansion cohort to evaluate safety, toxicity, and efficacy. Two cohorts have been completed in the U.S., and the study met its primary endpoint of safety. There were no SAEs or DLTs, and no patients were withdrawn because of adverse events. In Poland, the first four cohorts have finished, again without any SAEs or DLTs, and no patients were withdrawn because of adverse events. Intervention L-Annamycin IV over 2 hours qd × 3d Main outcome measures 1. Safety; 2. PR and CR according to International Working Group criteria for AML; 3. Assessment of any cardiotoxicity as determined by 3D echocardiography with strain analysis and cardiotoxicity biomarkers, including troponins levels. Results To date, the U.S. study has met its primary endpoint of safety. There were no SAEs or DLTs. No patient was withdrawn due to toxicity. There was one CRi and one PR in the six patients treated. The Polish study has enrolled the first four cohorts and the first patient in the fifth cohort. Of the 13 patients, 7 had relapsed AML, and 6 had primarily refractory AML. As in the U.S., there have been no SAEs, DLTs, or patients withdrawn. There have been no decreases in EF, and all cardiotoxicity markers have remained normal. Dose escalation continues to reach a therapeutic dose. Conclusions Absolutely no cardiotoxicity has been seen, along with preliminary evidence of efficacy. The investigational agent has received fast track and orphan designation in the U.S. A meeting with the FDA has been scheduled to ask for a single-arm phase 2 study for accelerated approval. If successful, this will be the first approved therapy for all patients with R/R AML agnostic of mutational status. Funding The study is supported by funding from the sponsor, Moleculin Biotech, LLC.

Abstract P2-15-03: Liposomal annamycin inhibition of lung localized breast cancer

Abstract Background and Rationale: Annamycin is an anthracycline antibiotic currently in clinical studies in AML patients. Our earlier studies showed that Annamycin is not cross-resistant with doxorubicin (DOX) and is a poor substrate for P-glycoprotein 1 (P-gp) [aka ATP-binding cassette sub-family B member 1 (ABCB1) or multidrug resistance protein 1 (MDR1)], a major mechanism of doxorubicin resistance in different types of cancer. Annamycin, in contrast to DOX, achieves relatively high levels of cellular accumulation, especially in multidrug resistant (MDR) cell lines, and induces significant DNA damage in MDR cancer cells. Clinically, Annamycin is administered in a liposomal formulation (L-Annamycin) the in vivo activity of which has been demonstrated in different tumor models. IND aimed preclinical studies revealed that in sharp contrast to DOX, Annamycin displays dramatically reduced to no cardiotoxicity. Subsequent pharmacokinetic and organ distribution studies revealed surprisingly high levels of Annamycin in lung tissue. Annamycin levels (AUC, 24 h) in lungs were over 10-fold greater than in plasma. In addition, when compared with DOX, lung levels of Annamycin were 6-fold higher than those achievable with DOX. Thus, studies aimed to assess efficacy of L-Annamycin in lung localized tumor models were initiated. Objective: The objective of this study was to assess the in vitro and in vivo activity of L-Annamycin in 4T1, a highly aggressive triple negative breast cancer (TNBC) lung colonization model. Methods: In vitro efficacy of Annamycin was investigated in a panel of TNBC cell lines including murine 4T1 and human MDA-MB 231, MDA-MB-468, BT20, and SUM149 cell lines. Uptake and subcellular distribution of Annamycin was assessed using fluorescent microscopy and FACS. Biodistribution of Annamycin was tested in naïve CD-1 mice after intravenous administration of the drug. Efficacy of L-Annamycin was tested in a syngeneic TNBC lung model, specifically, in immune-competent Balb/c mice injected intravenously with 4T1 cells expressing firefly luciferase. Results: Annamycin shows consistently high cytotoxicity across all tested cell lines with IC50s in the low nanomolar range. Uptake analysis performed on 4T1 cells indicated significantly higher uptake of the drug as compared to DOX. Annamycin shows distinct subcellular distribution with high cytosolic deposition, while DOX was mostly confined to the nucleus. In vivo efficacy studies in mice treated with L-Annamycin showed significantly increased survival when compared to vehicle treated animals. Delayed tumor progression was assessed by bioluminescent imaging and correlated with increased survival. Conclusion: Our in vitro studies demonstrate the high potency of L-Annamycin in vitro and in vivo. Its unique intracellular uptake and distribution might in part be responsible for L-Annamycin’s activity profile and lack of cardiotoxicity. The observed high in vivo efficacy of L-Annamycin in an aggressive TNBC lung metastasis model coupled with consistently high accumulation of L-Annamycin in lung parenchyma, may prove to open new therapeutic options for patients with tumors metastatic to the lung. Citation Format: Rafal Zielinski, Krzysztol Grela, Stanislaw Skora, Izabela Fokt, Matthew Sander, Adam Didluch, Waldemar Priebe. Liposomal annamycin inhibition of lung localized breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-15-03.

Abstract C094: Dose and schedule-dependent efficacy of liposomal Annamycin in pre-clinical models of acute myeloid leukemia

Abstract Annamycin (Ann) is a clinically evaluated anthracycline whose antitumor activity is not cross-resistant with that of doxorubicin (DOX). Formulated in liposomes, L-Annamycin (L-Ann) is currently in clinical studies in acute myeloid leukemia (AML) patients. In contrast to DOX, high levels of cellular accumulation of Ann were observed in multidrug resistant (MDR) cell lines, and consequently there was a significant degree of DNA damage and apoptosis. In vivo activity of L-Ann and free Ann was confirmed in different tumor models. In addition, preclinical toxicology studies revealed that both L-Ann as well as free Ann displayed dramatically reduced or no cardiotoxicity, in sharp contrast to DOX. In the current study, we assessed the anti-AML efficacy of Ann using multiple in vitro and in vivo models. In vitro efficacy was assessed in a panel of human AML cell lines including THP-1, OCI-AML3 and MV4;11. The uptake and subcellular distribution of Ann was evaluated using flow cytometry and fluorescence microscopy. In vivo biodistribution of Ann was tested in naïve B6 mice after intravenous administration of the drug followed by fluorescence microscopy. Anti-tumor efficacy was extensively tested in syngeneic AML-Turq 2 model in immunocompetent B6 mice, and subsequently in human OCI-AML3 model in immunosuppressed NSG mice. In the murine syngeneic AML-Turq 2 model, AML cells were visualized based on the mTurquoise fluorescence. AML disease progression was monitored using whole body bioluminescence imaging or flow cytometry, respectively. We observed a high potency of Ann against established human AML cell lines, with the IC50 in low nanomolar range. Saturation experiments indicated high uptake of Ann by the cancer cells. Interestingly, in addition to the expected uptake of Ann in the nucleus, Ann was also localized in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. In vivo studies confirmed anti-tumor efficacy of the Annamycin in both human and murine AML models. Based on bioluminescence imaging, the liposomal formulation of the drug significantly delayed AML progression in the human OCL-AML3/NSG model at 4 mg/kg with once weekly dosing. Similarly, significant dose-dependent reduction of peripheral blood AML blasts was observed in the murine AML-Turq2 model, and this reduction was strongly correlated with prolongation of animals survival. The median survival of mice receiving four doses of L-Ann once a week at 4 mg/ml was 37 days while mice receiving vehicle lived only 14 days (p=0.0002). Different doses and administration schedules of L-Ann were tested in effort to maximize survival benefits. In summary L-Ann is effective in AML, demonstrating significant activity in both in vitro and in vivo mouse models with a distinct pattern of intracellular uptake and organ distribution using a once a week schedule. This suggests that L-Ann with this profile, including a lack of cardiotoxicity and activity against DOX resistant tumors, may be an advantageous approach in the treatment of AML. Citation Format: Rafal Zielinski, Tomasz Zal, Krzysztof Grela, Stanislaw Skora, Izabela Fokt, Matthew Sander, Magdalena Remiszewski, Edward Felix, Meenakshi Shanmugasundaram, Waldemar Priebe. Dose and schedule-dependent efficacy of liposomal Annamycin in pre-clinical models of acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C094. doi:10.1158/1535-7163.TARG-19-C094

Phase I/II Trial of Nanomolecular Liposomal Annamycin in Adult Patients With Relapsed/Refractory Acute Lymphoblastic Leukemia

Treatment options for relapsed/refractory ALL in adult patients remain challenging. Annamycin is a highly lipophilic form of the anthracycline doxorubicin with the ability to bypass multidrug resistance mechanisms of cellular drug resistance. We performed a phase I/II multicenter, open-label, study to determine the maximally tolerated dose (MTD) of nanomolecular liposomal annamycin in adult patients with refractory ALL. Thirty-one patients were enrolled; the MTD was determined to be 150 mg/m(2)/d for 3 days. Other than tumor lysissyndrome, there were 3 grade 3 mucositis which comprised the MTD determination. There was also 1 case each of grade 3 diarrhea, typhlitis, and nausea. After determining the MTD, a 10-patient phase IIA trial was conducted. Eight of the patients completed 1 cycle of the 3 days of treatment at the MTD. Of these, 5 (62%) had an efficacy signal with complete clearing of circulating peripheral blasts. Three of these subjects also cleared bone marrow blasts with 1 subsequently proceeding onto successful stem cell transplantation. Single-agent nanomolecular liposomal annamycin appears to be well tolerated, and shows evidence of clinical activity as a single agent in refractory adult ALL.

Phase I Trial of Nanomolecular Liposomal Annamycin in Adult Patients with Refractory Acute Lymphoblastic Leukemia.

Abstract 4098There continues to be no effective second line therapy for refractory acute lymphoblastic leukemia (ALL) in adult patients and the cure rate with current therapy has not significantly improved in decades. Annamycin is a highly lipophilic form of the anthracycline doxorubicin with the ability to bypass multi-drug resistance (MDR-1) mechanisms of cellular drug resistance. A novel nanomolecular liposomal entity of annamycin was recently specifically synthesized which overcomes MDR with little to no cardiac toxicity and improved anti-tumor activity when compared to the original annamycin formulation. We performed a phase I multi-center, open-label, study to determine the maximally tolerated dose (MTD) of nanomolecular liposomal annamycin in adult patients with refractory ALL. The secondary objective was to study the MDR-1 encoded (permeability glycoprotein) PgP-170 glycoprotein expression in correlation with CD34 expression and MDR-1 mRNA levels in refractory ALL patients prior to and after receiving liposomal annamycin treatment. Thirty patients were enrolled on the study. The MTD was determined to be 150 mg/m2/day for 3 days. Other than the tumor lysis syndrome, there was only 1 severe adverse event (SAE) definitely related to the study drug consisting of grade 3 mucositis. There were also 3 other SAEs of grade 3-4 mucositis probably related to the study drug which comprised the MTD determination. There was no reported cardiac toxicity in any patients. After determining the MTD, a 10-patient phase IIA was conducted. Eight of the patients completed 1 cycle of the 3 days of treatment at the MTD. Of these, 5 (62%) had an efficacy signal with complete clearing of circulating peripheral blasts. Three of these subjects also cleared bone marrow blasts with one subsequently proceeding onto successful stem cell transplantation. The other 2 developed tumor lysis syndrome and unfortunately expired prior to response assessment. Nanomolecular liposomal annamycin appears to be well-tolerated with no cardiotoxicity and evidence of clinical activity as a single agent in refractory adult ALL. Given that the likely mechanism of action is overcoming innate drug resistance via MDR, PgP-170 mRNA and protein expression will be presented. We are currently testing liposomal annamycin in a phase I study in children and young adults with refractory ALL or AML, and planning a phase 2 registration trial in adult ALL Disclosures:Wetzler:Callisto Reserach Funding: Research Funding. Off Label Use: Annamycin. Refractory relapse to ALL. Wang:Callisto : Research Funding. Shepard:Callisto: Research Funding. Thomas:Callisto: Research Funding. Andreeff:Callisto: Research Funding. Kantarjian:Callisto: Research Funding.

Phase I study results of nanomolecular liposomal annamycin in refractory ALL

7066 Background: There continues to be no effective second-line therapy for refractory AML or ALL and the cure rate with current therapy has not significantly improved in decades. The first-line therapy for adult AML has remained the same 7 + 3 that it was a generation ago. Annamycin was specifically synthesized to overcome MDR and to have little to no cardiac toxicity when given as a nanomolecular liposomal entity. Methods: We performed a phase I multi-center, open-label, MTD study of nanomolecular liposomal annamycin in patients with refractory ALL. The secondary objective was to study the MDR-1 encoded P-1u70 glycoprotein expression in correlation with CD34 expression and MDR-1 MRP mRNA levels in refractory ALL patients before and after receiving liposomal annamycin. Results: Thirty patients were enrolled on the study. The MTD was determined to be 150 mg/m2/day for 3 days. Eight of the patients completed 1 cycle of the 3 days of treatment. Of these 8 subjects, 5 (62%) had an efficacy signal. All 5 completely cleared circulating blasts. Three subjects also cleared bone marrow blasts. Of these 3, 1 went on to a successful bone marrow transplant. The other 2 had tumor lysis syndrome and unfortunately expired. Other than the tumor lysis syndromes, there was only 1 SAE definitely related to the study drug which was a grade 3 mucositis but there were also 3 other SAEs of grade 3 or 4 mucositis probably related to the study drug which comprised the MTD determination. There were no reports of cardiac toxicity. Conclusions: Nanomolecular liposomal annamycin appears to be effective through its innate resistance to MDR even as a single agent in refractory adult ALL. We are now testing it in a phase I study in children and young adults with refractory ALL or AML. [Table: see text]

Treatment of Acute Lymphoblastic Leukaemia

Acute lymphocytic leukaemia (ALL) is a heterogeneous group of disorders that result from the clonal proliferation and expansion of malignant lymphoid cells in the bone marrow, blood and other organs. Distinct clinicopathological ALL entities have been identified, resulting in the adoption of risk-oriented treatment approaches. Advances in ALL therapy have led to long-term survival rates of >80% in children. However, only approximate, approximately 30-40% of adults achieve long-term disease-free survival. Contemporary ALL treatment programmes include induction, intensified consolidation, maintenance phases and CNS prophylaxis. The optimal treatment of Philadelphia chromosome-positive patients requires the addition of BCR-ABL tyrosine kinase inhibitors, such as imatinib, whereas allogeneic stem-cell transplantation remains the preferred approach for high-risk patients in first remission. Since only approximate, approximately 38% of adult ALL patients are free of disease 5 years after diagnosis and the outcome of salvage chemotherapy is very poor (complete remission rates of 20-30%, median survival of 3-6 months), novel agents are desperately required. Of those currently in clinical studies, the outlook for sphingosomal vincristine, pegylated asparaginase (pegaspargase), liposomal annamycin, ABT-751, pemetrexed, talotrexin, nelarabine and the novel BCR-ABL kinase inhibitors is discussed.

Phase I study of liposomal annamycin.

Annamycin is a highly lipophilic anthracycline with the ability to bypass the MDR-1 mechanism of cellular drug resistance. In this phase I study, annamycin entrapped in liposomes was administered by a 1- to 2-h intravenous infusion at 3-week intervals. Thirty-six patients with relapsed solid tumors were treated and 109 courses were administered at doses ranging from 3 to 240 mg/m2. The dose-limiting toxicity was thrombocytopenia. Five patients had a probable allergic reaction, requiring discontinuation of treatment in one. Treatment was well tolerated otherwise. No cardiac toxicity was seen on endomyocardial biopsy of four patients studied. There was limited gastrointestinal toxicity and no alopecia. No objective tumor responses were observed. Pharmacokinetic studies at 24, 120 and 240 mg/m2 showed a biexponential plasma concentration-versus-time profile. There was a linear relationship between the dose and the maximal plasma concentration with relatively constant plasma clearance values. The maximum tolerated dose (MTD) for liposomal annamycin defined in this study is 210 mg/m2. Because of a subsequent change in the formulation of the drug, future studies will use 190 mg/m2 as the MTD.

Modifications in High-Density Lipoprotein Lipid Composition and Structure Alter the Plasma Distribution of Free and Liposomal Annamycin

Recent studies have shown that changes in lipoprotein cholesterol and triglyceride concentration alters the plasma distribution of free (Ann.) and liposomal annamycin (LAnn) and that the majority of Ann. is associated with high-density lipoproteins (HDL) following the incubation in plasma of LAnn. To demonstrate that alterations in HDL lipid composition and HDL structure may influence the plasma distribution of Ann. and LAnn, Ann. and LAnn (20 ,μg/mL) were incubated in plasma pretreated with dithionitrobenzoate (DTNB, a compound which inhibits the conversion of free cholesterol to esterified cholesterol) 18h prior to the experiment or in untreated plasma for 60min at 37 °C. In addition, Ann. and LAnn were co-incubated with DTNB in plasma for 6min at 37 °C. Following incubation the plasma was separated into its HDL, low- density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and lipoprotein-deficient plasma (LPDP) fractions by ultracentrifugation and assayed for Ann. by fluorimetry. The HDL plasma cholesterol:triglyceride concentration ratio was significantly decreased following 18h of DTNB pretreatment compared to untreated plasma controls. No significant differences in LDL/VlDL plasma cholesterol:triglyceride concentration ratio following 18h of DTNB pretreatment was observed. An increased number of discoidal HDL particles were observed following 18h of DTNB pretreatment. When Ann. was incubated in plasma pretreated with DTNB for 18h the percentage of Ann. recovered in the HDL, LDL, and VLDL fractions significantly increased. However, the percentage of Ann. recovered within the LPDP fraction was significantly decreased. When LAnn was incubated in plasma pretreated with DTNB for 18h the percentage of Ann. recovered in the HDL fraction significantly decreased. The percentage of Ann. recovered in the LPDP fraction significantly increased when LAnn was incubated in plasma pretreated with DTNB for 18 h. No significant differences in Ann. lipoprotein distribution were observed when Ann. and LAnn were co-incubated with DTNB in plasma for 1 h. These findings suggest that the cholesterol:triglyceride concentration ratio and physical structure of HDL maybe important in defining the capacity of hDl to sequester Ann.

Blood and plasma lipoprotein distribution and gender differences in the plasma pharmacokinetics of lipid-associated annamycin.

The objectives of this study were to determine the lipoprotein distribution of unbound annamycin and liposomal annamycin within human and rabbit blood and plasma and to evaluate the plasma pharmacokinetics of liposomal annamycin in male and female rabbits following a single intravenous bolus of the compound. Annamycin and liposomal annamycin were incubated in human and rabbit blood and plasma for 60 min. at 37 degrees C. Following incubation blood and plasma samples were assayed by HPLC for drug in each of the lipoprotein and lipoprotein-deficient plasma fractions. To evaluate the plasma pharmacokinetics of liposomal annamycin in male versus female rabbits, a single intravenous bolus dose (5 mg/kg) of liposomal annamycin was administered to male and female rabbits. Sequential blood samples were obtained from the animals following the dose, analyzed for drug, and the pharmacokinetics determined using multicompartmental methods. The incorporation of annamycin into liposomes composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol resulted in no significant differences in blood versus plasma lipoprotein drug distribution. Furthermore, no differences in the plasma distribution of liposomal annamycin were observed when the drug was either incubated in vitro for 1 hr or 1 hr following intravenous administration into New Zealand male white rabbits. The plasma clearance and volume of distribution of liposomal annamycin were decreased and a increase in plasma AUC in female as compared to male rabbits following a single intravenous bolus of liposomal annamycin was observed. These findings suggest that the lipoprotein distribution of liposomal annamycin is not different when incubated in blood or plasma and that in vitro liposomal annamycin plasma distribution is similar to in vivo. Furthermore, it appears that the pharmacokinetics of liposomal annamycin are different following administration to male versus female rabbits.

Lyophilized preliposomal formulation of the non-cross-resistant anthracycline annamycin: effect of surfactant on liposome formation, stability and size.

We report a method of preparing a submicron and stable liposome formulation of the non-cross-resistant anthracycline annamycin. The lipids were dimyristoylphosphatidyl choline (DMPC) and dimyristoylphosphatidyl glycerol (DMPG) at a 7:3 molar ratio and the optimal lipid:drug ratio was 50:1 (w/w). The selected formulation was a preliposome lyophilized powder that contained the phospholipids, annamycin, and Tween 20. The liposome suspension was obtained on the day of use by adding normal saline at 37 degrees C (1 ml/mg annamycin) and hand shaking for 1 min. The presence of Tween 20 was essential in shortening the reconstitution step (from > 2 h to 1 min), avoiding the early formation of free drug crystals, and reducing the median particle size by tenfold (from 1.5 microm to 0.15 microm) without destroying the liposome vesicles. At room temperature, the preliposome powder was chemically stable for > 3 months, and the liposome suspension was chemically and physically stable for > 24 h. The in vitro cytotoxicity of the formulation was equivalent to that of the same lipid composition prepared by the standard evaporation method. The results of the study indicate that small amounts of surfactant may be used to enhance the reconstitution step and reduce the size of liposome suspensions obtained from lyophilized preliposome powders. The formulation described is being used for ongoing clinical trials with liposomal annamycin.

Distribution of Free and Liposomal Annamycin within Human Plasma Is Regulated by Plasma Triglyceride Concentrations but Not by Lipid Transfer Protein

Annamycin (Ann) is a lipophilic and non‐cross‐resistant anthracycline antibiotic currently in clinical development as a liposomal formulation (L‐Ann) composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). Previous studies have demonstrated that the incorporation of Ann into these liposomes prolongs its terminal serum half‐life and increases the tumor levels of the drug. However, an explanation for the altered pharmacokinetics and pharmacodynamics of doxorubicin and Ann when entrapped into these multi‐lamellar lipid vesicles remains unknown. Since the distribution of lipophilic compounds within plasma lipoproteins has been shown to influence the pharmacokinetics and organ distribution of a number of lipophilic compounds and this distribution appears to be regulated by lipid transfer protein (LTP), we studied the distribution of Ann and L‐Ann among plasma lipoproteins and the influence of LTP on the distribution of Ann and L‐Ann among plasma lipoproteins. Our results concluded that when Ann was incorporated into liposomes composed of DMPC and DMPG, over 65% of the initial Ann concentration would distribute into the high density lipoprotein (HDL) fraction and that free Ann and L‐Ann distribution within human plasma was independent of LTP activity. In addition, we observed that the increase in total plasma triglyceride (TG) concentrations (through the increase of very low‐density lipoproteins (VLDL)) resulted in the increase distribution of Ann and L‐Ann within the TG‐rich VLDL fraction. However, increasing the VLDL core TG/cholesterol ratio decreased Ann distribution into VLDL. These findings suggest that initial Ann distribution is regulated by a mechanism that does not involve LTP, but through its interaction with plasma VLDL—TG. Since many cancer patients exhibit lipid disturbances, including hypocholesterolemia and hypertriglyceridemia, these results may provide an explanation for the altered pharmacokinetics and pharmacodynamics seen with L‐Ann in these patients.