Paul S. Uster

Insertion of poly(ethylene glycol) derivatized phospholipid into pre‐formed liposomes results in prolonged in vivo circulation time

Transfer of MPEG1900‐DSPE from micellar phase to pre‐formed liposomes imparts long in vivo circulation half‐life to an otherwise rapidly cleared lipid composition. MPEG1900‐DSPE transfers efficiently and quickly in a time and temperature dependent manner. There is negligible content leakage and a strong correlation between assayed mol% MPEG1900‐DSPE, liposome diameter increase, and pharmacokinetic parameters such as distribution phase half‐life. Since a biological attribute (liposome clearance rate) can be modified by the insertion process, it suggests a simple and economical way to impart site‐specific targeting to a variety of liposome delivery systems. This method is also a convenient way to measure the ‘brush’ thickness of such conjugates directly.

Pharmacokinetics and Tissue Disposition in Monkeys of an Antisense Oligonucleotide Inhibitor of Ha-Ras Encapsulated in Stealth Liposomes

AbstractPurpose. This study examined the pharmacokinetics and tissue distribution of an antisense oligonucleotide ISIS 2503, formulated in stealth (pegylated) liposomes (encapsulated) or in phosphate-buffered saline (unencapsulated). Methods. Encapsulated or unencapsulated ISIS 2503 was administered to rhesus monkeys by intravenous infusion. The concentrations of ISIS 2503 and metabolites in blood, plasma, and tissue samples were determined by capillary gel electrophoresis. Results. Plasma concentrations of encapsulated ISIS 2503 decreased mono-exponentially after infusion with a mean half-life of 57.8 hours. In contrast, the concentration of unencapsulated ISIS 2503 in plasma decreased rapidly with a mean half-life of 1.07 hours. Both encapsulated and unencapsulated ISIS 2503 distributed widely into tissues. Encapsulated ISIS 2503 distributed primarily to the reticulo-endothelial system and there were few metabolites observed. In contrast, unencapsulated ISIS 2503 distributed rapidly to tissue with highest concentration seen in kidney and liver. Nuclease-mediated metabolism was extensive for unencapsulated oligonucleotide in plasma and tissues. Conclusions. The data suggest that stealth liposomes protect ISIS 2503 from nucleases in blood and tissues, slow tissue uptake, and slow the rate of clearance from the systemic circulation. These attributes may make these formulations attractive for delivering oligonucleotides to sites with increased vasculature permeability such as tumors or sites of inflammation.

Therapy of a xenografted human colonic carcinoma using cisplatin or doxorubicin encapsulated in long-circulating pegylated stealth liposomes

We compared the therapeutic effects of low doses of cisplatin and doxorubicin hydrochloride encapsulated in long-circulating liposomes composed of cholesterol/hydrogenated soy phosphatidylcholine-polyethylene glycol-distearoyl-phosphatidyl-ethanolamine. The encapsulation of cisplatin and doxorubicin in these liposomes made ineffectively low doses of the free drugs able to inhibit the growth of and affect cures of a human colonic carcinoma growing in nude mice. Liposome-encapsulated cisplatin had minor systemic toxic side effects indicated by an average 9% weight loss which was recovered 3-4 weeks after the last treatment. Toxicity was not observed in mice treated with liposome-encapsulated doxorubicin.

Pegylated liposome-encapsulated doxorubicin and cisplatin in the treatment of head and neck xenograft tumours

Purpose: To evaluate the in vitro and in vivo activity of unencapsulated doxorubicin (DOX) and cisplatin (CDDP) and their pegylated liposome encapsulated counterparts (PLED and PLEC) in a subcutaneous model of human squamous cell cancer of the head and neck. Methods: In vitro cytotoxicity was determined by means of the sulphorhodamine B assay and in vivo activity was assessed in terms of tumour growth delay following single intravenous doses of the various agents. Treatment-related toxicity was evaluated by means of serial weight measurement. Results: The IC50 values for DOX (12.1-fold) and CDDP (21.5-fold) were lower than for their liposome-encapsulated counterparts. When the two unencapsulated agents were compared, the IC50 value for DOX was 16-fold lower than that for CDDP. In the in vivo studies, liposomes containing DTPA (PLEDTPA) exerted no effect on KB xenograft tumours when compared to untreated controls (P > 0.1). PLED was significantly more effective than DOX at doses of 2 mg/kg, 4 mg/kg and 8 mg/kg (P < 0.001 for all comparisons). At the 8 mg/kg dose, 7/13 animals treated with PLED were free of disease at 60 days, compared to 0/12 treated with DOX. PLEC displayed superior activity in comparison to CDDP at the 4 mg/kg dose level (P < 0.001), although at doses of 2 mg/kg and 10 mg/kg this comparison only reached borderline statistical significance (0.1 > P > 0.05). The highest dose level of 20 mg/kg was fatal to all animals in the CDDP group but well-tolerated by the animals in the PLEC group. On the basis of serial weight measurements, both PLED and PLEC were shown to be tolerated better than DOX and CDDP. Conclusion: Both PLED and PLEC were shown to exert significant activity against head and neck xenograft tumours, with PLED showing particular efficacy.

Pegylated liposomal doxorubicin (DOXIL®, CAELYX®) distribution in tumour models observed with confocal laser scanning microscopy

The activity of doxorubicin hydrochloride in preclinical tumor models is markedly improved by encapsulation in pegylated liposomes (DOXIL, CAELYX). To understand this response in better detail, spatial and temporal drug distribution in frozen tumor sections was observed with confocal laser scanning (CLS) microscopy. Tumor tissue absorption and distribution phases were markedly altered by pegylated liposome encapsulation. The resultant tumor tissue area under the curve was increased at least several fold over an equivalent dose of free drug. The majority of visible fluorescence was in the nuclear compartment; encapsulated drug apparently leaves liposomes in the interstitial spaces within minutes and appears in the nucleus. Pilot experiments suggest that drug and lipid uncouple at an extracellular location. The increased nuclear exposure to drug is likely responsible for the enhanced therapeutic effect of encapsulation in pegylated liposomes.

Pharmacokinetics of a novel submicron budesonide dispersion for nebulized delivery in asthma

The objective of this study was to evaluate the safety and pharmacokinetics of unit dose budesonide (UDB), an aqueous dispersion of submicron-sized budesonide particles, and a commercially available budesonide suspension formulation. This was a randomized, double-blind, active-controlled, 4-period, 4-way crossover trial in 16 healthy, adult volunteers. Subjects received UDB 0.24, 0.12, and 0.06 mg or commercial budesonide 0.25 mg via a jet nebulizer. T(max) was significantly (p<0.05) earlier for UDB 0.06, 0.12, and 0.24 mg (4.5+/-3.3, 3.1+/-1.5, 3.7+/-1.5 min) vs. commercial budesonide (9.1+/-7.1 min). C(max) was significantly (p<0.05) higher for UDB 0.24 mg vs. commercial budesonide 0.25 mg (434.5+/-246.9 pg/mL vs. 303.5+/-177.4 pg/mL) but not between UDB 0.12 mg (239.9+/-140 pg/mL) and commercial budesonide 0.25 mg (p=0.448). AUC(0-infinity) was marginally, but significantly lower for UDB 0.24 mg than commercial budesonide 0.25 mg. AUCs for UDB 0.12 mg were lower than commercial budesonide 0.25 mg. UDB 0.24 mg was absorbed more rapidly and achieved higher peak concentrations than commercial budesonide 0.25 mg, but had a lower AUC(0-infinity). UDB 0.12 mg also was absorbed more rapidly but had lower C(max) and AUCs than commercial budesonide 0.25 mg.

The effect of vincristine-polyanion complexes in STEALTH liposomes on pharmacokinetics, toxicity and anti tumor activity

Abstract Poly(ethylene glycol) (PEG)-derivatized liposome vehicles improve antitumor effectiveness of entrapped anthracyclines and vinca alkaloids. However, the plasma clearance of entrapped vincristine is substantially faster than the lipid phase or other entrapped aqueous markers, suggesting leakage out of the liposome during transit in the blood compartment. We tested the effect of altering the drug’s in vivo leakage rate on pharmacokinetics, toxicity, and antitumor activity of entrapped drug in rodent models. Suramin, heparin, and dextran sulfate were tested for their ability to produce a precipitable complex in vitro. PEG-derivatized liposomes were prepared with the complexing agent inside, and vincristine was driven inside using an ammonium gradient. The resulting preparations were found to have plasma distribution half-lives significantly longer than the formulation without a complex-forming agent. There was no increase in acute lethality, and in the case of the suramin-vincristine complex, the acute lethality was significantly reduced at the highest does level. Anti-tumor activity against the mouse mammary carcinoma MC2 was tested in a multiple-dose study. Free vincristine did not affect the tumor growth rate significantly, but at the same dose level all PEG-coated liposome formulations inhibited tumor growth markedly. The suramin containing formulation was as effective as the formulation lacking polyanion, but the heparin and dextran sulfate containing formulations were less effective. Thus, compounds which form insoluble complexes with vincristine alter in vivo plasma distribution phase pharmacokinetics without increasing acute lethality, but without a corresponding increase in anti-tumor activity.

The effect of irradiation on the biodistribution of radiolabeled pegylated liposomes.

PURPOSE The effect of total-body irradiation (TBI) on the biodistribution and pharmacokinetics of (111)In-DTPA-labeled pegylated liposomes (IDLPL) was evaluated in tumor-bearing nude mice as part of an ongoing effort to develop liposome-targeted radiosensitizers. METHODS AND MATERIALS Mice received TBI (2 Gy or 5 Gy) according to two protocols: (1) to test the effect of radiation delivered 30 min before liposome injection on the time course of IDLPL biodistribution to tumor and normal tissues over 96 h; (2) to test the effect of radiation at times ranging from 72 h to 1 h before liposome injection on tumor and normal tissue uptake of IDLPL at 24 h. Tumor and tissue/organ levels of liposome uptake were measured by dissection and quantitation in a gamma counter. RESULTS For most tissues (tumor, liver, kidney, lung, skin, heart, and central nervous system), irradiation did not alter IDLPL biodistribution. Splenic uptake appeared to be increased by TBI, but further analysis revealed that this effect was due to reduced splenic weight in irradiated mice. IDLPL uptake was increased in the small intestine, stomach, musculoskeletal system, female reproductive tract, and adrenal glands in irradiated mice. CONCLUSION These findings suggest that concomitant administration of liposomal radiosensitizers during radical radiotherapy is likely to be safe. However, caution should be exercised in situations in which significant volumes of small intestine or hemopoietic tissue will be irradiated.

Single-fraction irradiation has no effect on uptake of radiolabeled pegylated liposomes in a tumor xenograft model

PURPOSE These studies were performed with the intention of examining the effect of single-fraction doses of radiotherapy (RT) on the tumor deposition of radiolabeled pegylated liposomes in an animal xenograft tumor model. METHODS AND MATERIALS Human KB head-and-neck xenograft tumors were established in female nude mice. The effect of single fraction tumor RT doses (5, 10, 15, and 20 Gy) on the tumor uptake of intravenously administered (111)In-DTPA-labeled pegylated liposomes (IDLPL) was examined using two protocols: (1) to test the effect of RT delivered 30 min before liposome injection on the time course of tumor uptake over a 96-h period; (2) to test the effect of RT at times ranging from 72-h to 1-h before liposome injection on the levels of liposome uptake at 24 h. Tumor and normal tissue/organ (blood, liver, spleen, lung, and kidney) liposome uptake was determined by dissection and quantitation in a gamma counter. RESULTS There was no demonstrable effect of RT on tumor uptake of IDLPL (p > 0.1 for all comparisons). Reassuringly, neither was there an effect of RT on the pharmacokinetics and biodistribution of radiolabeled liposomes to normal tissues. CONCLUSIONS Single fraction doses of RT appear to have no effect on tumor or normal tissue biodistribution and pharmacokinetics of radiolabeled pegylated liposomes in this animal model.

Tumor Uptake and Therapeutic Effects of Drugs Encapsulated in Long-Circulating Pegylated Stealth® Liposomes

Abstract In this study, tumor uptake and clearance of doxorubicin were determined for two formulations of the drug: the free form in aqueous solution and the encapsulated form in polyethylene glycol-coated (pegylated, STEALTH®) liposomes composed of cholesterol/hydrogenated soy phosphatidylcholine/ polyethylene glycol-distearoyl-phosphatidyl-ethanolamine (Doxil®). The determinations used confocal laser scanning microscopy in a pancreatic carcinoma model in nude mice. The movement of pegylated liposomes containing doxorubicin from blood vessels into tumors was studied using confocal microscopy combined with autoradiography of liposomes containing a tritium-labeled phospholipid. Laser microscopy measurements showed that the lipo-some-encapsulated doxorubicin remained in the tumor longer than the free drug and produced a six-fold increase in the area under the concentration-time curve (AUC). Autoradiography showed that the extravasated tritium-labeled lipid had entered the nuclei as well as the cytoplasm of tumor cells. The authors also compared the therapeutic effects of intravenous cisplatin, doxorubicin hydrochloride, vincristine sulfate, and vinorelbine tartrate, each in the aqueous free form or encapsulated in pegylated liposomes. In this pancreatic carcinoma model, the liposome-encapsulated drugs were all more effective than the free drugs in inhibiting tumor growth and in producing cures. Except for cisplatin, all of the free drugs had toxic systemic side effects indicated by an average weight loss of 3 to 5%, which was recovered by 2 to 4 weeks after the last treatment. The liposome-encapsulated drugs did not cause weight loss.