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Purpose: The pharmacokinetics (PK), biodistribution and therapeutic efficacy of cisplatin encapsulated in long-circulating pegylated (Stealth®) liposomes (SPI-077) were compared with those of nonliposomal cisplatin in two murine (C26 colon carcinoma and Lewis lung) tumor models. Methods: In therapeutic effectiveness studies, mice bearing murine C26 or Lewis lung tumors received multiple intravenous doses of SPI-077 or cisplatin in a variety of treatment schedules and cumulative doses. In the PK and biodistribution study, mice received a single intravenous bolus injection of 3 mg/kg of either SPI-077 or cisplatin 14 days after inoculation with 106 C26 tumor cells. Plasma and tissues were analyzed for total platinum (Pt) content by graphite furnace (flameless) atomic absorption spectrophotometery (GF-AAS). Results: Efficacy studies showed that SPI-077 had superior antitumor activity compared to the same cumulative dose of cisplatin. When lower doses of SPI-077 were compared to cisplatin at its maximally tolerated dose in Lewis lung tumors, equivalent SPI-077 antitumor activity was seen at only half the cisplatin dose. Higher cumulative doses of SPI-077 were well tolerated and had increased antitumor effect. SPI-077 PK were characterized by a one-compartment model with nonlinear (saturable) elimination, whereas cisplatin PK were described by a two-compartment model with linear elimination. SPI-077 had a 55-fold higher volume of distribution, 3-fold higher peak plasma levels, and a 60-fold larger plasma AUC compared with cisplatin. In addition, SPI-077-treated animals displayed a 4-fold reduction in Pt delivered to the kidneys (primary target organ of toxicity) relative to cisplatin, but a 28-fold higher tumor AUC than cisplatin. Conclusions: Based on the results of our studies, encapsulation of cisplatin in long-circulating pegylated liposomes has overcome limitations experienced with other liposomal cisplatin formulations. SPI-077 has a prolonged circulation time and increased tumor Pt disposition, and its antitumor effect is significantly improved compared to cisplatin in murine colon and lung cancer models.

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.

A study of the plasma pharmacokinetics, tumor localization, and safety of a single dose of doxorubicin encapsulated in liposomes containing surface-bound polyethylene glycol (PEG-liposomal doxorubicin) was conducted in patients with Kaposi's sarcoma (KS) as a manifestation of acquired immune deficiency syndrome (AIDS). Eighteen patients with AIDS-KS diagnosed by examination of biopsy specimens were randomly assigned to receive either standard doxorubicin or PEG-liposomal doxorubicin. Consecutive participants were entered at three dose levels (10, 20, and 40 mg/m2) in ascending fashion. Clearance of PEG-liposomal doxorubicin was 0.034 L/h/m2 to 0.108 L/h/m2, volume of distribution (Vd) was 2.2 L/m2 to 4.4 L/m2, and half-lives (t1/2) of the initial decline in the plasma concentration-time curve and of the terminal decline were 3.77 hours and 41.3 hours, respectively. Seventy-two hours after administration, doxorubicin levels observed in lesions of patients receiving PEG-liposomal doxorubicin were 5.2 to 11.4 times greater than those found in patients given comparable doses of standard doxorubicin. PEG-liposomal doxorubicin and standard doxorubicin were roughly equipotent in producing toxicity. Encapsulation in liposomes containing surface-bound PEG significantly limits the distribution and elimination of doxorubicin, results in greater accumulation of the drug in KS lesions 72 hours after dosing than does standard doxorubicin, and may improve drug efficacy and therapeutic index in the treatment of AIDS-KS.

Vincristine is used clinically for the treatment of various types of cancer. Recent significant therapeutic improvements obtained by entrapping anthracyclines in sterically stabilized liposomes raised the question whether the therapeutic index of vincristine can be similarly increased by formulation into such long-circulating liposomes. Encapsulation of vincristine in sterically stabilized liposomes (SL-VCR) prolonged the drug's distribution phase plasma half-life in rats from 0.22 to 10.5 hr. There was no significant difference in LD50 (⪋ 2.5 mg/kg, i.v.), but mice given sublethal doses of SL-VCR experienced significantly less weight loss than those given the same dose of free drug. Compared to free drug, SL-VCR was most effective against i.p. or s.c. implanted tumors. However, i.v. tumor inoculation nullified the therapeutic advantage of encapsulation. A single i.v. 2 mg/kg dose of SL-VCR increased the life span of mice bearing i.v. implanted P388 cells by only 44%, while the life span of i.p. P388 implanted mice was increased by 199%. In an s.c. implanted murine colon carcinoma, multiple doses of free drug did little to slow the growth of the tumors, but SL-VCR was able to produce long-term survivors in several dose regimens. These results indicate that prolonged circulation time increases the therapeutic index of VCR entrapped in liposomes against s.c. or i.p. implanted tumors, but does not improve the drug's activity against rapidly growing i.v. disseminated leukemias. © 1995 Wiiey-Liss, Inc.

Ligand attachment to polyethylene glycol (PEG) grafted, long circulating liposomes at the polymer terminus is of interest for targeting but the effect of positively charged groups is unknown. Amino-polyethylene glycol-phosphatidylethanolamine (AminoPEG-PE), prepared in four steps from alpha-amino-omega-hydroxy-PEG, was tested for influence on liposome interactions in vivo: blood circulation and biodistribution. Despite surface amines on each liposome conferring cationic behavior, in vivo properties are comparable to those obtained with methoxy-PEG-PE. The consequences are profound for targeting and possibly systemic delivery of cationic lipidic-polynucleotide complexes.

Advanced liposomal therapeutics has been attained by liposome surface modification, initially with specific glycolipids and subsequently with surface-grafted PEG, reducing in vivo rapid recognition and uptake, giving prolonged blood circulation, and providing selective localization in tumors and other pathological sites, as described in recent reviews. The result is improved efficacy of encapsulated agents. The surface PEG may produce a steric barrier, as described for colloids. Reduced in vivo uptake may result from inhibition of plasma-protein adsorption, or opsonization, by the steric coating. Several physical studies support this mechanism, including electrophoretic mobility (zeta potential). Our previous results for 2000-dalton PEG indicated a coating thickness about 5 nm, in agreement with independent measurements. We report here results for 750 to 5000-dalton PEGs. The calculated coating thickness increases with molecular weight in a nonlinear fashion. The dependence of blood circulation and tissue distribution on PEG molecular weight correlates with zeta-potential estimates of PEG-coating thickness. Effects on tissue distribution are reported for liver and spleen, the major phagocytic organs. The biological properties of these liposomes depend on the surface polymer rather than the lipid bilayer, yielding important advantages for lipid-mediated control of drug interaction and release without affecting the biodistribution.

To investigate the safety, tolerability, and pharmacokinetics (PKs) of topical SAR 1118 Ophthalmic Solution in healthy adults. SAR 1118 is an investigational small molecule lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18; αLβ2) antagonist that inhibits LFA-1 binding to intercellular adhesion molecule-1 (ICAM-1; CD54) targeting T-cell-mediated inflammation.A randomized, double-masked, placebo-controlled, dose-escalation study of SAR 1118 was performed in 4 cohorts with 7 randomized subjects per cohort (2 placebo: 5 active drug subjects; 0.1%, 0.3%, 1.0%, 5.0%) in 28 healthy adults. Dosing was divided into 3 periods each separated by a 72-h treatment-free observation: once-daily (QD) × 1, twice-daily (BID) × 10, and thrice-daily (TID) × 10 days. Data obtained at the beginning and end of each period included: slit-lamp, best-corrected visual acuity (BCVA), Schirmer tear test (STT) without anesthesia, tear film break-up time (TBUT), intraocular pressure (IOP), and tear/plasma samples for PK analysis.All subjects completed the study; there were no tolerability issues or missed treatments (total, 1,428 administered doses). No serious ocular or nonocular adverse events (AEs) occurred over 1,148 subject study days (41 days/subject) and no significant abnormalities were identified on ocular exam. There were 38 ocular AEs (N = 11 subjects) and 21 nonocular AEs (N = 11 subjects). Most AEs were mild in severity and occurred in the 0.3% and placebo groups. No changes were observed in CD3, CD4, and CD8 blood lymphocyte counts. Tear PK profiles support a QD/BID dosing schedule. Plasma levels of SAR 1118 in the 0.1% and 0.3% groups were below level of quantitation (BLQ;0.50 ng/mL) at all time points and transiently detected within the first 5 min to ∼1 h following administration in the 1.0% and 5.0% groups.SAR 1118 Ophthalmic Solution appears safe and well-tolerated up to 5.0% TID in healthy adult subjects. PK analysis shows adequate ocular exposure with minimal systemic exposure.

Adsorption of block copolymers onto colloids is a promising method to reduce their biological interactions leading to phagocytosis that, for liposomes in particular, is challenged by recent competition through covalent coating with polyethyleneglycol (PEG). The interaction of block copolymer emulsifying agents and PEG lipid derivatives was investigated for evidence of adsorption though dynamic light scattering measurements of mean particle size distribution and compared with those of entrapped aqueous label efflux. The results show increases in hydrodynamic radius are less than about 3.5 nm, regardless of liposome composition or preparation method. At the same time, efflux of entrapped aqueous label increases indicating that adsorption does occur. Efflux was reduced but not eliminated when the liposomes were prepared with high temperature phase transition lipids. In contrast, efflux was not observed upon addition of the PEG lipid derivative. In vivo studies of liposomes treated with the block copolymer, F-108, showed a moderate increase in blood circulation time, but less than that when the PEG lipid derivative was incorporated.

Efforts to overcome rapid uptake of liposomes by cells of the mononuclear phagocytic system (MPS) have identified that lipids derivatized with the hydrophilic polymer poly(ethylene glycol) (PEG) have many advantages. The structure-function relationship of PEG-derivatized phosphatidylethanolamine (PEG-PE) has been examined by studies of blood lifetime and tissue distribution in both mice and rats. Liposomes composed of phosphatidylcholine (PC), cholesterol, and 7.5 mol% of PEG-PE show prolonged circulation and reduced MPS uptake when the PEG has a molecular weight in the range of 1000 to 5000. Up to 35% of the injected dose remains in the blood and less than 10% is taken up by the MPS (liver plus spleen) after 24 h in the best cases as compared to 1% and 40%, respectively, for liposomes without PEG-PE. Prolonged circulation with PEG-PE is independent of cholesterol, degree of saturation in either the PC or the PE lipid anchor, lipid dose, or addition of other negatively charged lipids, phosphatidylglycerol or cholesterol sulfate. This versatility in lipid composition and dose without alteration of blood lifetime or tissue distribution is essential for controlling drug dosage and release properties in a liposome-based therapeutic agent.

The value of novel systemically long-circulating liposomes to prolong the duration of an antidiuretic hormone, arg8-vasopressin (VP), was investigated as a representative of low molecular weight pep-tides with rapid clearance. Cholesterol content was found to have a controlling effect on VP release in serum. Three types of liposomes were selected for urine production measurements in VP deficient Brattleboro rats. One contained phosphatidylserine (PS), which was rapidly cleared from the circulation. In the other two liposomes, the PS component was replaced by either phosphatidylglycerol or a novel phospholipid derivatized with polyethylene glycol (PEG); both showing prolonged circulation. Free VP (up to 8 µg/kg) gave reduced urine production for less than 24 hr. The PG formulation exhibited a dose-dependent prolonged duration of bioactivity of up to 4 days. Substitution of PEG-PE resulted in a 2-day delay followed by a prolonged duration of bioactivity for over 4 days. The duration of the prolonged bioactivity was not dose dependent but the amplitude was. This is attributed to VP release from liposomes which have distributed intact to another compartment without having been taken up by the RES. By balancing liposome circulation time, release rate, and dose, long-circulating liposomes can be applied to prolong the biological activity of a therapeutic peptide.

The purpose of this study was to identify the pharmacokinetics of Amphotericin B Colloidal Dispersion in patients undergoing bone marrow transplantations with systemic fungal infections and to assess the influence of ABCD on renal function. Seventy-five patients (42 females, 33 males) with a median age of 34.5 years and median weight of 70.0 kg were enrolled in the study. The plasma concentration data was available in 51/75 patients and was best described by a two-compartment model; both plasma clearance and volume of distribution increased with escalating doses; the overall average terminal elimination half-life was 29 h. Serum creatinine values over the duration of therapy were available in 59/75 patients. Overall, there was no net change in renal function over the duration of therapy.

The toxicity of cisplatin encapsulated in pegylated, long-circulating liposomes (SPI-077) was compared with nonliposomal cisplatin in male and female cynomolgus monkeys (n = 2-4 per sex per group) treated with intravenous infusions of 2.5 or 25 mg/kg SPI-077, 2.5 mg/kg cisplatin, placebo liposomes, or saline once every 3 weeks for total of five treatments. All animals survived until scheduled necropsy at 3 days after the final treatment or after a treatment-free 4-week recovery period. Emesis occurred after each treatment in all cisplatin-treated monkeys, but only once in one monkey treated with high-dose SPI-077. Dose-related mild decreases in red blood cell (RBC) count, hemoglobin, and hematocrit to or slightly below low normal range occurred in the high-dose SPI-077 and placebo liposome treatment groups after each treatment, with partial to complete recovery between treatments and no signs of correlating bone marrow toxicity. Decreases were similar in cisplatin-treated monkeys, but resolved only slightly between treatments and after the end of treatment (continuing to decrease in females) and were accompanied by bone marrow hypocellularity. Indirect, but not direct, bilirubin levels were cyclically elevated in the high-dose SPI-077 and placebo-treated animals, but not in the other treatment groups. Levels had either fully resolved or were near baseline and/or saline group values prior to the next treatment. Serum cholesterol levels were cyclically increased in SPI-077- and placebo liposome-treated animals, and minimally increased numbers of foam cells were seen in the liver, spleen, kidney, and other organs; both were considered related to the lipid dose administered. Cisplatin-treated monkeys exhibited sensory polyneuropathy and moderate irreversible toxic tubular nephrosis, but no neuropathy or nephrotoxicity was seen in either SPI-077 treatment group. Microscopically, treatment-related cell death was seen in dorsal root ganglia (DRG), affecting 15% of the cells in cisplatin-treated animals, compared to 8 and 12% in the low- and high-dose SPI-077 treatment groups. Neither drug was ototoxic. In summary, repeated administration of SPI-077 produced minimal, reversible effects related to the lipid dose administered, mostly limited to the 25 mg/kg dose group. The most notable effects in this group were cyclical decreases in hematology parameters thought to be related to increased recycling of a small fraction of RBCs and limited cell death in the DRG in the absence of any neurophysiological changes. Animals treated with a 10-fold lower dose of cisplatin (2.5 mg/kg), in contrast, exhibited myelo-, nephro-, and neurotoxicity, including sensory neuropathy, and were emetic after every dose. The SPI-077 liposomal formulation of cisplatin may provide a less toxic alternative to standard cisplatin solution.

Immunoliposomes composed of hydrogenated soy phosphatidylcholine, cholesterol, methoxypoly(ethylene glycol)-distearoyl phosphatidylethanolamine (mPEG-DSPE), and hydrazide-PEG-DSPE (mole ratio, 57:38:3.3:1.7) linked to periodate-oxidized chimerized mouse IgG (C225, anti-human epidermal growth factor receptor) were prepared by an optimized aggregation-free procedure. The antigen-binding activity of the immunoliposomes was well preserved. When injected intravenously into naive rats, the immunoliposomes (approximately 18 IgG per 100 nm liposome) exhibited long circulation times (MRT = 8.5 h, Cl = 0.2 ml/h). Subsequent injections of the immunoliposomes into the same animals resulted in rapid clearance (MRTor = 0.7 h, Clor = 7 ml/h), which was accompanied by a significant increase in anti-C225 specific titers. Upon repeated injection or coinjection with the parent liposomes free C225 consistently exhibited prolonged circulation without any increase in C225-specific antisera, but was cleared quickly when administered into animals that had been pretreated with the immunoliposomes. Screening of the immunoliposome induced antisera against human polyclonal IgG and C225-derived Fab' fragment revealed that the immune response was specifically triggered by the constant human region of C225. These results demonstrate that the preparations of PEG-grafted immunoliposomes are more immunogenic than the free IgG component, which is of profound importance to the antibody-mediated liposomal drug delivery effort.

Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.

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 objective of this Phase 3 study was to confirm the efficacy and safety of recombinant human arylsulfatase B (rhASB) treatment of mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome), a rare, fatal lysosomal storage disease with no effective treatment.Thirty-nine patients with MPS VI were evaluated in a randomized, double-blind, placebo-controlled, multicenter, multinational study for 24 weeks. The primary efficacy variable was the distance walked in a 12-minute walk test (12MWT), whereas the secondary efficacy variables were the number of stairs climbed in a 3-minute stair climb (3MSC) and the level of urinary glycosaminoglycan (GAG) excretion. All patients received drug in an open-label extension period for an additional 24 weeks.After 24 weeks, patients receiving rhASB walked on average 92 meters (m) more in the 12MWT (p=.025) and 5.7 stairs per minute more 3MSC (p=.053) than patients receiving placebo. Continued improvement was observed during the extension study. Urinary GAG declined by -227+/-18 microg/mg more with rhASB than placebo (p.001). Infusions were generally safe and well tolerated. Patients exposed to drug experienced positive clinical benefit despite the presence of antibody to the protein.rhASB significantly improves endurance, reduces GAG, and has an acceptable safety profile.