Thomas P. Johnston

Sustained Delivery of Interleukin-2 from a Poloxamer 407 Gel Matrix Following Intraperitoneal Injection in Mice

Parenteral delivery of recombinant biologic response modifiers (BRMs) remains a challenge because of the brief intravascular half-life of most recombinant proteins and their associated rapid clearance from the circulation. Recombinant derived interleukin-2 (rIL-2) was formulated with Pluronic F-127, N.F. (poloxamer 407, N.F.) and the biological activity determined vs time at 4, 22, and 37°C. As assessed by rIL-2-induced peripheral blood lymphocyte (PBL) uptake of [3H]thymidine, storage of rIL-2/poloxamer 407 (33% w/w) for 72 hr at 4 and 22°C did not result in an overall negative slope of the [3H]thymidine vs time profiles. However, storage of an rlL-2/poloxamer formulation at 37°C for 72 hr resulted in an approximate 15% reduction in the biological activity as assessed by [3H]thymidine incorporation. As assessed by bioassay ([3H]thymidine uptake), the cumulative percentage rIL-2 released in vitro at 22°C after 8 hr from rIL-2/poloxamer 407 matrices containing either 30% (w/w) or 35% (w/w) poloxamer 407 was 81.8 ± 1.7 and 82.1 ± 4.7%, respectively. When ELISA was used to determine the amount of rIL-2 released vs time, the corresponding values for the cumulative percentage rIL-2 released were 82.6 ± 10.1 and 40.9 ± 8.8%. Cytotoxicity of rIL-2-stimulated PBLs cultured with poloxamer 407 (0.17%, w/w) toward malignant Daudi cells was significantly (P < 0.05) enhanced compared to controls. Finally, mice injected with the rIL-2/poloxamer 407 formulation (1 × 105 U/inj. q.d. × 3 days) demonstrated a bioequivalent effect of rIL-2-induced natural killer (NK) cell activity in vitro toward malignant murine YAC-1 cells at one-half the standard exogenously administered dose of rIL-2 known to generate enhanced NK lytic activity in mice (1 × 105 U/inj. b.i.d. x 3 days). No untoward systemic side effects were observed for mice injected i.p. with polymer vehicle alone (30%, w/w) (0.15 ml q.d. × 3 days), pH 7 phosphate-buffered saline (PBS) (0.15 ml q.d. × 3 days), rIL-2 formulated with poloxamer 407 (30%, w/w) (1 × 105 U/0.15 ml q.d. × 3 days and 0.5 × 105 U/0.15 ml q.d. × 3 days), or rIL-2 dissolved in PBS (1 × 105 U/0.15 ml b.i.d. × 3 days). Thus, poloxamer 407, N.F., did not denature rIL-2 when the latter was stabilized with human serum albumin (HSA), enhanced the in vitro lytic ability of human rIL-2-stimulated PBLs against malignant Daudi cells, and served as a sustained-release parenteral vehicle for rIL-2 when injected i.p. into mice. Thus, based on these preliminary findings, it appears that poloxamer 407, N.F., may potentially be useful for the formulation and sustained delivery of select protein pharmaceuticals following extravascular administration.

Mechanism of poloxamer 407-induced hypertriglyceridemia in the rat

One 300 mg i.p. injection of the nonionic surfactant poloxamer 407 (Pluronic F-127) produces a significant increase above control of both circulating cholesterol and triglyceride (TG) concentrations. The present study was conducted to determine the effect of poloxamer 407 (P-407) on the capacity to hydrolyze circulating TG by lipoprotein lipase (LPL) in an attempt to determine the mechanism of action of P-407. The concentration of TG in the rat following a single 300 mg i.p. injection of P-407 was marked, increasing from 84 +/- 10 to 3175 +/- 322 mg/dL at 24 hr. The maximal rate of TG accumulation (5.74 mg/dL/min) in the plasma of P-407-injected rats occurred between 2 and 4 hr post-injection. In vitro incubation of LPL with P-407 significantly inhibited enzyme activity with an inhibitory concentration at which 50% of the enzymatic activity was lost of approximately 24 microM. Concentrations of P-407 exceeding 350 microM in vitro completely inhibited LPL activity. The effects of P-407 on the enzymatic activity of LPL in post-heparin plasma obtained following a single 300 mg dose of P-407 to rats demonstrated greater than 95% suppression of LPL activity 3 hr post-injection compared with controls. Inhibition of LPL activity was greater than 90% as long as 24 hr following a single i.p. injection of P-407. However, while the heparin-releasable fraction of capillary-bound LPL was inhibited in the plasma, LPL activity significantly increased in cardiac and skeletal muscle in poloxamer-injected animals compared with sham-injected controls. Although there was no significant change in LPL activity in adipose tissue, testes, and lung resulting from P-407 treatment, LPL activity increased by 37% in myocardium, 69% in soleus, and 66% in gastrocnemius muscle in P-407-injected rats when compared with controls. Our studies would suggest that the predominant mechanism by which P-407 induced an increase in circulating TG was by a reduction in the rate at which TG was hydrolyzed due to inhibition of heparin-releasable LPL by the surfactant.

The P-407-induced murine model of dose-controlled hyperlipidemia and atherosclerosis: a review of findings to date.

Abstract: We are continuing to both elucidate underlying mechanisms and identify clinical applications for a chemically induced murine model of dose-controlled hyperlipidemia and atherosclerosis. This murine model neither utilizes genetically modified mice nor a high-fat, cholate-containing diet, although simultaneous ingestion of a high-fat, cholate-enriched diet potentiates the hyperlipidemic response and the number and size of aortic atherosclerotic lesions formed in C57BL/6 mice. The chemical agent used to induce hyperlipidemia is poloxamer 407 (P-407), a nonionic surface-active-agent. To date, we have investigated the effect of P-407 on the biologic activity of a variety of key enzymes involved with lipid metabolism and transport. These enzymes include 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lipoprotein lipase (LPL), cholesterol 7&agr;-hydoxylase (C7&agr;H), sterol 27-hydroxylase (S27H), lecithin cholesterol acyltransferase (LCAT), cholesteryl-ester-transfer-protein (CETP), hepatic lipase (HL), and endothelial lipase (EL). P-407 directly inhibits the heparin-releasable fraction of LPL and HL and indirectly increases the biologic activity of CETP and LCAT. Long-term (> 4 months) administration of P-407 to C57BL/6 mice appears to have no effect on the biologic activity of S27H and HMG-CoA reductase, but decreases the activity of C7&agr;H. This would suggest that hypertriglyceridemia and hypercholesterolemia result from inhibition of LPL and C7&agr;H, respectively, while the biologic activity of CETP and LCAT are indirectly increased to compensate for the increased cholesterol burden. The above model has proven useful for predicting the therapeutic efficacy of existing and possibly newer statin drugs, as well as evaluating the potential of one statin drug (atorvastatin calcium) to cause the regression of P-407–induced atherosclerotic lesions in mice. The P-407–induced murine model of atherogenesis represents an alternative to the use of either genetically modified or diet-induced models and may also prove beneficial for the evaluation of newer classes of antihyperlipidemic agents such as antioxidants, CETP inhibitors, and liver X receptor (LXR) agonists.

Sustained-release interleukin-2 following intramuscular injection in rats

A potential sustained-release recombinant interleukin-2 (rIL-2) formulation was evaluated following intramuscular (i.m.) injection in rats. Poloxamer 407 (Pluronic® F-127) is a block copolymer comprised of polyoxyethylene and polyoxypropylene segments which exhibits the property of reverse thermal gelation. Thus, an rIL-2/poloxamer 407 preparation was injected i.m. in rats as a viscous mobile solution with subsequent gelation in vivo. Resultant plasma rIL-2 concentration-time data indicated absorption rate-limited disposition of rIL-2 following i.m. injection. The mean values of the absorption rate constant (kclim were 0.64 ± 0.073 and 0.21 ± 0.019 h−1 following i.m. injection of an rIL-2 aqueous solution and the HL-2 gel formulation, respectively. The mean values of the elimination rate constant (kelim) were 1.76 ± 0.22 and 1.21 ±0.079 h−1 following administration of rIL-2 as an aqueous solution or gel formulation, respectively. The blood sampling time point at which the greatest plasma rIL-2 concentration (Cmax.) was observed was 2 h for rats injected i.m. with rIL-2 formulated in poloxamer 407 compared to 1 h for rats injected i.m. with an rIL-2 aqueous solution. The mean value of the Cmax. was significantly (p < 0.05) less in rats injected i.m. with the rIL-2 gel formulation (Cmax= 12500 ± 1450 pg/ml) compared to rats injected i.m. with an aqueous solution of rIL-2 (Cmax = 19 600 ± 2650 pg/ml). The bioavailability of rIL-2 when injected i.m. as an rIL-2/poloxamer gel formulation relative to an i.m. injection of an rIL-2 aqueous solution was approx. 1.0. Cumulative amounts of rIL-2 recovered in the urine 48 h after an i.m. injection of either an rIL-2 aqueous solution or rIL-2 gel formulation were less than 1 percent of the administered dose. Since the rIL-2/poloxamer formulation evaluated in this study resulted in a decrease in the maximum blood concentration of rIL-2 achieved and an increase in the time required to reach a maximum blood concentration, without a reduction in the bioavailability of the protein, the rIL-2 gel formulation may represent an alternative, sustained-release mode of rIL-2 administration.

The poloxamer 407-induced hyperlipidemic atherogenic animal model.

We are attempting to develop a chemically-induced murine model for the study of atherosclerosis. Injection of poloxamer-407 (P-407) into rats and mice causes significant dose-dependent hypercholesterolemia and hypertriglyglyceridemia. The elevated triglycerides (TG) seem to result primarily from the compounds inhibition of lipoprotein lipase. P-407 also indirectly stimulates the activity of the rate limiting enzyme in cholesterol (CHOL) biosynthesis, HMG CoA reductase. In addition, P-407 promotes changes in the concentration of hepatic CHOL content. These date indicate that the hyper CHOL could be the result of increased CHOL synthesis, as well as a clearing of CHOL from the liver. Chronic injection into mice of P-407 for 145 d produced atherogenic lesions in the aortas of C57BL/6 mice. The response was equivalent to that seen in animals eating a high CHOL diet for 145 d. Cholic acid potentiated the P-407-induced atherogenesis. These data suggest that P-407 could be used as an agent for the study of hyperlipidemia-induced atherogenesis.

Effect of Poloxamer 407 on the Activity of Microsomal 3-hydroxy-3-methylglutaryl Coa Reductase in Rats

A single 300-mg i.p. injection of poloxamer 407 (P-407, also called Pluronic F-127) in rats produces a marked hypercholesterolemia for a minimum of 96 h. The purpose of this investigation was to determine mechanisms by which P-407 causes hypercholesterolemia. The enzyme targeted for investigation is the rate-limiting enzyme in cholesterolgenesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Injection of P-407 in fasted rats resulted in a significant (p < 0.05) elevation in plasma cholesterol (61.2 +/- 4.2 mg/dl) as soon as 1 h after injection compared with sham-injected controls (50.1 +/- 3.7 mg/dl). Plasma cholesterol (CHO) 24 h after injection of P-407 was 449 +/- 57 mg/dl, with the fastest rate of accumulation occurring from 1 to 12 h (approximately 16.6 mg/dl/h). Over the concentration range of 0-5 mM, P-407 did not appear significantly to affect the activity of HMG-CoA reductase in vitro. However, the enzymatic activity assayed in microsomal fractions isolated from the livers of P-407-injected rats reached a maximum of 262 +/- 42.6 pmol mevalonate/min/mg approximately 15 h after injection, with a subsequent decline to control activity (94.1 +/- 8.7 pmol/min/mg) at approximately 40 h after injection. At 48 h after injection of P-407, the activity of HMG-CoA reductase significantly (p < 0.05) decreased below control values with a mean activity of 9.4 +/- 1.2 pmol/min/mg. The CHO concentrations in hepatic tissue were significantly (p < 0.01) increased at 2 h (3.26 +/- 0.19 mg/g) and 4 h (3.75 +/- 0.38 mg/g) and significantly (p < .01) reduced at 15 h (1.56 +/- 0.19 mg/g) after injection of P-407 compared with tissue CHO concentrations determined in control animals (2.65 +/- 0.18 mg/g). However, the hepatic CHO content appeared to return to control values by 24 h (mean +/- SEM, 2.61 +/- 0.08 mg/g) after injection. These data suggest that the activity of HMG-CoA reductase is regulated by some indirect mechanism(s) after injection of P-407 in rats.

Potential downregulation of HMG-CoA reductase after prolonged administration of P-407 in C57BL/6 mice

This study investigated the potential alteration in the amount of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase messenger RNA (mRNA) and lipoprotein lipase (LPL) mRNA in the livers of C57BL/6 mice after long-term (200 days) treatment with the nonionic surfactant called poloxamer 407 (P-407). Previously, P-407 has been used to produce a dose-controlled hyperlipidemic state in C57BL/6 mice with subsequent formation of atherosclerotic lesions. Five groups of mice were studied; controls (C); mice fed a standard chow diet enriched with only cholic acid (CH); mice fed the high-cholesterol, high-fat Paigen diet (HF); mice treated with 0.5 g/kg P-407 every third day (P); and mice administered 0.5 g/kg P-407 every third day while consuming a diet identical to that of mice in group CH (PC). Neither a significant (p < 0.05) weight loss nor alteration in liver enzymes (AST and ALT) were observed for any group throughout the study when compared with the control mice. Total plasma cholesterol (CHOL) was significantly elevated compared with controls for mice in groups HF, P, and PC, whereas total plasma triglycerides (TG) were significantly increased for mice in only groups P and PC. Long-term ingestion of a high-fat diet or a diet enriched in cholic acid resulted in a significant (p < 0.05) reduction in HDL-CHOL when compared with controls. Plasma samples assayed at 200 days for mice in groups HF and P showed a shift in the lipoprotein fraction distribution primarily to VLDL-CHOL as compared with mice in group C in which, as expected, most of the CHOL was contained in the HDL fraction. The biologic activity of HMG-CoA reductase assayed in hepatic microsomal homogenates was significantly reduced for mice in groups CH (p < 0.01), HF (p < 0.01), and PC (p < 0.05), but not for mice in group P, when compared with control. A statistical analysis of the data demonstrated significant (p < 0.05) reductions in the HMG-CoA reductase mRNA levels in hepatic tissue for all treatment groups relative to mRNA levels determined for mice in group C. In contrast, no treatment group demonstrated a significant difference in hepatic LPL mRNA levels when compared with mRNA levels determined for control animals. These data demonstrate that P-407 administration to C57BL/6 mice significantly decreased the amount of HMG-CoA reductase mRNA detected in liver.

Controlled release implants for cardiovascular disease

Abstract The systemic therapy of many cardiovascular diseases is often hampered by adverse drug effects. The present paper examines the use of controlled release implants as a means for optimizing drug concentrations at the affected site in the cardiovascular system, while using a relatively low systemic dose. Controlled release systems have been prepared by combining a drug of choice with either a non-degradable polymer, such as a silicone rubber, polyurethane, and ethylene vinylacetate, or a biodegradable compound such as poly(glycolic-lactic acid) or a high molecular weight polyanhydride. Controlled release matrices containing ethylenehydroxydiphosphonate (EHDP), when implanted next to a bioprosthetic heart valve leaflet, prevented pathologic calcification. Similarly, controlled release matrices containing lidocaine-HCl have been used experimentally as epicardial implants to convert ventricular tachycardia to normal sinus rhythm in dogs. A matrix system containing gentamicin has been used by others [35] to prevent experimental valvular endocarditis. Other workers have used a dexamethasone-releasing cardiac pacing lead in clinical studies, to prevent scar tissue formation, which leads to elevated electrical pacing threshold [15,16]. Future controlled release systems for cardiovascular use will very likely incorporate innovative design features including: a reservoir configuration to replenish or change drug therapy, modulatable drug release to vary drug dosing as desired, and closed-loop feedback to increase or decrease release rates in response to disease status.

Effects of Nicotinic Acid on Poloxamer 407‐Induced Hyperlipidemia

We attempted to determine the mechanism(s) of poloxamer (P)‐407‐induced hyperlipidemia in rats by administering a lipid‐lowering drug with a known mechanism of action. Five weight‐matched animals were assigned to each of four treatment groups. Two groups received P‐407 300 mg/ml and two received saline 1 ml. One of the P‐407 and one of the saline groups were administered nicotinic acid 100 mg/kg by intraperitoneal injection at 6–96 hours after blood sampling. Blood samples were collected at 7 points from time zero to 120 hours and analyzed for triglyceride and cholesterol concentrations. The detergent produces hypertriglyceridemia (HTG) increasing from 53.4 ± 7.0 mg/dl (time zero) to 4026.9 ± 42.1 mg/dl by 24 hours. The HTG response was significantly attenuated by nicotinic acid (at t = 24 hrs). This, however, was followed by an average triglyceride concentration increase of 2.8‐fold from 72 to 120 hours. The detergent produces a dramatic hypercholesterolemia (HCHO), increasing cholesterol from 47.5 ± 1.8 mg/dl to 468.5 ± 27.9 mg/dl by 48 hours. The HCHO was significantly affected by nicotinic acid administration during the accumulation phase. Nicotinic acid reduced cholesterol concentration from 364.4 ± 16.1 mg/dl to 276.8 ± 16.4 mg/dl at 24 hours (p<0.05). It is a potent antilipolytic agent, limiting the free fatty acids available for the synthesis of triglyceride and cholesterol. These data suggest that P‐407 may act by stimulating the release of free fatty acids from the adipocyte for at least 24 hours after injection.

Controlled release of 1-hydroxyethylidene diphosphonate: in vitro assessment and effects on bioprosthetic calcification in sheep tricuspid valve replacements

Abstract Calcification (CALC) is the most frequent cause of the clinical failure of bioprosthetic valves (BHVs). Controlled-release (paravalvar) administration of the anticalcification agent ethanehydroxydiphosphonate (EHDP), as either Na 2 EHDP or in combination (1:1) with the less soluble CaEHDP, from a silicone rubber matrix (20% w/w EHDP) was studied both in vitro and in vivo for the prevention of BHV CALC. Seventeen sheep (6–7 months old, male, Suffolk) underwent tricuspid valve replacement using Hancock I, 25 mm porcine aortic bioprostheses. BHV explant evaluation after 16–20 weeks revealed that two of the 7 control BHV were calcified (139 ± 20.8 μg Ca 2+ /mg of tissue), while none of the 9 BHV retrieved from animals receiving controlled release EHDP demonstrated CALC (4.41 ± 1.09 μg Ca 2+ /mg of tissue). No adverse effects of EHDP on bone or calcium metabolism were noted. The cumulative percent of EHDP released per electron microprobe analysis was 40.4% ± 9.68 (Na, CaEHDP) to 79.0% ± 4.82 (Na 2 EHDP) in vivo compared to 35.7% ± 7.72 and 78.6 ± 11.1 in vitro, respectively. Assessment of the Youngs modulus (Y) using thermomechanical analysis (TMA) revealed a 1.5-fold (Silastic Q7-4840) to 9.5-fold (Silastic 382) increase in Y following drug loading. The Y for explanted, Silastic Q7-4840 polymer matrices ranged from 2.84 × 10 4 to 5.57 × 10 5 dyne/cm 2 . In vitro osmotic related matrix swelling of the Na 2 EHDP loaded, unsealed matrices (20% w/w) after 75 days was minimized to a 35.8% increase in weight due to coincorporation of CaEHDP with Na 2 EHDP in a 1:1 ratio and was further reduced (22.2% increase in weight) by sealing 76% of the releasing surface, compared to Na 2 EHDP matrices which demonstrated a 414% and 141% increase in weight, respectively.