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.     

Repeated 28-day oral toxicity study of vinclozolin in rats based on the draft protocol for the “Enhanced OECD Test Guideline No. 407” to detect endocrine effects

We performed a 28-day repeated-dose toxicity study of vinclozolin, a widely used fungicide, based on the draft protocol of the “Enhanced OECD Test Guideline 407” (Enhanced TG407) to investigate whether vinclozolin has endocrine-mediated properties according to this assay. Seven-week-old SD rats were administered with vinclozolin daily by oral gavage at dose rates of 0, 3.125, 12.5, 50 and 200 mg/kg/day for at least 28 days. The vinclozolin-treated male rats showed a reduction of epididymis and accessory sex organ weights and an alteration of hormonal patterns. A slight prolongation of the estrous cycle and changes in the estrogen/testosterone ratio and luteinizing hormone level were observed in vinclozolin-treated female rats. Thyroxin concentrations were decreased and thyroid-stimulating hormone concentrations were increased in both sexes; however, there were no compound-related microscopic lesions in the thyroid gland or changes in the thyroid weight. The endocrine-related effects of vinclozolin could be detected by the parameters examined in the present study based on the OECD protocol, suggesting the Enhanced TG407 protocol should be a suitable screening test for the detection of endocrine-mediated effects of chemicals.     

Gene transfer mediated by sphingosine/ dioleoylphosphatidylethanolamine liposomes in the presence of poloxamer 188

A cationic liposome system consisting of sphingosine (SP) and dioleoylphosphatidylethanolamine (DOPE) was developed for in vitro and in vivo gene transfer. A nonionic surface active agent of poloxamer 188 was incorporated in the formulations to stabilize the DNA/liposome complex. Comparison of the results obtained from systems with and without the effect of poloxamer 188 was made to investigate the efficiency of gene expression. In vitro transfection study of the DNA/liposome complex showed that with the effect of poloxamer 188, gene transfer into some cell lines was enhanced. In vivo systemic delivery of the DNA/liposome complex with poloxamer 188 demonstrated gene expression with improved luciferase activity in all major organs including lung, spleen, heart, liver, and kidney. High level transgene activity was found in lung and spleen with prolonged gene expression. This was attributed to poloxamer 188 that stabilized the liposome system and produced homogeneous DNA/liposome complex for enhancement of gene delivery.     

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4′-butylidenebis(2-tert-butyl-5-methylphenol) and 3-(dibutylamino)phenol, based on the OECD draft protocols

We performed a uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study [enhanced Organization for Economic Co-operation and Development (OECD) test guideline No. 407] of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) and 3-(dibutylamino)phenol, based on the OECD draft protocols. In the uterotrophic assay of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol), female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and no changes were observed. In the Hershberger assay of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol), the test chemical was orally administered to castrated male SD rats at doses of 0, 50, 200, and 1,000 mg/kg/day for ten consecutive days beginning on postnatal day 56, and no changes were observed. When this chemical was orally administered at doses 0, 5, 25, and 125 mg/kg/day for at least 28 days in the subacute oral toxicity study, an increase in thyroid weight was observed in the female rats in the 125 mg/kg group, an increase in serum thyroid-stimulating hormone (TSH) values in the male and female rats in the 125 mg/kg group, and a decrease in serum T3 and T4 values in the male rats in the 125 mg/kg group, and thyroid follicular epithelial cell hypertrophy was observed in some of the female rats in the 125 mg/kg group. These findings were concluded to be the result of endocrine-mediated effects of the chemical on thyroid function. In addition, increased liver weight, abnormal histological findings in the liver, and abnormal biochemical parameters related to liver function were observed in male and/or female rats in 5 mg/kg group and higher dose groups. The no-observed-effect level for 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) was concluded to be <5 mg/kg/day. In the uterotrophic assay of 3-(dibutylamino)phenol, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and no changes were observed. In the Hershberger assay of 3-(dibutylamino)phenol, the test chemical was orally administered at doses of 0, 50, 200, and 400 mg/kg/day to castrated male SD rats for ten consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when this test chemical was orally administered at doses 0, 30, 100, and 300 mg/kg/day for at least 28 days in the subacute oral toxicity study, thyroid weight increased in the male rats in the 300 mg/kg group, thyroid follicular epithelial cell hypertrophy was observed in a small number of male rats in the 300 mg/kg group, serum T3-values decreased in the female rats in the 300 mg/kg group, and a tendency for TSH-values to increase was observed in the male and female rats in the 300 mg/kg group. Therefore, 3-(dibutylamino)phenol was also concluded to have slight anti-thyroid acting effects as the endocrine-mediated effects. On the other hand, increased hemosiderin deposition in the spleen, increased spleen weight, hematological abnormalities, and squamous epithelial hyperplasia of the forestomach were detected in male and/or female rats in the 100 and/or 300 mg/kg groups, and thus the no-observed-effect level for 3-(dibutylamino)phenol was concluded to be 30 mg/kg/day.     

The pharmacokinetics of dronedarone in normolipidemic and hyperlipidemic rats

The objectives of the current study were to characterize the pharmacokinetic profile of dronedarone in the rat, and to examine the effect of hyperlipidemia on its pharmacokinetics. Single doses of dronedarone were administered to rats intravenously (4 mg/kg), orally (55 mg/kg) and intraperitoneally (65 mg/kg). To induce hyperlipidemia, some of the rats were administered intraperitoneal doses of poloxamer 407 before giving an oral dose of dronedarone. After intravenous doses of 4 mg/kg dronedarone, plasma clearance and volume of distribution at steady‐state were 25.1 ± 8.09 mL/min/kg and 10.8 ± 4.77 L/kg, respectively. After oral doses the maximum plasma concentrations (Cmax) and their median time of attainment (tmax) were 1.87 ± 1.65 mg/mL and 3.5 h, respectively. Intraperitoneal administration of 65 mg/kg dronedarone base yielded plasma Cmax and median tmax of 0.816 ± 0.611 mg/mL and 3 h, respectively. Protein binding was high in NL and HL plasma. Dronedarone is extensively distributed with high volume of distribution in the rat. The drug showed poor bioavailability (<20%) after oral and intraperitoneal administration. The increased plasma concentrations after oral dosing to hyperlipidemic rats appears to be attributable to a direct effect on metabolizing enzymes or transport proteins. Copyright © 2016 John Wiley & Sons, Ltd.     

Thermally Reversible In Situ Gelling Carbamazepine Liquid Suppository

Carbamazepine (CBZ), indicated for the control of epilepsy, undergoes extensive hepatic first-pass elimination after oral administration. A rectal dosage form of CBZ is not commercially available, although it is of particular interest when oral administration is impossible. Conventional suppositories can cause patient discomfort and may reach the end of the colon; consequently, the drug can undergo the first-pass effect. Mucoadhesive liquid suppositories of CBZ were prepared by adding carbopol to formulation of thermally gelling suppositories that contain 20% poloxamer 407 and either 15% poloxamer 188 or 1% methylcellulose. Gellan gum was also tried instead of 20% poloxamer. All formulations contained 10% CBZ. The characteristics of the suppositories differed depending on the formulation. The formula containing 20% poloxamer 407, 1% methylcellulose, and 0.5% carbopol showed reasonable gelation temperature, gel strength and bioadhesive force. The analysis of release mechanism showed that CBZ released from the suppositories by Fickian diffusion. In vivo evaluation of the same formulation showed higher peak plasma concentration of CBZ compared with the orally administered suspension containing the equivalent amount of drug. However, there was no statistical significant difference (p > 0.05) in extent of bioavailability between the liquid suppository and oral suspension as indicated by the values of AUC_{0 - ∝}, 17.9 and 18.8 μ g.h/ml, respectively. These results suggested that mucoadhesive in situ gelling liquid suppository could be an effective and convenient delivery system of carbamazepine.     

Preparation of ibuprofen-loaded liquid suppository using eutectic mixture system with menthol

To prepare an ibuprofen-loaded liquid suppository using eutectic mixture with menthol, the effects of menthol and poloxamer 188 (P 188) on the aqueous solubility of ibuprofen were investigated. The physicochemical properties such as gelation temperature, gel strength and bioadhesive force of various formulations composed of ibuprofen, menthol and P 188 were investigated. Then, the pharmacokinetic study of ibuprofen delivered by the liquid suppositories composed of P 188 and menthol were then performed. In the absence of P 188, the solubility of ibuprofen increased until the ratio of menthol to ibuprofen increased from 0:10 to 4:6 followed by an abrupt decrease in solubility above the ratio of 4:6, indicating that four parts of ibuprofen formed eutectic mixture with six parts of menthol. In the presence of P 188, the solutions with the same ratio showed abrupt increase in the solubility of ibuprofen. Furthermore, the solution with ratio of 4:6 showed more than 2.5- and 6-fold increase in the solubility of ibuprofen compared with that without additives and that without menthol, respectively. The poloxamer gel with menthol/ibuprofen ratio of 1:9 and higher than 15% poloxamer 188 showed the maximum solubility of ibuprofen, 1.2mg/ml. Ibuprofen increased the gelation temperature and weakened the gel strength and bioadhesive force of liquid suppositories. However, menthol did the opposite due to forming the eutectic mixture with ibuprofen. The ibuprofen-loaded liquid suppository [P 188/menthol/ibuprofen (15/0.25/2.5%)] with the maximum ibuprofen solubility of 1.2mg/ml was administered easily to the anus and to remain at the administered site without leakage after the dose. Furthermore, it gave significantly higher initial plasma concentrations, Cmax and AUC of ibuprofen than did solid suppository, indicating that the drug from poloxamer gel could be more absorbed than that from solid one in rats. Thus, the liquid suppository system with P 188 and menthol, a more convenient and effective rectal dosage form for ibuprofen will be expected to enhance the rectal bioavailability of ibuprofen.     

Synergistic encapsulation of the anti-HIV agent efavirenz within mixed poloxamine/poloxamer polymeric micelles

This study investigated the synergistic performance of mixed polymeric micelles made of linear and branched poly(ethylene oxide)-poly(propylene oxide) for the more effective encapsulation of the anti-HIV drug efavirenz. The co-micellization process of 10% binary systems combining different weight ratios of a highly hydrophilic poloxamer (Pluronic F127) and a more hydrophobic poloxamine counterpart (Tetronic T304 and T904) was investigated by means of dynamic light scattering, cloud point and electronic spin resonance experiments. Then, the synergistic solubilization capacity of the micelles was shown. Findings revealed a sharp solubility increase from 4 μg/ml up to more than 33 mg/ml, representing a 8430-fold increase. Moreover, the drug-loaded mixed micelles displayed increased physical stability over time in comparison with pure poloxamine ones. Overall findings confirmed the enormous versatility of the poloxamer/poloxamine systems as Trojan nanocarriers for drug encapsulation and release by the oral route and they entail a relevant enhancement of the previous art towards a more compliant pediatric HIV pharmacotherapy.

From the Clinical Editor

In this study, the authors demonstrate the versatility of poloxamer/poloxamine systems as Trojan nanocarriers for anti-HIV drug encapsulation and release by the oral route. A highly relevant stability and solubility enhancement is shown, which may ultimately lead to more compliant anti-HIV pharmacotherapy.     

Effects on splenic, hepatic, hematological, and growth parameters following high-dose poloxamer 407 administration to rats

The nonionic surfactant poloxamer 407, NF (PIuronic ^® F-127, NF) has previously been shown to produce marked hyperlipidemia in rats at a dose of 1.5 g/kg for greater than 96 h following a single intraperitoneal (i.p.) injection (Wout et al. J. Parenter. Sci. Technol., 46 (1992) 192–200). In an effort to characterize any potential toxicity of the polymeric vehicle to various organ systems in the rat following multiple i.p. injections, a dose of 0.33 g/kg per day (10% w/w solution) or 1.0 g/kg per day (30% w/w solution) of poloxamer 407 was administered once daily for 4 consecutive days. When compared to control (non-injected) animals, rats injected with 0.33 g/kg per day of poloxamer 407 did not show a significant (p > 0.05) increase or decrease in spleen, liver, or total body weight. A complete blood count (CBC) with a white blood cell (WBC) differential was performed on blood samples collected on day five from rats injected with poloxamer 407 at both doses. The CBC with WBC differentia] was conducted to assess any changes in the WBC count, percent lymphocytes (LY), percent monocytes (MO), percent granulocytes (GR), red blood cell (RBC) count, hemoglobin (HGB), percent hematocrit (HCT), and the mean corpuscular volume (MCV) following administration of poloxamer 407. Rats injected i.p. with a dose of 0.33 g/kgper day of poloxamer 407 for 4 days demonstrated a significant (p < 0.05) increase in the number of MO when compared to controls. Administration of 1.0 g/kg per day of poloxamer 407 to rats for 4 days demonstrated distinct splenomegaly when compared to non-injected control animals. In addition, a significant (p < 0.05) reduction in body weight and significant (p < 0.05) decrease in the percent LY, RBCs, HGB, and percent HCT were noted. Lastly, a significant (p < 0.05) increase in the number of WBCs and the percent MO was observed in this same group of rats. However, rats administered 1.0 g/kg per day of poloxamer 407 for 4 days were observed to have no detectable changes in the values of the MCV, the percent GR, or liver-to-body weight ratio when compared to control animals. Thus, repetitive i.p. injections of poloxamer 407 to rats at a dose of 1.0 g/kg per day for four days results in splenomegaly and a reduction in total body weight. Splenomegaly in rats administered poloxamer 407 at a dose of 1.0 g/kg per day resulted from red pulp expansion due to infiltration of macrophages which contained phagocytized lipids.     

Pharmacological characterization of bradykinin B1 and B2 receptors in IMR-90 and INT-407 human cell lines using a microphysiometer

1. In the present study, we used a microphysiometer to measure bradykinin-induced acidification responses in IMR-90, a human lung fibroblast cell line, and INT-407, a human colonic epithelial cell line. Furthermore, we investigated the effect of 24 h exposure of transforming growth factor (TGF)-alpha on the bradykinin response in INT-407 cells. 2. Bradykinin (0.1-100 nmol/L) was potent in producing acidification responses in IMR-90 cells (pEC50 8.79+/-0.13; Hill slope 0.96+/-0.04) and INT-407 cells (pEC50 8.90+/-0.04; Hill slope 1.00+/-0.07). These responses were competitively antagonized by the bradykinin B2 receptor antagonist icatibant in both IMR-90 cells (apparent pKB = 8.54+/-0.15; Hill slope = 1.09+/-0.13 and 1.66+/-0.26 in the absence and presence of 10 nmol/L icatibant, respectively) and INT-407 cells (pKB = 8.12+/-0.07 (3, 10 and 30 nmol/L icatibant); Hill slope = 1.06+/-0.04). However, the bradykinin B1 receptor antagonist des-Arg9Leu8-bradykinin (3 micromol/L) had no effect on the bradykinin responses. 3. The non-peptide bradykinin B2 receptor antagonist FR173657 selectively antagonized bradykinin-induced acidification responses in INT-407 cells in a competitive manner (pKB = 8.76+/-0.10; Hill slope = 0.92+/-0.05) at lower concentrations (1 and 3 nmol/L) but in an insurmountable manner at higher concentrations (10 nmol/L; Hill slope = 1.04+/-0.09). This compound, at concentrations of 10 and 100 nmol/L (Hill slope = 1.38+/-0.15), also proved to be an insurmountable antagonist in IMR-90 cells. 4. The bradykinin B1 receptor selective agonist Lys0des-Arg10-bradykinin (0.1 nmol/L to 0.1 micromol/L) failed to produce acidification responses in IMR-90 cells, even after 24 h pre-incubation with bacterial lipopolysaccharide (0.1 microg/mL). 5. A 24 h pre-incubation of INT-407 cells with TGF-alpha (1, 10 and 100 ng/mL) caused a significant concentration-dependent decrease in maximal bradykinin response without affecting the pEC50. 6. In addition to this study being the first to use a microphysiometer to characterize bradykinin B2 receptors in cultured IMR-90 human lung fibroblast cells and INT-407 human colonic epithelial cells, we also showed that pre-incubation of INT-407 cells with TGF-alpha caused a significant decrease in maximal acidification response mediated by bradykinin B2 receptors.