Pharmacokinetic and Pharmacodynamic Evaluation of Different PEGylated Human Interleukin-11 Preparations in Animal Models.

Treating thrombocytopenia induced by chemotherapy remains an unmet-medical need. The use of recombinant human interleukin-11 (rhIL-11) requires repeated injections and induces significant fluid retention in some patients. Modification of human interleukin-11 with chemically inert polyethylene glycol polymer (PEG) may extend the peripheral circulation half-life leading to an improved pharmacokinetic and pharmadynamic profile. In this study, a number of rhIL-11 PEG conjugates were created to determine the optimal approach to prolong circulating half-life with the most robust pharmacological effect. The lead candidate was found to be a single 40-kDa Y-shaped PEG linked to the N-terminus, which produced a long-lasting circulating half-life, enhanced efficacy and alleviated side effect of dilutional anemia in healthy rat models. This candidate was also shown to be effective in myelosuppressive rats in preventing the occurrence of severe thrombocytopenia while ameliorating dilutional anemia, compared to rats receiving daily administration of unmodified rhIL-11 at the same dose. These data indicated that a single injection of the selected modified rhIL-11 for each cycle of chemotherapy regimen is potentially feasible. This approach may also be useful in treating patients of acute radiation syndrome when frequent administration is not feasible in a widespread event of a major radiation exposure.

The pharmacokinetic and pharmacodynamic properties of site-specific pegylated genetically modified recombinant human interleukin-11 in normal and thrombocytopenic monkeys.

In order to improve the pharmacokinetic and pharmacodynamic properties of recombinant human interleukin-11 mutein (mIL-11) and to reduce the frequency of administration, we examined the feasibility of chemical modification of mIL-11 by methoxy polyethylene glycol succinimidyl carbonate (mPEG-SC). PEG-mIL-11 was prepared by a pH controlled amine specific method. Bioactivity of the protein was determined in a IL-11-dependent in vitro bioassay, its pharmacodynamic and pharmacokinetic properties were investigated by using normal and thrombocytopenic monkey models. N-terminus sequencing and peptide mapping analysis revealed that Lys33 is the PEGylated position for PEG-mIL-11. Bioactivity of PEG-mIL-11 assessed by B9-11 cell proliferation assay was comparable to that of mIL-11. More than 79-fold increase in area-under-the curve (AUC) and 26-fold increase in maximum plasma concentration (Cmax) was observed in pharmacokinetic analysis. Single dose administration of the PEG-mIL-11 induced blood platelets number increase and the effect duration were comparable to that of 7 to 10 consecutive daily administration of mIL-11 to the normal and thrombocytopenic monkey models. PEG-mIL-11 is a promising therapeutic for thrombocytopenia.

Anti-adipogenic activity of an olive seed extract in mouse fibroblasts / Actividad anti-adipogénica de un extracto de huesos de aceituna en fibroblastos de ratón

The administration of different polyphenols protects against increased body weight and fat accumulation. The aim of the study was to determine the anti-adipogenic activity of an olive-seed polyphenolic extract, by means of mouse fibroblast cell line 3T3-L1 adipocyte differentiation. Material and methods: cells were incubated and differentiated (6000 cells/cup) in the presence of olive-seed extract at 10 and 50 mg/l biosecure concentrations of polyphenols, and with no extract in the control sample. After 5 to 7 days mature adipocytes are formed. The fat clusters are quantified by means of red-oil staining, 490 nm absorbance, and the expression of the leptin and PPARg genes, and then compared to the values obtained in the cultures before and after adipocyte differentiation. Results: the control samples, with no extract, presented an accumulation of fat of 100%. By contrast, the addition of 50 mg/l of olive-seed extract polyphenols resulted in a 50% accumulation of fat, similar to that of the non-differentiated cells. A 10 mg/l extract concentration had no effect. Anti-adipogenic activity is thus confirmed, as the expression of the PPARg and leptin genes is reduced in adipocyte differentiation in the presence of extract at 50 mg/l. In conclusion, both the formation of fatty substances characteristic of adipogenesis, and the expression of the adipogenic PPARg and leptin genes are found to be inhibited by the prior addition of olive-seed extract polyphenols at a 50 mg/l concentration (AU)

La administración de diferentes polifenoles protege contra el incremento de peso y la acumulación de grasa. Objetivo: comprobar la actividad anti-adipogénica de un extracto polifenólico de huesos de aceituna, utilizando la diferenciación a adipocitos de la línea celular 3T3-L1 de fibroblastos de ratón. Material y métodos: se cultivan y diferencian las células (6.000 células/pocillo) en presencia del extracto de huesos de aceitunas a 10 y 50 mg/l de polifenoles, concentraciones bioseguras, y sin extracto como control. A los 5-7 días se forman los adipocitos maduros. Se cuantifican los cúmulos de grasa formados mediante tinción con OilRed y medida de la absorbancia a 490 nm y la expresión de los genes de leptina y PPARg, relacionándolos con los valores en los cultivos antes y después de diferenciarse a adipocitos. Resultados: las muestras control, sin extracto, se consideran el 100% de acumulación de grasas. En contraste, la adición de 50 mg/l de extracto de polifenoles de huesos de aceituna muestra un cúmulo de grasa de alrededor del 50%, semejante a las células no diferenciadas. Con 10 mg/l de extracto no se muestra efecto. Se confirma la actividad antiadipogénica, observándose disminución en la expresión de los genes PPARg y de leptina en la diferenciación a adipocitos en presencia del extracto a 50 mg/l. En conclusión, la formación de los cuerpos grasos característicos de la adipogénesis queda inhibida previa adición de 50 mg/l de polifenoles de extracto de huesos de aceituna, así como la expresión de los genes adipogénicos PPARg y de leptina (AU)

Improvement of biological and pharmacokinetic features of human interleukin-11 by site-directed mutagenesis.

Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for its use in chemotherapy-induced thrombocytopenia (CIT). However, due to its serious side effects, its clinical use has been limited. The current work presents significantly improved efficacy of rhIL-11 via knowledge based re-designing process. The interleukin-11 mutein (mIL-11) was found to endure chemical and proteolytic stresses, while retaining the biological activity of rhIL-11. The improved efficacy of mIL-11 was evident after subcutaneous administration of mIL-11 and rhIL-11 in the rodent and primate models. More than three-fold increase in maximum plasma concentration (Cmax) and area-under-the curve (AUC) was observed. Furthermore, three-fold higher increase in the platelet counts was obtained after seven consecutive daily subcutaneous mIL-11 injections than that with rhIL-11. The mIL-11 demonstrated not only improved stability but also enhanced efficacy over the currently used rhIL-11 regimen, thereby suggesting less toxicity.

Phase I/II dose escalation study of recombinant human interleukin-11 following ifosfamide, carboplatin and etoposide in children, adolescents and young adults with solid tumours or lymphoma: a clinical, haematological and biological study.

Thrombocytopenia remains the major dose-limiting toxicity of myelosuppressive chemotherapy in children with solid tumours. Recombinant human interleukin-11 (rhIL-11) has been approved by the Food and Drug Administration as treatment for adults with solid tumours and lymphomas with severe chemotherapy-induced thrombocytopenia. We conducted a phase I/II trial of rhIL-11 following ifosfamide, carboplatin and etoposide (ICE) chemotherapy in children with solid tumours or lymphomas. Patients received ifosfamide 1800 mg/m(2)/d for 5 d, carboplatin 400 mg/m(2)/d for 2 d and etoposide 100 mg/m(2)/d for 5 d with rhIL-11 subcutaneous (s.c.) at 25-125 microg/kg/d on days 6-33. Forty-seven patients with median age 10.5 years (range, 0.7-26 years) were studied. Median days to absolute neutrophil count >/=0.5 x 10(9)/l, platelet count >/=50 x 10(9)/l and platelet transfusions were 23, 18, 18, 16.5 and 18.5, 21, 20, 18 and 3, 3, 4, and 2 d at doses 25, 50, 75 and 100 Schulteg/kg respectively. There was a dose-dependent increase in C(max) (7.6-25.5 ng/ml), AUC(0-rho) (57-209 ng.h/ml) and T(1/2) (4-8.2 h) respectively. There was a 4% incidence of anti-IL-11 antibody formation. Clinically important adverse events to rhIL-11 were papilloedema and periosteal bone formation. In summary, rhIL-11 was well tolerated at doses of

A multiple-dose, safety, tolerability, pharmacokinetics and pharmacodynamic study of oral recombinant human interleukin-11 (oprelvekin).

A study in healthy men and women was performed to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of orally administered recombinant human interleukin-11 (oprelvekin) (OAO). Four cohorts of 10 subjects each received 3, 5, 10 or 30 mg (8:2/OAO:placebo ratio), first as a single dose with a 7-day washout period, then 7 consecutive daily doses. Safety was assessed by ongoing evaluation of adverse events (AEs) and laboratory values. PK samples were collected on the first and last day of dose administration. The established effects of subcutaneous oprelvekin on C-reactive protein (CRP, upward arrow), platelet count (upward arrow), fibrinogen (upward arrow) and hemoglobin (downward arrow), were evaluated. PK analysis showed that most subjects (27/34, 79%) had undetectable serum levels of IL-11. PD measures showed no changes from baseline between any OAO group and the placebo group. Orally administered oprelvekin was safe and well tolerated at all doses. A total of five AEs (abdominal pain, diarrhea, headache, rhinitis, grade 3 alanine aminotransferase elevation) were reported across all groups. Evaluations of serum IL-11 levels indicate that OAO is not systemically absorbed at levels above the lower limit of the bioanalytic assay. These data in addition to the lack of effect on PD measures suggest that there is a decreased potential of systemic adverse events with OAO.

Pharmacokinetics of [125I]-recombinant human interleukin-11: 1. Absorption, distribution and excretion after subcutaneous administration to male rats.

Absorption, distribution, metabolism and excretion of [125I]-rhIL-11 (recombinant human interleukin-11) after subcutaneous administration in rats were investigated. After a single administration, the concentration of radioactivity in the tissues was 2-6-fold higher in the liver and kidneys, and slightly higher in the gastrointestinal tract as compared to the plasma concentration. However, since the concentration in the other tissues was lower than the plasma concentration, the transport of rhIL-11 into tissues appeared to be low. Tissue radioactivity rapidly diminished, thus accumulation of rhIL-11 in tissues was thought to be low. Excretion of radioactivity into urine and feces was almost complete 72 h after administration, with 88.5% of the dosed radioactivity being found in urine and 7.9% in feces. When [125I]-rhIL-11 was administered to bile-duct cannulated rats, 44.4% of the dosed radioactivity was excreted into bile up to 48 h after administration. Most radioactivity in bile and urine was found in the TCA supernatant and low molecular weight fraction by HPLC analysis, indicating that rhIL-11 was eliminated from the body by metabolism.

Pharmacokinetics of [125I]-recombinant human interleukin-11: 2. Placental transfer and excretion into milk after subcutaneous administration to rats.

Placental transfer and excretion into milk of [125I]-rhIL-11 (recombinant human interleukin-11) after subcutaneous administration in female rats were investigated. After administration of [125I]-rhIL-11 to rats on the 14th day of gestation, radioactivity in the kidney was the highest among excised tissues, being 3 times higher than that in the plasma at 1.5 h. Radioactivity in other tissues, including the mammary gland, ovary, uterus, placenta and amniotic fluid, was lower than that in the plasma. Although radioactivity in fetuses was detected 6 h after administration, the level was only 2% of the plasma concentration in dams, and the radioactivity was not found in fetal-derived TCA precipitates. These results indicate that rhIL-11 does not readily pass through the placenta into the fetus. After subcutaneous administration of [125I]-rhIL-11 to lactating rats 14 days after delivery, radioactivity in milk was 1.1-1.6 times that in the plasma of dams. Radioactivity in clotted milk in the stomachs of suckling infants was almost equal to that in the dam's milk; however, only a small amount of radioactivity was detected in infant kidneys.

Hepatic disposition characteristics of recombinant human interleukin-11 (rhIL-11) in the perfused rat liver.

The hepatic disposition characteristics of recombinant human interleukin-11 (rhIL-11) were investigated in perfused rat liver to clarify the mechanism of hepatic clearance which is a major contributor to the rapid clearance of rhIL-11 in vivo. We analyzed the disposition characteristics of [(111)In]-labeled rhIL-11 using a single-pass constant infusion mode at different concentrations of rhIL-11. The venous outflow rapidly reached a steady-state condition at every concentration. Liver extraction ratio at steady-state (Ess) was decreased with increase in the concentration, suggesting that there is a saturable interaction between the liver cells and rhIL-11 molecule. Cellular localization experiments demonstrated that rhIL-11 was taken up by both liver parenchymal and nonparenchymal cells depending on their surface area, suggesting that this uptake was mediated by electrostatic interaction due to cationic charges in the cytokine.

Pharmacokinetics of recombinant human interleukin-11 (rhIL-11) in healthy male subjects.

AIMS: To study the pharmacokinetics of recombinant human interleukin-11 (rhIL-11) in healthy male volunteers following subcutaneous (s.c.) and intravenous (i.v.) administration. METHODS: RhIL-11 was infused intravenously at 10-50 micrograms kg-1 for 1 or 3 h, or administered subcutaneously at 3-50 micrograms kg-1 to volunteers. RhIL-11 was also administered at 3 micrograms kg-1 s.c. once daily for 7 days. Plasma and urinary concentrations were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: RhIL-11 showed linear pharmacokinetics after both intravenous infusion and s.c. administration. Comparison of t1/2 and MRT values after i.v. administration with those after s.c. administration indicated that rhIL-11 pharmacokinetics after s.c. administration were absorption rate-limited. Bioavailability after s.c. administration was about 65%. Since RhIL-11 was not detected in urine after a single 50 micrograms kg-1 s.c. dose, rhIL-11 was considered to be eliminated by metabolism. There was no significant change in the pharmacokinetic profile of rhIL-11 following repeated s.c. administration. CONCLUSIONS: RhIL-11 demonstrated linear pharmacokinetics at these dose ranges after single and repeated s.c. administration or constant-rate i.v. infusion in healthy volunteers.

Renal disposition of recombinant human interleukin-11 in the isolated perfused rat kidney.

PURPOSE: To clarify the mechanism of the renal clearance of recombinant human interleukin- 11 (rhIL- 1), we investigated the renal disposition characteristics of rhIL-11 in the perfused rat kidney. METHODS: The disposition characteristics of (111)In-labeled rhIL-11 were analyzed using a single-pass indicator dilution technique and statistical moment analysis in the perfused rat kidney under filtering and nonfiltering conditions. RESULTS: Steady-state distribution volume (Vd) calculated from the venous outflow patterns of rhIL-11 at the doses of 0.3 to 10 microg/kidney was between 0.35 and 0.40 ml/g kidney. However, Vd at the highest dose decreased to a value almost identical to that of bovine serum albumin, suggesting that there is a reversible and saturable interaction between the capillary wall and rhIL-11 molecule. In filtering kidney, a remarkable accumulation of rhIL-11 was observed while its urinary excretion was highly restricted at all doses. In nonfiltering kidney, rhIL-11 showed a decreased but still significant renal uptake. Taken together, the marked renal uptake of rhIL-11 may be explained by both efficient tubular reabsorption and significant uptake from the capillary side. These processes were not saturable within the tested dose range. These characteristics of rhIL-11 are likely based on non-specific electrostatic interaction with the tissues due to its cationic charge in the cytokine. CONCLUSIONS: The renal disposition processes of rhIL-11 were clarified at organ level in a quantitative manner. These findings agree well with previous observations in an in vivo disposition study in mice.

Disposition characteristics of recombinant human interleukin-11 after a bolus intravenous administration in mice.

The pharmacokinetics and disposition characteristics of recombinant human interleukin-11 (rhIL-11) after systemic administration of 10 to 1000 micrograms/kg was investigated in mice. After a bolus i.v. injection of 100 micrograms/kg of 111In-labeled rhIL-11, radioactivity disappeared rapidly from the circulation after a biexponential function. Plasma clearance profiles based on immunoreactivity and biological activity were identical to the disappearance pattern of radioactivity in the early phase after injection. In the range of 10 to 100 micrograms/kg, pharmacokinetic parameter estimates such as the T1/2 alpha and total body clearance were almost constant, suggesting that pharmacokinetics of rhIL-11 were linear within this dose range. 111In-labeled rhIL-11 distributed mainly to the kidney and liver; within 10 min, 40 and 20% of the dose, respectively, accumulated. On the other hand, the urinary excretion ratio was very small (ca., 1% of the dose), suggesting that rhIL-11 was cleared rapidly by glomeruler filtration followed by efficient tubular reabsorption. Pharmacokinetic analysis of the tissue distribution data demonstrated large kidney and hepatic uptake clearances. At the highest dose (1000 micrograms/kg), total body clearance significantly decreased, due primarily to saturation of hepatic uptake. These findings will provide useful information for the development of rhIL-11.