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AIM To investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of the highly selective oral p38alpha/beta mitogen-activated protein (MAP) kinase inhibitor Org 48,775-0 in a first-in-human study. METHODS In the single ascending dosing (SAD) study, an oral dose of Org 48,775-0 (0.3-600 mg) was evaluated in healthy males. In the multiple ascending dosing (MAD) study, levels of 30, 70 and 150 mg were dosed for six consecutive days, twice daily. Both studies were performed in a double-blind, randomized, placebo-controlled, cross-over fashion and evaluated pharmacokinetics, pharmacodynamics (ex vivo inhibition of lipopolysaccharide [LPS]-induced tumor necrosis factor (TNFα) release) and routine clinical and laboratory data. Pharmacokinetic and pharmacodynamic parameters of Org 48,775-0 were compared between healthy males and postmenopausal females, and the effect of a standardized fat meal was evaluated. RESULTS All adverse events observed in the SAD (16; dizziness and headache, diarrhoea and catheter-related phlebitis) and MAD (43; mainly somnolence, dizziness, headache and nasopharyngitis) cohorts were mild, transient and completely reversible. Pharmacokinetics were linear up to single doses of 400 mg. Median Tmax ranged from 0.5 to 1.8 hours, geometric mean for T1/2 from 7.0 to 14.4 hours. Org 48,775-0 doses equal to and greater than 30 mg significantly inhibited LPS-induced TNFα release (42.3%; 95% CI = -65.2, -4.3) compared to placebo. In the MAD study, Org 48,775-0 treatment inhibited LPS-induced TNFα release during the entire steady-state period. Levels of inhibition amounted 30-75% for 30 mg, 53-80% for 70 mg and 77-92% for 150 mg Org 48,775-0. CONCLUSION Org 48,775-0 has the capacity to significantly inhibit MAP kinase activity in humans without safety concerns.

A clinical pharmacokinetic study was performed in 12 healthy women to evaluate systemic exposure to aluminum following topical application of a representative antiperspirant formulation under real-life use conditions. A simple roll-on formulation containing an extremely rare isotope of aluminum (26 Al) chlorohydrate (ACH) was prepared to commercial specifications. A 26 Al radio-microtracer was used to distinguish dosed aluminum from natural background, using accelerated mass spectroscopy. The 26 Al citrate was administered intravenously (i.v.) to estimate fraction absorbed (Fabs ) following topical delivery. In blood samples after i.v. administration, 26 Al was readily detected (mean area under the curve (AUC) = 1,273 ± 466 hours×fg/mL). Conversely, all blood samples following topical application were below the lower limit of quantitation (LLOQ; 0.12 fg/mL), except two samples (0.13 and 0.14 fg/mL); a maximal AUC was based on LLOQs. The aluminum was above the LLOQ (61 ag/mL) in 31% of urine samples. From the urinary excretion data, a conservative estimated range for dermal Fabs of 0.002-0.06% was calculated, with a mean estimate of 0.0094%.

Arginine-vasopressin (AVP) is a physiological co-activator of the hypothalamus–pituitary–adrenal (HPA) axis, together with corticotrophin releasing hormone (CRH). A synthetic analogue of AVP, desmopressin (dDAVP), is often used as a pharmacological tool to assess co-activation in health and disease. The relation between dDAVP’s neuroendocrine, cardiovascular, pro-coagulatory, anti-diuretic and non-specific stress effects has not been studied. A randomized, double-blind, placebo-controlled, three-way crossover study was performed in 12 healthy male and female volunteers (6 : 6). dDAVP was administered intravenously as a 10 μg bolus (over 1 min) or a 30 μg incremental infusion (over 60 min). Neuroendocrine, cardiovascular, pro-coagulatory, anti-diuretic effects and adverse events (AEs) were recorded, and autonomic nervous system (ANS) activation evaluated. The incremental infusion reached 1.8-fold higher dDAVP concentrations than the bolus. Neuroendocrine effects were similar for the 10 μg dDAVP bolus and the 30 μg incremental infusion, while cardiovascular and coagulatory effects were greater with the 30 µg dose. Osmolality and ANS activity remained uninfluenced. AEs corresponded to dDAVP’s side-effect profile. In conclusion, the neuroendocrine effects of a 10 μg dDAVP bolus administered over 1 min are similar to those of a 30 μg incremental infusion administered over one hour, despite higher dDAVP concentrations after the infusion. Cardiovascular and coagulatory effects showed clear dose-related responses. A 10 μg dDAVP bolus is considered a safe vasopressinergic function test at which no confounding effects of systemic or autonomic stress were seen.

Background: Sugammadex, a modified γ-cyclodextrin, facilitates rapid reversal of rocuronium- and vecuronium-induced neuromuscular blockade (NMB). Cyclodextrins are known for their ability to form inclusion complexes with various drugs. Theoretically, molecules with a high affinity for sugammadex could interact and displace sugammadex from the sugammadex-rocuronium or sugammadex-vecuronium complex, potentially resulting in the recurrence of NMB due to recirculation of free rocuronium or vecuronium. Objective: This study aimed to evaluate whether the administration of high doses of flucloxacillin or diclofenac can result in recurrence of NMB through displacement of sugammadex from its complex with rocuronium or vecuronium, following successful reversal of NMB by a suboptimal dose of sugammadex 2 mg/kg. Flucloxacillin has previously been identified using a modelling approach as a drug with displacement potential, while diclofenac was assessed due to its common intravenous use in the peri-operative and post-surgery setting. Methods: This was a randomized, open-label, parallel, single-centre study conducted at SGS Life Services-CPU, Antwerp, Belgium. Twenty-four healthy, propofol-anaesthetized, adult volunteers were randomized to either rocuronium 0.6 mg/kg or vecuronium 0.1 mg/kg, followed by a suboptimal dose of sugammadex 2 mg/kg 15 minutes after induction of NMB. Five minutes after successful sugammadex reversal, subjects received either diclofenac 75 mg (15-minute infusion) or flucloxacillin 2 g (5-minute infusion) according to randomization. The suboptimal dose of sugammadex and relatively high doses of diclofenac and flucloxacillin were applied to create favourable conditions for the potential displacement of sugammadex from the sugammadex-rocuronium or sugammadex-vecuronium complex, and thus possible recurrence of NMB due to recirculation of free rocuronium or vecuronium. Possible recurrence of NMB was assessed by neuromuscular monitoring, performed with acceleromyography, and was continued until ∼90 minutes after the start of diclofenac or flucloxacillin administration. Recurrence of NMB was concluded if three consecutive train-of-four (TOF) ratios were

Free (0.6 mg), and liposome encapsulated chloroquine (0.6, 3 mg), were injected intraperitoneally, intramuscularly and subcutaneously in mice. Intraperitoneal administration of liposome-encapsulated chloroquine resulted in high and long lasting concentrations of chloroquine in the blood compared with intraperitoneal administration of free chloroquine. After administration of the liposome-encapsulated chloroquine the concentrations in the spleen were also higher, indicating that chloroquine liposomes reached the blood compartment intact after intraperitoneal administration. After intramuscular and subcutaneous administration the chloroquine liposomes acted as a local depot, giving a slower release from the subcutaneous fat layer than from the muscle depot. After the 0.6 mg dose a burst effect was found at about 7 h in most of the animals; this was not found after the 3 mg dose. This finding and the slower release after the 3 mg dose than after the 0.6 mg dose could be explained by the formation of aggregates after the injection.

To fully understand the potential and limitations of drug-laden liposomes in therapy, it is necessary to have information available on the behaviour of both drug and liposome in the body after administration. For two cytostatics (doxorubicin and cisplatin) the effect of liposome encapsulation on the therapeutic index was studied in tumor-bearing rats. Data were collected on the fate of liposome and drug in the rats. Analysis of these data provided a possible explanation for the observation that liposome encapsulation increased the therapeutic index for doxorubicin, but not for cisplatin. In the second part of this article the beneficial effect of liposome encapsulation of chloroquine (CQ) on the treatment and prophylaxis of murine malaria is demonstrated. This effect could be ascribed to sustained release of CQ from the intraperitoneally injected liposomes. Finally, examples of successful drug targeting with CQ containing immunoliposomes, or successive administration of antibodies and CQ containing liposomes, in a murine malaria model are presented.

In conventional drug targeting approaches attempts are made to accumulate a drug-carrier-homing device combination at the target site. Three important issues should be dealt with appropriately to make this approach successful: (1) access of the combination to the target site, (2) timing of the delivery of the drug and (3) retention of the drug at the target site (Tomlinson, 1987). Most target cells or tissues are localized in a fixed position in the body. The drug-carrier-homing device combination needs to gain access to the target sites, adhere to them and release its load, either inside or in the close proximity of the target. Unfortunately, accumulation of relatively large fractions of the drug at target sites outside the circulation (e.g. solid tumors) after intravenous administration has been found to be more the exception than the rule. This applies both to macromolecular carriers and to particulate carrier systems. Access to target sites present in the blood circulation (e.g. certain endothelial cells and macrophages, and blood clots) is (in principle) less restricted. Successful delivery of drugs to macrophages with particulate systems, such as liposomes laden with immunomodulating agents, antibiotics, antiparasitic or antiviral agents, has been described extensively in reviews (e.g. Emmen and Storm, 1987; Alving, 1988; Nassander et al., 1990; Storm et al., 1991). Huang and co-workers demonstrated that immunoliposomes bearing antibodies with the proper specificity for mouse lung endothelial cells indeed accumulate to a high degree in mouse lungs upon intravenous injection (Hughes et al., 1989).

The interaction between (immuno)liposomes and different (target and nontarget) cells was investigated in vitro. Maleimidophenylbutyrate-phosphatidylethanolamine (MPB-PE)-containing reverse-phase evaporation vesicles (REV-MPB-PE) were used; Fab' (polyclonal) fragments against mouse red blood cells (RBC) were selected as the homing device. Unwanted, nonspecific interactions were observed. these could be overcome by blocking the free reactive maleimide group of MPB-PE after Fab' coupling with dithiothreitol (DTT), or by storing the immunoliposomes for a period of 1 week before use. the specific interaction between immunoliposomes and target cells was maintained during storage. Storage of MPB-PE liposomes before Fab' coupling to REV-MBP-PE, however, reduced the coupling capacity considerably.

An overview of the literature concerning in vivo studies with immunoliposomes is presented. The design of immunoliposomes, their fate in vivo in the light of physiological constraints as well as potential applications are discussed. It appears that the therapeutic utility is restricted to a limited number of applications. General rules for a successful deployment of immunoliposomes are slowly emerging.

SUMMARYIn this study, the advantage of the use of chloroquine (CQ) containing liposomes (lipCQ) over free CQ in the chemotherapy of murine malaria (Plasmodium berghei) was demonstrated. The maximum permissible dose per intraperitoneal injection was 0·8 and 10 mg for CQ and lipCQ, respectively. An increase in therapeutic and prophylactic efficacy of lipCQ in comparison with free CQ at a 0·8 mg CQ dose level was found. It was possible to obtain 100% efficacy (injection at day 5 after infection; parasitaemia 4–8%) with one single intraperitoneal injection of 6 mg lipCQ. Moreover, the ability to increase the doses of CQ per injection after liposome encapsulation allowed successful treatment of infections with CQ-resistantPlasmodium bergheiwhich could not be cured by a 7-day course with the maximum tolerable dose of free CQ of 0·8 mg/mouse/day.

In a previous report (P. A. M. Peeters, C. W. E. M. Huiskamp, W. M. C. Eling, and D. J. A. Crommelin. Parasitology, 1989, in press) an increase in therapeutic and prophylactic potential was found when chloroquine (CQ) was encapsulated in fluid-state liposomes (lipCQ) and tested in Plasmodium berghei-infected mice in comparison to intraperitoneal (i.p.) administration of the free drug. In this study, the same model was used to demonstrate that encapsulation of CQ into gel-state liposomes further increased the preventive and therapeutic effect considerably. CQ determinations in whole blood, plasma, and red blood cells (RBC) after i.p. administration of fluid- or gel-state lipCQ revealed a prolonged availability of the drug in comparison to administration of free CQ. The CQ concentrations were related to the CQ levels needed for prevention or therapy of Plasmodium berghei infections in mice.

Immunoliposomes were made by covalently linking Fab' fragments (from rabbit antimouse erythrocyte IgG) to reverse-phase evaporation vesicles (REV) via maleimido-4-(p-phenylbutyrate) phosphatidylethanolamine (MPB-PE) as anchor molecule. These immunoliposomes were characterized in terms of size, charge, stability and antigen binding capacity. The effect of the liposomal Fab' density on the interaction with the target cell was studied. Two isolation methods were tested to separate free Fab' from liposomally bound Fab'. The necessity of deactivation of remaining reactive sites with dithiothreitol preincubation to increase the specificity of immunoliposome target cell interactions was demonstrated.

In this report a model to study the fate of target cells in the blood circulation after injection of appropriate immunoliposomes is discussed. The effect of intravenous administration of antimouse RBC immunoliposomes, F(ab′) 2 or IgG on the fate of intravenously injected 51 Cr-labelled mouse RBC (Cr-mRBC) in the mouse and, particularly, in the rat was studied. The immunoliposome was of the Fab′-MPBPE-REV type (Fab′-fragments covalently linked to reverse phase evaporation vesicles by maleimido-4-( p -phenylbutyrate)phosphatidylethanolamine). In the rat model a high blood level (80%) of the injected dose of target cells, Cr-mRBC, was maintained for several hours. The elimination by Fab′-liposomes, F(ab′) 2 or IgG of Cr-mRBC, and subsequent uptake into liver and spleen was dose dependent. Administration of Fab′-liposomes or F(ab′) 2 resulted in a preferential uptake into the spleen (above a certain dose also, but much lower, uptake into the liver was observed), while after IgG administration 51 Cr-label was mainly recovered in the liver. At equal protein doses (± 130 μg) Fab′-liposomes induced a faster elimination of the Cr-mRBC and a higher uptake into the spleen than F(ab′) 2 . The potential advantage of the use of drug-loaded immunoliposomes to eliminate target cells from the blood stream and to induce a certain pharmacological effect in the target cells, in comparison with the free antibody administration of F(ab′) 2 or IgG is discussed.

The potential therapeutic application of chloroquine-containing immunoliposomes (Fab′-lipCQ) in a Plasmodium berghei malaria model was studied. Extending a previously described in vivo model (Peeters et al. (1988) Biochim. Biophys. Acta 943, 137–147) it was demonstrated that injection of antimouse red blood cell (anti-mRBC) Fab′-lipCQ was significantly more effective than liposome-encapsulated chloroquine (lipCQ) or free chloroquine in delaying or preventing a patent infection after intravenous injection of parasitized mouse red blood cells (p-mRBC) in rats. The results could be improved by injecting synchronized infected cells instead of non-synchronous p-mRBC in order to minimize the presence of free parasites which could easily infect rat RBC. It was further demonstrated that sequential injection of anti-mRBC IgG and lipCQ or chloroquine resulted in complete inactivation of the injected parasitized cells while Fab′-lipCQ administration resulted in a maximum score of 50% at an equal chloroquine, protein and phospholipid dose. In this report the potential of the concept of drug targeting for the effective treatment of a disease, which manifests in blood cells, was demonstrated.