Cationic poly-L-lysine 3H-dendrimers with either 16 or 32 surface amine groups (BHALys [Lys]4 [3H-Lys]8 [NH2]16 and BHALys [Lys]8 [3H-Lys]16 [NH2]32, generation 3 and 4, respectively) have been synthesized and their pharmacokinetics and biodistribution investigated after intravenous administration to rats. The species in plasma with which radiolabel was associated was also investigated by size exclusion chromatography (SEC). Rapid initial removal of radiolabel from plasma was evident for both dendrimers (t(1/2) < 5 min). Approximately 1 h postdose, however, radiolabel reappeared in plasma in the form of free lysine and larger (but nondendrimer) species that coeluted with albumin by SEC. Plasma and whole blood pharmacokinetics were similar, precluding interaction with blood components as a causative factor in either the rapid removal or reappearance of radioactivity in plasma. Administration of monomeric 3H L-lysine also resulted in the appearance in plasma of a radiolabeled macromolecular species that coeluted with albumin by SEC, suggesting that biodegradation of the dendrimer to L-lysine and subsequent bioresorption may explain the pharmacokinetic profiles. Capping the Lys8 dendrimer with D-lysine to form BHALys [Lys]4 [3H-Lys]8 [D-Lys]16 [NH2]32 resulted in similar, and very rapid, initial disappearance kinetics from plasma when compared to the L-lysine capped dendrimer. Since significant extravasation of these large hydrophilic molecules seems unlikely, this most likely reflects both elimination and extensive binding to vascular surfaces. Capping with "non-natural" D-lysine also appeared to render the dendrimer essentially inert to the biodegradation process. For the L-lysine capped dendrimers, radiolabel was widely distributed throughout the major organs, with no apparent selectivity for organs of the reticuloendothelial system. In contrast, a greater proportion of the administered radiolabel was recovered in the organs of the reticuloendothelial system for the D-lysine capped system, as might be expected for a nondegrading circulating foreign colloid. To our knowledge this is the first data to demonstrate the biodegradation/bioresorption of poly-L-lysine dendrimers and has significant implications for the utility of these systems as drugs or drug delivery systems.

The impact of PEGylation on the pharmacokinetics and biodistribution of (3)H-labeled poly l-lysine dendrimers has been investigated after intravenous administration to rats. The volumes of distribution, clearance and consequently the plasma half-lives of the PEGylated dendrimers were markedly dependent on the total molecular weight of the PEGylated dendrimer, but were not specifically affected by the PEG chain length alone. In general, the larger dendrimer constructs (i.e. >30 kDa) had reduced volumes of distribution, were poorly renally cleared and exhibited extended elimination half-lives ( t 1/2 1-3 days) when compared to the smaller dendrimers (i.e. <20 kDa) which were rapidly cleared from the plasma principally into the urine ( t 1/2 1-10 h). At later time points the larger dendrimers concentrated in the organs of the reticuloendothelial system (liver and spleen); however, the absolute extent of accumulation was low. Size exclusion chromatography of plasma and urine samples revealed that the PEGylated dendrimers were considerably more resistant to biodegradation in vivo than the underivatized poly l-lysine dendrimer cores. The results suggest that the size of PEGylated poly l-lysine dendrimer complexes can be manipulated to optimally dictate their pharmacokinetics, biodegradation and bioresorption behavior.

BackgroundTopical microbicides, used by women to prevent the transmission of HIV and other sexually transmitted infections are urgently required. Dendrimers are highly branched nanoparticles being developed as microbicides. However, the anti-HIV and HSV structure-activity relationship of dendrimers comprising benzyhydryl amide cores and lysine branches, and a comprehensive analysis of their broad-spectrum anti-HIV activity and mechanism of action have not been published.Methods and FindingsDendrimers with optimized activity against HIV-1 and HSV-2 were identified with respect to the number of lysine branches (generations) and surface groups. Antiviral activity was determined in cell culture assays. Time-of-addition assays were performed to determine dendrimer mechanism of action. In vivo toxicity and HSV-2 inhibitory activity were evaluated in the mouse HSV-2 susceptibility model. Surface groups imparting the most potent inhibitory activity against HIV-1 and HSV-2 were naphthalene disulfonic acid (DNAA) and 3,5-disulfobenzoic acid exhibiting the greatest anionic charge and hydrophobicity of the seven surface groups tested. Their anti-HIV-1 activity did not appreciably increase beyond a second-generation dendrimer while dendrimers larger than two generations were required for potent anti-HSV-2 activity. Second (SPL7115) and fourth generation (SPL7013) DNAA dendrimers demonstrated broad-spectrum anti-HIV activity. However, SPL7013 was more active against HSV and blocking HIV-1 envelope mediated cell-to-cell fusion. SPL7013 and SPL7115 inhibited viral entry with similar potency against CXCR4-(X4) and CCR5-using (R5) HIV-1 strains. SPL7013 was not toxic and provided at least 12 h protection against HSV-2 in the mouse vagina.ConclusionsDendrimers can be engineered with optimized potency against HIV and HSV representing a unique platform for the controlled synthesis of chemically defined multivalent agents as viral entry inhibitors. SPL7013 is formulated as VivaGel® and is currently in clinical development to provide protection against HIV and HSV. SPL7013 could also be combined with other microbicides.

Circulating MPs can convert pCRP to pro-inflammatory mCRP in patients following MI, demonstrating for the first time mCRP generation in vivo and its detection in circulating blood. MPs can bind to cell membranes and transfer mCRP to the cell surface, suggesting a possible mCRP transport/delivery role of MPs in the circulation.

Dendrimers have potential for delivering chemotherapeutic drugs to solid tumors via the enhanced permeation and retention (EPR) effect. The impact of conjugation of hydrophobic anticancer drugs to hydrophilic PEGylated dendrimer surfaces, however, has not been fully investigated. The current study has therefore characterized the effect on dendrimer disposition of conjugating R-carboxyl protected methotrexate (MTX) to a series of PEGylated 3 H-labeled poly-L-lysine dendrimers ranging in size from generation 3 (G3) to 5 (G5) in rats. Dendrimers contained 50% surface PEG and 50% surface MTX. Conjugation of MTX generally increased plasma clearance when compared to conjugation with PEG alone. Conversely, increasing generation reduced clearance, increased metabolic stability and reduced renal elimination of the administered radiolabel. For constructs with molecular weights >20 kDa increasing the molecular weight of conjugated PEG also reduced clearance and enhanced metabolic stability but had only a minimal effect on renal elimination. Tissue distribution studies revealed retention of MTX conjugated smaller (G3-G4) PEG 570 dendrimers (or their metabolic products) in the kidneys. In contrast, the larger G5 dendrimer was concentrated more in the liver and spleen. The G5 PEG 1100 dendrimer was also shown to accumulate in solid Walker 256 and HT1080 tumors, and comparative disposition data in both rats (1 to 2% dose/g in tumor) and mice (11% dose/g in tumor) are presented. The results of this study further illustrate the potential utility of biodegradable PEGylated poly-L-lysine dendrimers as long-circulating vectors for the delivery and tumor-targeting of hydrophobic drugs.

Tritium-labeled poly- l-lysine dendrimers displaying 8 or 16 surface lysines have been capped with benzene sulfonate (BS), benzene disulfonate (BDS), or succinate (Succ) groups, and the intravenous pharmacokinetics and disposition profiles of the resulting dendrimers (Lys(8)(BS)(16), Lys(16)(BS)(32), Lys(16)(BDS)(32), Lys(16)(Succ)(32)) have been evaluated. Lys(16)(Succ)(32) was rapidly removed from the plasma primarily via renal elimination. Lys(16)(BS)(32) and Lys(16)(BDS)(32) were opsonized, resulting in more prolonged plasma elimination kinetics and increased uptake by the liver. Data obtained at higher doses suggested some evidence of nonlinear pharmacokinetics. Lys(8)(BS)(16) had reduced affinity for plasma proteins and was cleared more rapidly than the larger Lys(16)(BS)(32) or Lys(16)(BDS)(32) dendrimers. Lys(8)(BS)(16) and Lys(16)(BS)(32) were metabolized in vivo, resulting in the production of a low molecular weight species (possibly the cleavage product Lys(BS) (2)) that was extensively renally eliminated and accounted for almost all of the radioactivity recovered in urine ( approximately 20-45% of administered (3)H). In contrast, only 3-5% of the administered (3)H was recovered in the urine of rats administered Lys(16)(BDS)(32), suggesting increased resistance to in vivo degradation. The plasma clearance, distribution, and metabolic profiles of lysine dendrimers are therefore significantly influenced by the structure and charge of the capping groups. In particular, larger arylsulfonate-capped lysine dendrimers are rapidly opsonized and initially cleared from the plasma by the reticuloendothelial organs. The degree of metabolism is subsequently dictated by the nature of the surface capping group with BDS surfaces seemingly more resistant to breakdown. In contrast, smaller arylsulfonate-capped dendrimers are less readily opsonized and phagocytozed but are metabolically labile, and succinate-capped dendrimers are rapidly eliminated by the kidneys.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.Type 2 diabetes (T2D) is highly prevalent, with an estimated 370 million affected individuals worldwide, and this is predicted to double by 2030 1,2 . Despite the presence of several well-established drug classes for treating T2D, there is still a considerable unmet need for a drug that halts or reverses disease progression. The gp130 receptor cytokines IL-6 and CNTF modify food intake and body weight and improve insulin resistance in mice and humans [3][4][5][6] . Axokine, the human variant of CNTF, underwent human clinical trials for the treatment of amyotrophic lateral sclerosis, but the drug was repurposed to treat obesity and T2D 7-9 . After showing promise, the clinical development of Axokine was discontinued when some treated patients developed antibodies 9 , because of the fear that this could interfere with the neuroprotective action of endogenous CNTF. Although IL-6 protects against obesity and insulin resistance 10 , it is also pro-inflammatory owing-in part-to its 'trans-signalling' effects 11,12 , which limits its therapeutic utility.The gp130 cytokines signal by binding to the cytokine α-receptors, which are the IL-6 receptor (IL-6R) and CNTF receptor (CNTFR) for IL-6 and CNTF, respectively. Binding initiates the recruitment and dimerization of two transmembrane β-receptors: the IL-6-IL-6R complex binds two gp130 receptors as a homodimer, whereas the CNTF-CNTFR complex binds gp130 receptor and the LIF receptor (LIFR) as a heterodimer 13 . By transferring the LIFR-binding module from CNTF to IL-6, we engineered the chimeric protein IC7 14 , which predominantly consists of IL-6 residues, rendering it far less likely to induce an immune response compared with CNTF, because IL-6 circulates freely whereas CNTF is intracellular, and lacks a signal sequence peptide. Because IC7 is a chimaera with a unique sequence

Respiratory infections caused by human rhinovirus are responsible for severe exacerbations of underlying clinical conditions such as asthma in addition to their economic cost in terms of lost working days due to illness. While several antiviral compounds for treating rhinoviral infections have been discovered, none have succeeded, to date, in reaching approval for clinical use. We have developed a potent, orally available rhinovirus inhibitor 6 that has progressed through early clinical trials. The compound shows favorable pharmacokinetic and activity profiles and has a confirmed mechanism of action through crystallographic studies of a rhinovirus−compound complex. The compound has now progressed to phase IIb clinical studies of its effect on natural rhinovirus infection in humans.