Gregory Gregoriadis

... ATTEMPTS TO IMPROVE LOCALIZATION IN TUMOUR TISSUES Gregory Gregoriadis, Diane E. Neerunjun and Ruth Hunt Clinical Research Centre, Watford Road, Harrow, Middx., II.K. (Received in final form June 27 ... L. KRUPP, AV CHOBANIAN and PI BRECHER, Biochem. ...

Previously, we showed that CD206-targeted liposomal delivery of co-encapsulated immunodominant myelin basic protein (MBP) sequences MBP46-62, MBP124-139 and MBP147-170 (Xemys) suppressed experimental autoimmune encephalomyelitis in dark Agouti rats. The objective of this study was to assess the safety of Xemys in the treatment of patients with relapsing-remitting multiple sclerosis (MS) and secondary progressive MS, who failed to achieve a sustained response to first-line disease-modifying therapies. In this phase I, open-label, dose-escalating, proof-of-concept study, 20 patients with relapsing-remitting or secondary progressive MS received weekly subcutaneously injections with ascending doses of Xemys up to a total dose of 2.675 mg. Clinical examinations, including Expanded Disability Status Scale score, magnetic resonance imaging results, and serum cytokine concentrations, were assessed before the first injection and for up to 17 weeks after the final injection. Xemys was safe an...

Haloperidol (Hal), a highly hydrophobic drug, was complexed with two β-cyclodextrin (β-CD) derivatives. Hal solubility was increased 20-fold in the presence of a 10-fold excess of methyl β-CD (Meβ-CD) and 12-fold in the presence of a 10-fold excess of 2-hydroxypropyl β-CD (HPβ-CD). The stoichiometries and stability constants of Hal–Meβ-CD (1:1 and 2345 M−1 at 27°C) and Hal–HPβ-CD (1:1 and 2112 M−1 at 27°C) complexes were calculated by the continuous variation and phase solubility methods respectively. Differential scanning calorimetry and 1H-NMR were used to confirm the formation of inclusion complexes. Moreover, the enthalpy and entropy of the complexation process were calculated for both complexes in order to obtain such information as the main `driving force' and whether or not complex formation is thermodynamically favoured. This was achieved by monitoring the isothermic solubility lines at various temperatures.

Human blood lymphocytes were coated with increasing amounts of human kappa chain (2-85mug/10(7) cells) through the linking reagent CrCl(3). These cells were then exposed to small unilamellar liposomes composed of egg phosphatidylcholine, cholesterol and phosphatidic acid (molar proportions 7:7:1) containing carboxyfluorescein and/or (111)In-labelled bleomycin and bearing (131)I-labelled affinity chromatography-purified or non-purified anti-(kappa-chain) immunoglobulin G (IgG) [see the preceding paper, Gregoriadis, Meehan & Mah (1981) Biochem. J.200, 203-210]. In some experiments liposomes contained [(14)C]phosphatidylcholine. (1) Lymphocytes (10(7)) coated with 2-85mug of kappa chain and exposed to liposomes devoid of IgG or bearing non-purified anti-(kappa chain) IgG bound only a small proportion of the liposomal markers. Even with liposomes bearing the purified anti-(kappa chain) IgG, uptake of the labels improved only slightly for cells coated with up to 10mug of kappa chain. However, with higher concentrations of the antigen on the cell surface, binding was improved considerably to reach values of 31% ((111)In-labelled bleomycin) and 43% ((131)I-labelled IgG) of added liposomes for cells coated with 85mug of kappa chain. (2) Lymphocytes coated with kappa chain were exposed to liposomes bearing increasing amounts (0-180mug/0.9mg of egg phosphatidylcholine) of purified anti-(kappa chain) IgG. It was found that under the present conditions, binding of all three markers ((111)In-labelled bleomycin, (131)I-labelled IgG and [(14)C]phosphatidylcholine) was directly proportional to the concentration of IgG on the liposomal surface. However, uptake values remained unchanged above 90mug of IgG. (3) Antibody-mediated uptake of liposomes by cells coated with the corresponding antigen without loss of their metabolic activities may provide a method of efficient targeting.

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination 'co-delivery' and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell--a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.

In experiments designed to study the co-adjuvant action of interleukin-2, a model antigen (tetanus toxoid) was passively entrapped in, or covalently coupled to multilamellar liposomes in the presence or absence of interleukin-2 (IL-2). When present, IL-2 was either co-entrapped with the toxoid, entrapped alone in liposomes with toxoid coupled to their surface, or coupled to the surface of liposomes with entrapped toxoid. The role of spatial localization of IL-2 within the liposomal structure (vis a vis that of the toxoid) was studied in terms of its immunoadjuvant action in vivo. Male CD-1 mice were injected intramuscularly twice with a variety of toxoid-containing liposomal preparations in the absence or presence of IL-2 incorporated in the same liposomes as above. In some experiments mice were immunized with liposomal toxoid mixed with separately entrapped IL-2. Results show that IL-2 augments significantly secondary immune responses (IgG1, IgGa, IgG2b subclasses) against the liposomal toxoid (up to 15-fold compared with the liposomal toxoid alone), regardless of cytokine and antigen mode of accommodation in the liposomal structure but only when both are present in the same vesicles. It is suggested that liposomal IL-2 may prove useful as a co-adjuvant for vaccines which are weak or ineffective.

Tetanus toxoid was incorporated into liposomes composed of equimolar phospholipid and cholesterol. The toxoid was either passively entrapped into multilamellar vesicles prepared by the dehydration-rehydration procedure (DRV) or covalently coupled by diazotization to the surface of multilamellar vesicles (MLV) prepared by the classical procedure. Up to 82.3% of the antigen used was entrapped in neutral, negatively and positively charged DRV composed of a variety of unsaturated and saturated phospholipids and 63.1% was coupled to MLV composed of egg phosphatidylcholine. After freeze-drying of toxoid-incorporating DRV and MLV and subsequent rehydration, up to 93.5% of the antigen was recovered with liposomes and, in the case of MLV, retained its external localization. Upon freeze-drying in the presence of 0.25 M trehalose, up to 96.1% of the antigen was recovered with the DRV liposomes. In immunization studies using Balb/c mice, DRV composed of equimolar egg phosphatidylcholine and cholesterol were shown to act as immunological adjuvants to the entrapped tetanus toxoid. In addition, there was no difference in immune responses between DRV and MLV of identical composition but bearing the toxoid on their surface. Comparison of immune responses to the toxoid entrapped in DRV made of phospholipids with varying gel to liquid crystalline transition temperature (Tc) revealed a reduction in responses to very low or nil values for DRV made of distearoyl phosphatidylcholine (Tc 54 degrees C).

The non-linear least-squares model for calculation of the stability constant (Kst) of a drug-cyclodextrin complex has been used in fluorimetry studies. Complexation of riboflavin with beta-cyclodextrin (beta-CyD) was monitored fluorimetrically by measuring changes in the fluorescence intensity of the vitamin in the presence of various amounts of beta-CyD. Formation of an inclusion complex was confirmed in the solid state by differential scanning calorimetry (DSC) and in aqueous solution by proton nuclear magnetic resonance spectroscopy (1H NMR). The experimental Kst value (2112 M-1) derived from the fluorimetry studies appeared to fit well to a 1:1 drug-to-cyclodextrin molar ratio according to the non-linear mathematical model. The model is particularly suitable for fluorescent compounds of which fluorescence intensity is influenced by the presence of cyclodextrins.

Abstract Yeast β-fructofuranosidase (invertase) or 131 I-labelled albumin were entrapped into liposomes composed of phosphatidylcholine, cholesterol and phosphatidic acid. Of the β-fructofuranosidase activity in the liposomal preparations 96–100% was latent. The ...

Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration-rehydration method into Lipodine liposomes composed of 16 micro moles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 micro moles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 micro moles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1 : 0.5 : 0.25). Incorporation efficiency was high (89-93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration-rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV-DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 micro g liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 micro g of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine) may be a useful system for the oral delivery of DNA vaccines.

A number of SH-containing proteins or protein derivatives were coupled to small unilamellar liposomes. These were composed of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol (1:1, phospholipid/cholesterol molar ratio) and activated (DPPE moiety) with the heterobifunctional reagents N-hydroxysuccinimide ester of iodoacetic acid (hydroxysuccinimide iodoacetate), N-succinimidyl-4-(2-bromoacetylamino)benzoate (SBAB) or N-succinimidyl-3-(2-pyridyldithio)proprionate (SPDP). DPPE was activated with the reagents before or after its incorporation into liposomes. Protein coupling values varied widely depending on the reagent and the protein used, but were highest in the case of SPDP-activated liposomes and SPDP-modified immunoglobulin G (IgG). Monoclonal anti-Thy1 125I-IgG1-bearing liposomes (SPDP- or SBAB-activated) containing quenched carboxyfluorescein were incubated under a variety of conditions with mouse AKR-A cells expressing the cross-reactive Thy 1.1 antigen. The following observations were made; (a) binding of intact liposomes to the cells at 4 degrees C reached plateau values after about 1 h with at least 70% of the liposomes used being capable of associating with the target cells; (b) binding of liposomes to AKR-A cells was much more pronounced than when using another cell line (EL4-Tc); (c) binding to AKR-A cells could be effected with as little as 1.3 molecules (average) of IgG1 per vesicle; (d) binding was inhibited only modestly by the presence of 50% mouse plasma; (e) stability of IgG1-bearing liposomes in terms of entrapped solute and IgG1 retention in the presence of plasma at 37 degrees C was maintained quantitatively for at least 5.5 h, and by 24 h, 54% of the IgG1 was still associated with the liposomes. AKR mice were injected intravenously with 99mTc-labelled AKR-A cells and 2.5 min later with anti-Thy1 125I-IgG1-bearing liposomes containing quenched carboxyfluorescein and 111In-Ca-DTPA or with similar liposomes devoid of IgG1. In parallel experiments, AKR mice received either of the liposome preparations without previous injection of cells. On the basis of patterns of quenched carboxyfluorescein, 111In and 125I-clearance from the circulation, of 99mTc levels in the blood and of values of 111In in the liver and spleen, it appeared that IgG1-bearing liposomes were capable of binding to their target cells in the vasculature. Such binding accelerated the clearance of interacting moieties (i.e., AKR-A cells and liposomes). The present results suggest that targeting of liposomes to circulating in vivo is feasible.

ABSTRACT Abstract A procedure is reported for the high-yield entrapment of a wide variety of solutes in small liposomes. The procedure entails mixing of preformed “empty” (water-filled) small unilamellar vesicles (SLFV) (60-80 nm diameter) with sucrose and the solute destined for entrapment. Using appropriate sugar to lipid mass ratios (eg. 1-5), dehydration of the mixture and subsequent controlled rehydration leads to the formation of vesicles (90-200 nm diameter) entrapping considerable proportions (up to 87%) of the solute. Results with solutes as diverse as penicillin, riboflavin, doxorubicin, desoxyfructo-seroto-nin, and epidermal growth factor suggest that entrapment values depend on the sugar to lipid mass ratio and the identity of the solute but not on the liquid crystalline transition temperature of the phospholipid component of the SUV.

... 11035 Characterization and Photoprotection Studies of a Model y-Cyclodextrin-Included Photolabile Drug Entrapped in Liposomes Incorporating Light Absorbers Yannis L. Loukas? ... (Poole, Dorset, UK). Deuterium oxide (99.9%) was from Fluka (Poole, Dorset, UK). ...

BackgroundThe need for lifelong, daily insulin injections can have a dramatic effect on patient compliance, can be painful, and runs the risk of local infections. Furthermore, needle-stick injuries are common, and the issue of needle disposal is troublesome. Injecting a long-acting insulin analog with needle-free administration would be a significant improvement for diabetic subjects, but is not currently feasible. To achieve a constant, reliable delivery of a novel, long-acting insulin analog, Lipoxen's SuliXen® (polysialylated insulin) in a solid dosage form capable of being delivered without a needle has been developed. The aim of this study was to evaluate the feasibility of Lipoxen's SuliXen delivery with the Glide solid dose injector, Glide SDI®.Materials and MethodsA formulation containing 14 kDa polysialic acid (PSA)-recombinant human insulin conjugate was manufactured at Lipoxen PLC and transferred to Glide Pharma. The PSA–insulin conjugate solution was incorporated into different excipients at Glide Pharma (excipients 1 and 2), and formulations were manufactured containing implants with doses of 0.3 and 1.0 IU of insulin, respectively. Two different polymeric excipients were investigated for their suitable release profiles. The physicomechanical properties of the formulations were characterized in terms of solid dosage form strength (via three-point bend and compression) and disintegration time at 37°C. A preclinical efficacy study was performed in a nondiabetic rat model (Sprague-Dawley).ResultsThe study demonstrated successful incorporation of PSA–insulin conjugate into formulations compatible for use with the solid dose injector. Physicochemical characterization indicated that each formulation produced was physically robust. For excipient 1, the compressive stress and three-point-bend-test values recorded for the 0.3 IU formulation were 106.99 ± 14.3 MPa and 30.6 ± 1.4 N (force in newtons), respectively. Corresponding values for the 1.0 IU dose were 53.10 ± 10.2 MPa and 16.66 ± 1.0 N. For excipient 2, the compressive stress and three-point-bend-test values recorded for the 0.3 IU dose were 53.10 ± 10.2 MPa and 7.64 ± 0.9 N, respectively, whereas the corresponding values recorded for the 1.0 IU dose were 41.61 ± 7.4 MPa and 13.18 ± 1.3 N. Each formulation successfully penetrated a laboratory substrate, achieving 100% penetration in each case. In vivo analysis demonstrated that PSA–insulin conjugate shows prolongation of activity (at least two-fold more compared to insulin) for more than 5 hours in the rat model.ConclusionEven though additional work may be required, for example, to develop several fixed dose formulations, the preliminary results show that solid dosage forms incorporating PSA–insulin conjugate maintained the prolongation of PSA–insulin conjugate activity in the rat model. Convenient and easy to use, the solid dose injector will not only ensure diabetic patient compliance and trust but also provide cost-effective solutions for safe, reliable, and controlled needle-free injection of PSA–insulin conjugate.