Liposome: Encapsula's Scientific Blog

Measuring the size of the liposomes

2012-02-06T12:37:00.005-06:00

Methods for measuring the size of the liposomes may vary in complexity. The most precise method for determining the size of the liposomes is electron microscopy. Each individual liposomes can be viewed by electron microscope and with enough patience, time and skills to avoid numerous artifacts, one can obtain very accurate information about the profile of a liposome population over the whole range of sizes. The main problem is that the technique can be very time consuming (at least 400 liposomes should be counted for each batch of liposome) In addition to that electron microscopes are quite expensive and can only be operated by a highly trained microscopist.

Laser light scattering analysis is fairly rapid and simple to perform, however it measures an averaged property of the bulk of the liposomes. Laser light scattering analysis will only provide useful information on the size distribution for liposomes up to 1 um. The size of the liposome particles that are larger than one micron can be determined by a Coulter counter that uses laser diffraction (e.g. Mastersizer) or uses obstruction techniques (e.g Accusizer).

In the next blog, the principle of liposome size measurement by photon correlation spectroscopy will be explained in detail.

Depletion of alveolar macrophages by Clodrosome® (Clodronate encapsulated liposome)

2011-12-17T00:12:00.002-06:00

Lung macrophages play a significant role in the biology of lung. They release over one hundred known secretory products such as cytokines, arachidonic acid metabolites and oxygen radicals. Lung macrophages are involved in diverse function such as the maintenance of lung sterility, immune modulation, identification and killing of cancer cells and prevention of allergy. Lung macrophages may also contribute to lung injury. As an example, cigarette smoking stimulates the generation of oxygen radicals by macrophages. Lung macrophages function as mobile unicellular endocrine and paracrine that are strategically located to monitor and modify the micro-environment of the lung.

Usually the term "lung macrophage" and "alveolar macrophage (AM)" are used synonymously, however this is not completely accurate. Pleural macrophages are also another sub-population of lung macrophages. Alveolar macrophages obtain energy predominantly by aerobic metabolism, since they reside in alveoli where the concentration of oxygen is high. Pleural macrophages, on the other hand, rely on glycolytic pathway to replenish their energy stores, because the concentration of oxygen in the pleural space is relatively low.

One way to investigate the in vivo function of a particular cell type is to deplete these cells in the laboratory animals such as mice and rats and to note alterations in response to experimental manipulations.

In this blog entry, we discuss the use of tracheal insufflation of liposome encapsulated clodronate to selectively deplete alveolar macrophages in laboratory animals. Clodronate liposomes target phagocytic macrophages, while minimizing exposure and possible injury to other cells that do not take up liposomes. Many papers have reported a significant depletion of alveolar macrophages in mice and rats lasting for more than 5 days after a single insufflation of clodronate liposomes. These alveolar macrophage depleted animals showed a markedly reduced ability to recruit neutrophils and to release tumor necrosis factor (TNF) into the alveolar space on endotoxin challenge. Insufflation of free and non-encapsulated clodronate drug also causes alveolar macrophage depletion. However free clodronate-induced alveolar macrophage depletion was not specific since ultra-structural studies revealed that it is also caused damage to the alveolar epithelial cells. Thus, encapsulation of clodronate into the liposomes is necessary to specifically target clodronate to macrophages.

The fact that clodronate causes lysis of alveolar macrophages buy only cytoplasmic edema of alveolar epithelial cells suggests that clodronate is preferentially more toxic to macrophages. Administration of clodronate liposomes leads to ingestion of liposomes by macrophages, which are then destroyed after phospholipase-mediated disruption of the liposomal membranes and release of encapsulated clodronate. Previous studies have shown that intravenous injection of free and non-encapsulated clodronate do not deplete hepatic and splenic macrophages. However, it has been observed that tracheal insufflation of free and non-encapsulated clodronate also depleted alveolar macrophages. The reason for this discrepancy is due to rapid clearance of clodronate which has a plasma half-life of a few minutes after intravenous injection. Because of the tight alveolar epithelial barrier the clearance of free and non-encapsulated clodronate from the alveolar space after tracheal insufflation is expected to be slow. As a result, alveolar macrophages are exposed to free and non-encapsulated clodronate for a much more longer period of time. To determine whether depletion of alveolar macrophages was depend on encapsulation of clodronate in liposomes, the effects of plain PBS liposomes and free and non-encapsulated clodronate on tracheal insufflation was studied by Breg et al (Journal of Applied Physiology, June 1993, vol. 74, no. 62812-2819). The study showed that insufflation of plain PBS liposomes (80 ul) had no effect on the number of rat alveolar macrophages, whereas insufflation of free and non-encapsulated clodronate at the dosage equivalent to the amount present in 80 ul of clodronate liposome caused a similar degree of alveolar macrophage depletion and PMN influx into the alveolar space as clodronate liposomes did.

Many studies have suggested that maximum depletion of alveolar macrophages occurred 3 days after tracheal insufflation of clodronate liposome. A single insufflation of clodronate liposome caused a dosed dependent reduction of alveolar macrophages reaching a maximal depletion of >70%at a dose of 120 ul (5 mg/ml encapsulated clodronate) of clodronate liposomes. A dose dependent increase in the number of PMNs also occurred. A time course study using 80 ul and 120 ul of clodronate liposomes shows that a significant depletion of alveolar macrophages occurred 1 day after insufflation and lasted for 7 days. However, by day 9, the number of alveolar macrophages was almost back to the baseline level. 120 ul of clodronate liposomes resulted in a higher PMN influx than did 80 ul of clodronate liposomes. PMN influx is not desirable and therefore the best dosage is the one that depletes the macrophages and minimizes the influx of PMN (See figure 1).

Figure 1- Effect of tracheal insufflation of clodronate liposomes on alveolar macrophages and neutrophils on rats (data was reproduced from the study done by Berg et al published in Journal of Applied Physiology, June 1993, vol. 74, no. 62812-2819)

How does Clodrosome® (Clodronate encapsulated liposome) kill macrophages?

2011-12-16T23:57:00.002-06:00

Mechanism of action

After Clodrosome®has been dosed to the animal by the chosen route, the clodronate liposomes will come into contact with macrophages and other phagocytic cells. The phagocyte immediately recognizes the liposomes as foreign particles and proceeds with destroying these invading particles. The first step in this destruction is phagocytosis in which the liposomes are engulfed by the cell into an internal vesicle known as a phagosome as shown in the figure below. Lysosomes, which contain many types of destructive enzymes, including phospholipases, fuse with the phagosome forming a phagolysosome. The lysosomal membrane also contains proton pumps which will lower the internal pH of the phagolysosome. The low pH, phospholipases and other macromolecular interactions all contribute to compromising the liposomal membrane thus releasing the encapsulated clodronate. The low internal pH of the phagolysosome may contribute to the ability of the clodronate to cross the phagolysosomal membrane into the macrophage’s cytosol.

Once in the cytosolic medium, clodronate is mistakenly recognized as cellular pyrophosphate and used by several Class II aminoacyl-tRNA synthetases to produce a non-hydrolyzable ATP analog, adenosine 5?-(β, γ-dichloromethylene) triphosphate (AppCCl₂p)^1–3. The exact mechanism by which AppCCl₂p causes cell death remained elusive for some time until Lehenkari and coworkers generated data which supported the hypothesis described in the lower figures. Basically, their hypothesis involves cytosolic AppCCl₂p crossing the mitochondrial outer membrane and irreversibly binding to the ATP/ADP translocase which transverses the mitochondrial inner membrane. Inhibiting this enzyme initiates pore openings in the mitochondrial inner membrane. Loss of mitochondrial inner membrane integrity, in turn, results in depolarization and allows molecular signals to be released from the mitochondrion which initiate cell death via apoptosis⁴.

Note that the ability to internalize clodronate from the external medium is unique to osteoclasts, therefore free clodronate is toxic only to these cells in the bone when dosed to animals. The only proven effective method for delivering clodronate intracellularly to other cell types in vivo is via liposomes as described above.

Figure 1- Phagocytosis and degradation of Clodronate liposomes

Figure 2-In a functional mitochondrion, the TCA cycle generates high-energy protons in its matrix which are pumped into the inner membrane space along the electron transport chain (requires cytochrome C as a cofactor). The resulting proton gradient provides energy for the f₁f₀ ATP synthase to generate ATP from ADP and P_i. ATP is transported to the cytosol via ATP/ADP translocase. The translocase only functions when both ATP and ADP are bound to the enzyme, therefore one ADP is removed from the cytosolic ADP pool for each ATP leaving the mitochondrial matrix.

Figure 3- However, when AppCCl₂p diffuses from the cytosol into the inner membrane space, it binds to an ATP-binding site on the ATP/ADP translocase inhibiting the transport activity. This inhibition initiates a cascade which eventually opens the PT pore depleting the electrochemical and proton gradients as well as releasing mitochondrial proapoptotic factors, such as cyt C, into the cytosol. See reference 4 for further detail.

References:

1. Rogers, M.J., Russell, R.G.G., Blackburn, G.M., Williamson, M.P. & Watts, D.J. Metabolism of halogenated bisphosphonates by the cellular slime mould dictyostelium discoideum. Biochemical and Biophysical Research Communications 189, 414-423 (1992).
2. Rogers, M.J. et al. Inhibitory effects of bisphosphonates on growth of amoebae of the cellular slime mold dictyostelium discoideum. Journal of Bone and Mineral Research 9, 1029-1039 (2009).
3. Frith, J.C., Mönkkönen, J., Blackburn, G.M., Russell, R.G.G. & Rogers, M.J. Clodronate and Liposome-Encapsulated Clodronate Are Metabolized to a Toxic ATP Analog, Adenosine 5′-(β,γ-Dichloromethylene) Triphosphate, by Mammalian Cells In Vitro. Journal of Bone and Mineral Research 12, 1358-1367 (1997).
4. Lehenkari, P.P. et al. Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite. Mol. Pharmacol. 61, 1255-1262 (2002).

Cytotoxicity of Bisphosphonates such as Clodronate, Pamidronate and Zelodronate

2011-12-15T10:32:00.016-06:00

Many experiments have shown that the bisphosphonates ,such as clodronate, pamidronate and zoledronate have cytotoxic effect on endothelial as well as on human and mouse tumor cells, all representing component of the tumor stroma. The IC 50 values of all bisphosphonates are in the mM range. Clodronate shows the lowest efficiency in all cases. The nitrogen containing bisphosphonates such as pamidronate and zoledronate have higher activity compare to non-nitrogen containing bisphosphonates such as clodronate. Human Umbilical Vein Endothelial Cells (HUVEC) have shown the highest sensibility for all bisphosphonates tested, especially for zoledronate the cytotoxic activity is twice as high as for tumor cells. The enhanced cytotoxicity of zoledronate on HUVECs compared to other bisphosphonates has already been reported by several groups. This is due to the difference in mechanism of action of nitrogen containing bisphosphonates (e.g. zelodronate and pamidronate) compare to non-nitrogen containing bisphosphonate such as Clodronate.

Both groups of bisphosphonates (non-nitrogen containing and nitrogen containing) inhibit bone resorption by induction of osteoclast apoptosis. However, at the molecular level the mechanism of action of these molecules differ. Non-nitrogen containing bisphosphonates such as Clodronate are metabolically incorporated into analogues of ATP that are resistant to hydrolysis by ATP-dependent metabolic enzymes. On the other hand, nitrogen containing bisphosphonates such as pamidronate and zoledronate are inhibitors of the mevalonate pathway and thereby they prevent prenylation of small GTPase signaling protein required for osteoclast function.

The table below summarizes the toxicity of free and encapsulated bisphosphonates that were reported in various scientific papers.

Table 1- IC50 values (after 4 hours of incubation) of free and liposomal encapsulated clodronate, pamidronate and zoledronate in human rhabdomyosarcoma (A673), murine teratocarcinoma cells (F9) and human umbilical vein endothelial cells (HUVECs) and non activated and activated mouse peritonial macrophages. (Source: Comparison of cytotoxic properties of free and liposomal bisphosphonates in vitro, Renate Frei, Thesis, Swiss Federal Institute of Technology Zurich, 2005)

** The IC50 unit is mM.

The Future of Nano-Medicine

2011-12-03T11:31:00.004-06:00

See a larger view of the image here:

http://the-scientist.com/wordpress/wp-content/uploads/2011/10/10_11_Swallowing-the-surgeon.jpg

Read the whole story here:

http://the-scientist.com/2011/10/01/a-small-revolution/

Lipid movements in liposomes

2011-10-20T14:29:00.001-05:00

Liposomes are dynamic systems. The lipids in the liposome bilayers move in various ways.

Lateral diffusion refers to the lateral movement of lipids in the membrane. Lipids are generally free to move laterally if they are not restricted by certain interactions. Lateral diffusion is a fairly quick and spontaneous process. The first part of the animation shows the lateral movement of a red color lipid.

Lipid rotation around its axis: The whole lipid molecule is free to rotate around its vertical axis. This motion is slower: it takes few microseconds to complete one rotation.

Lipid tail wagging: The non-polar tails undergo a "wagging" motion due to the rotation around the C-C single bonds. These motions are rapid (several times in a nanosecond) because the barrier for internal rotation around the C-C bond is low. However, the configuration around the cisdouble bond remains unchanged.

Flip-Flop from one half of the bilayer to the other half of the bilayer is normally very slow. Flip-flop would require the polar head-group of a lipid to traverse the hydrophobic core of the membrane. The last part of the animation shows the flip-flop of a red color lipid from one layer to another.

Doxorubicin Liposomes: A journey through nano-particle engineering

2011-10-06T20:42:00.002-05:00

The following presentation is for educational purposes only. Graduate students and university professors are allowed to use the material however they should mention Encapsula NanoSciences as the source of the material.

Direct link to the slide view:

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You need to click on the mouse to change the slides.

Immunoliposomes

2011-09-29T17:06:00.001-05:00

Immunoliposomes are generated by conjugating antibodies either directly to lipid bilayer of liposomes in presence or absence of PEG chains (type I immunoliposomes) or to the distal end of the PEG chain (type II immunoliposomes).

Conjugation of antibodies directly to the lipid bilayer of a PEG containing liposomes (type I immunoliposomes) can result in reduced or even diminishes antigene bonding, depending on the amount of incorporated PEG and the length of the PEG chains. However antigen binding properties of immunoliposomes can be restored by conjugating the antibody to the terminus of the PEG chain and therefore most of the recently developed immunoliposomes are based on type II immunoliposomes.

Target cell recognition by immunoliposomes is influenced by two factors:

1) The type of the antibody molecule (ie, whole antibody or antibody fragments)
2) The chemistry of conjugation (eg, random coupling vs directional coupling)

It has been extensively shown that whole antibodies coupled to liposomes are highly immunogenic. These liposomes are rapidly eliminated through Fc-mediated phagocytosis by macrophages of the liver and spleen, and also by tumor localized macrophages.

Random coupling methods (eg, using thiolated antibody coupled to maleimide PEG lipids or using modified amino reactive PEG lipids) risk antibody inactivation and liposome aggregation by cross linking. The disadvantages of using whole antibody can be circumvented by the use of antibody fragments such as fragment antigen binding (Fab') or single chain fragment variable (scFv).

Fab' fragments

Fab' fragments are generated by pepsin digestion of IgG molecules and subsequent mild reduction. Fab' fragments can also be produced in recombinant form by being expressed in prokaryotic systems. Fab' fragments have an average molecular weight of 50 kDa and expose one or several free sulfhydryl groups, depending on the method of production. Fab' type II immunoliposomes have reduced immunogenicity compared to IgG type II immunoliposomes. IgG type II immunoliposomes are cleared faster than Fab' type II immunoliposomes. A study by Pastorino et al published in Cancer Res (2003) 63 (1):86-92 shows that Fab' type II immunoliposomes have approximately two fold reduced immunogenicity compared with IgG type II immunoliposomes. The rate of elimination was three fold faster for IgG type II immunoliposomes compared with Fab' type II immunoliposomes.

scFv fragments

scFv fragments are the smallest fragments to contain the entire antigen binding site of an antibody. They are formed by connecting the variable heavy and light chain domains with a short peptide linker with 15-20 aminoacids. scFv molecules have an average molecular weight of 25 kDa and can be produced in bacteria.

In order to conjugate scFv fragments to liposomes, one or more additional cysteine residues are attached to the C terminus of scFv fragments. This allows for site-directed conjugation with the reactive sulfhadryl groups located opposite the antigen binding sites and therefore similar to conjugation of Fab' fragments, conjugation of scFv' fragments does not interfere with target cell recognition.

Expression of scFv' fragments in bacteria normally results in a mixture of monomeric and dimeric molecules. The dimeric molecules are the oxidation products of two monomeric molecules. In order to achieve efficient coupling, scFv' preparations have to be reduced under mild conditions prior to conjugation.

Coupling of antibodies to preformed liposomes

In direct coupling method the liposomes containing reactive groups are formed, and then antibodies and other chemicals are added in order to achieve conjugation. However, direct coupling can result in lower coupling efficiencies. Coupling efficiencies of 20-80 percent was reported when scFv' molecules were directly conjugated to Mal-PEG liposomes.

Post insertion method

In post insertion method antibodies are first conjugated to Mal-PEG micelles. A study by Nielsen et al. published in Biochim Biophys Acta (2002) 159 (1-3): 109-118 has shown coupling efficiencies up to 95% and preserving more than 80% of immunoreactivity using this method.

Factors influencing therapeutic efficacy

Drug targeting with immunoliposomes is highly complex and influenced by various parameters and both the target site and the liposome.

The targeting efficiency of immunoliposomes is influenced by antigen density on the target cells. A high density of target antigens increases drug delivery and anti-tumor activity. However, antigen density on target cells is often low.

Drug loaded type II immunoliposomes containing antibodies that target an internalized antigen such as CD19 showed much more potency compared to immunoliposomes containing antibodies that target a non internalized antigen such as CD20. This study shows that internalization of immunoliposomes is a prerequisite for the induction of efficient cytotoxicity.

Therapeutic efficacy of immunoliposomes similar to regular liposomes is also dependent on the rate of release of drug and the lipid composition of the liposomes.

Unfortunately, the major problem with immunoliposomes is their poor extravasation properties. Extravasation is antibody independent and it is the rate limiting step in the tumor cell targeting of solid tumors. This will restrict the applications of immunoliposomes to those cancers in which tumor cells are readily accessible such as hematological malignancies or minimal residual diseases.

To circumvent the problems associated with poor extravasation of immunoliposomes new approaches are developed to combine immunoliposomes with vascular targeting. In this approach liposomes are targeted to cells associated with neovascularization in tumor tissue. This is a clever strategy because all solid tumors depend on neovascularization in order to grow and also tumor blood vessels are easily accessible for immunoliposomes. Endothelial cells are genetically stable and should not become resistant to immunoliposomes therapy. Several antibodies and antibody fragments recognize antigens associated with endothelial cell activation and proliferation. These antigens include E-selectin, vascular endothelial growth factor receptor-2, the fibronectin splice variant ED-B, endoglin (CD105), vascular cell adhesion molecule-1 and tumor endothelial marker 1. In vivo study of anti-ED-B scFv immunoliposomes showed 62-90% reduction of tumor growth in F9 teratocarcinoma bearing mice in comparison to animals treated with untargeted control liposomes.

This questions was also answered on Quora:

http://www.quora.com/What-are-immunoliposomes

Lipid Shape Theory and pH sensitive liposomes

2011-09-28T19:59:00.000-05:00

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Immunoliposomes: Different strategies for conjugating antibodies to liposomes

2011-09-28T15:17:00.001-05:00

Over the past two decades various techniques have been developed for conjugation of antibodies and peptides to the surface of liposomes. In this article we will be briefly describe some of these methods.

Methods that involve using a modified antibody

1) Attachment of an antibody to liposomes using a modified antibody (biotinylated antibody) and biotinylated DOPE lipid through avidin-biotin reaction

2) Attachment of an antibody to a liposome using a modified antibody (through an oxidized sugar on the Fc region) and Hydrazide-PEG-PE lipid

3) Attachment of an antibody to a liposome using either a thiolated antibody or a reduced antibody (Fab') using a MPB-PE lipid

4) Attachment of an MPB modified antbody to the liposomes using PDP-PE (DOPE or DSPE) lipid

5) Attachment of an antibody to a liposome using either a thiolated antibody or a reduced antibody (Fab') using liposomes containing NHSIA attached to DOPE

6) Attachment of an antibody to a liposome using either a thiolated antibody or a reduced antibody (Fab') using liposomes containing reactive lipid maleimide

7) Attachment of an antibody to a liposome using either a thiolated antibody or a reduced antibody (Fab') using MPB-PE liposomes

8) Attachment of modified liposomes (by NHSIA) to modified antibodies (by SATA)

9) Attachment of an antibody to a liposome using either a thiolated antibody or a reduced antibody (Fab') using PDP-PE liposomes

10) Attachment of liposomes (reacted with SPDP) to antibodies (reacted with SPDP and DTT)

Methods that involve using a non-modified antibody

1) Attachment of antibodies to liposomes containing P-NP group

2) Attachment of a reactive liposome containing carboxyl group to -NH2 group in antibody using EDC/Sulfo-NHS chemistry

3) Attachment of a reactive liposome containing amine group (PE) to -COOH group in antibody using EDC/Sulfo-NHS chemistry

This question was also answered on Quora:

http://www.quora.com/What-methods-are-used-for-conjugation-of-antibodies-to-liposomes?q=liposome

Clodrosome Database

2011-06-23T13:05:00.014-05:00

Within the next month we are going to launch www.clodrosome.com website which will contain comprehensive information about Clodronate liposomes and macrophage depletion. In the mean time the following database can be used which summarizes all the Clodronate liposome research papers from 1994 to June 2011.

Encapsula NanoSciences Data_Clodronate liposomes

How can one maximize the chemical and physical stability of liposomes?

2011-03-25T16:48:00.002-05:00

There are many ways to increase the chemical and physical stability of liposomes.

1. One way is to avoid using unsaturated phospholipids. Unsaturated phospholipids are subject to peroxidation. However this might not always be doable and the use of unsaturated lipids might be necessary for some liposomes formulations. If unsaturated phospholipids are used then it is important to do the following:

* Maintain an oxygen free environment during the manufacturing process and storage
* Add tocopherols or other membrane active antioxidants
* Limit the light exposure during the manufacturing process and storage

2. Store the liposomes in 4 C.

3. NEVER freeze the liposomes.

4. Buffer and maintain the pH of liposomes at 6-6.5 during the manufacturing and storage to avoid both acid- and base- catalysed hydrolysis.

5. Most buffers enhance hydrolysis; ensure minimal concentration of buffer sufficient to maintain pH.

6. Add ethylenediaminetetraacetic acid (EDTA) to ensure the absence of multivalent cations. The presence of multivalent cations may cause aggregation and bilayer destabilization.

7. Control the temperature or select lipids so that lipid phase transitions do not occur during storage.

8. Monitor the aggregation state of the liposomes and adjust the aqueous phase or lipid composition accordingly.

9. Avoid using lipid compositions which spontaneously fuse (i.e. DSPC SUV) or increase fusion activity upon accumulation of hydrolysis products.

10. Incorporation of cholesterol into liposomes can reduce destabilizing effects of hydrolysis products.

To see the original Q&A on Quora click on this link:

http://www.quora.com/How-can-one-maximize-the-chemical-and-physical-stability-of-liposomes

Is it necessary to use PEGylation in order to formulate and commercialize a liposome based drug?

2011-03-16T14:48:00.002-05:00

It is not necessary to use PEGylation in order to formulate an effective liposome based drug.

As a matter of fact, out of 15 promising liposome based drugs that are commercialized or are in phase III only 4 of them are PEGylated. These four drugs are: Doxil, Lipodox, Thermodox and Lipoplatin. See here:

http://www.liposomes.org/2011/03/how-many-liposome-based-drugs-have-been.html

Doxil and Lipodox (both PEGylated form of liposomal doxorubicin) have significantly more side effects than Myocet (the non PEGylated form of liposomal doxorubicin) and this is mainly due to the long circulation properties of PEGylated liposomes.

See the original Q&A here: http://www.quora.com/Is-it-necessary-to-use-PEGylation-in-order-to-formulate-and-commercialize-a-liposome-based-drug

Is it necessary to add cholesterol to a liposome formulation?

2011-03-16T00:00:00.002-05:00

The presence of cholesterol exerts a profound influence on the properties of the lipid bilayers of the liposomes. It has been known for several decades that the addition of cholesterol to a fluid phase bilayer (mainly unsaturated lipids) decreases its permeability to water. A liposome that is made from 100% unsaturated lipid in "fluid phase" can not hold its encapsulated content and the encapsulated water soluble drugs will leak out over time and therefore the addition of cholesterol is necessary in order to prevent the leakage of the encapsulated drug from the liposomes. Cholesterol molecules fill in the free space that was formed due to the kink in the chain of the unsaturated lipids and this will decrease the flexibility of the surrounding lipid chains. This interaction also increases the mechanical rigidity of fluid bilayers and decreases their lateral diffusion. In contrast, the addition of cholesterol to gel phase bilayers (mainly saturated lipids) disrupts local packing orders and increases the diffusion coefficient and decreases the elastic modulus. Liposomes made from 100% saturated lipids are leak-proof in the absence of the cholesterol. One of the main reasons for addition of cholesterol to liposomes that are made from saturated lipids is to decrease the phase transition temperature. The phase transition temperature of the mixture of the lipids in the liposomes will be decreased, but not eliminated, by the addition of cholesterol.

"Trigger release liposomes" such as temperature sensitive liposomes or radiation sensitive liposomes should be engineered in a way to have phase separated domains. The existence of the phase separated domains make the liposomes unstable upon trigger and therefore the liposomes release their content upon trigger.

Phase separation is necessary for engineering of the radiation sensitive liposomes because the polymerizable lipids should be adjacent to each other for the polymerization to happen. The addition of cholesterol molecules will disrupt the formation of the polymerizable domains because cholesterol molecules sit between the polymerizable molecules and prohibit the lipid molecules to exchange electrons and get polymerized upon radiation.

Temperature sensitive liposomes also demonstrate enhanced release of the encapsulated drug via grain boundary permeabilization when heated to their phase transition temperature. Addition of the cholesterol will also disrupt the formation of the grain boundary domains. Thermodox-a temperature sensitive liposomes at phase III- is a cholesterol free liposome.

Radiation sensitive liposomes and temperature sensitive liposomes are formulated by using saturated lipids. The addition of cholesterol to gel phase liposomes will increase the fluidity of the membrane and therefore all the gaps and imperfections that are formed in the lipid bilayers due to the trigger (radiation or heat) will be healed immediately and this will prevent the liposomes to leak more of their content upon trigger and this is undesirable. Trigger release liposomes should release as much of their content as possible upon trigger.

To see the original Q&A on Quora click here:

http://www.quora.com/Is-it-necessary-to-add-cholesterol-to-a-liposome-formulation

What is the effect of the phase transition temperature of the lipid on the liposome formulation?

2011-03-13T03:53:00.001-05:00

Physicochemical properties of liposomes depend on many factors such as ionic strength, pH and temperature. Usually liposomes have low permeability to the encapsulated molecules (unless the molecule is membrane permeable). The permeability of the membrane changes with temperature. One of the most important properties of the lipid bilayer is the relative fluidity and mobility of each individual lipid molecule in the bilayer. The mobility of the lipids change with the temperature. At a given temperature a lipid bilayer can exist in a solid (gel) or liquid phase. In both phases the lipid molecules are constrained to the two dimensional plane of the membrane but in liquid phase the lipid molecules can diffuse much more freely within the plane. At a given temperature a lipid molecule will exchange location with its neighboring lipid molecules millions of times a second and go through the process of random walk.

Phase transition temperature of phospholipids and lipid bilayers depends on the following:

1) The length of the acyl chain in the lipid
2) The degree of saturation of the hydrocarbon chains in lipid
3) The ionic strength of the suspension medium
4) The type of the polar head group

The phase behavior of the lipid bilayer is determined by the Van der Waals interactions between adjacent lipid molecules. The interaction is mainly governed by two factors:

1) The length of the acyl chain in the lipid
2) The packing of the lipids in the bilayer

Longer tail lipids have more area to interact. This will increase the strength of the interaction and consequently decrease the mobility of the lipid. Therefore at a given temperature a short tailed lipid will be more fluid than an otherwise identical long-tailed lipid.

The degree of unsaturation of the lipid tails can effect the packing of the lipids in the bilayer. An unsaturated double bond can produce a kink in the alkane chain. This kink will create extra free space within the bilayer which allows additional flexibility in the adjacent chains. Unsaturated lipids have a significantly lower transition temperature compare to saturated lipids.

Liposome made from pure phospholipids (in the absence of cholesterol) will not form at temperatures below the phase transition temperature of phospholipid. If the encapsulated molecule is temperature sensitive (e.g. a protein) then pure long chain saturated lipids can not be used in the liposomes because the lipids have to be heated up and the protein can not tolerate high temperatures.

Most lipid bilayers are not composed of a single type of lipid. In nature lipid membranes are usually a complex mixture of various lipid molecules. If some of the lipids in the mixture are liquid at a given temperature while other lipids are in gel phase, then the two phases will exist in spatially separated populations. This phenomenon is called "phase separation".

"Trigger release liposomes" such as temperature sensitive liposomes or radiation sensitive liposomes should be engineered in a way to have phase separated domains. The existence of the phase separated domains make the liposomes unstable upon trigger and therefore the liposomes release their content upon trigger.

The presence of cholesterol exerts a profound influence on the property of the lipid bilayers. It has been known for the past four decades that the addition of cholesterol to a fluid phase bilayer decreases its permeability to water. Cholesterol molecules fill in the free space that was formed due to the kink in the chain of the unsaturated lipid and this will decrease the flexibility of the surrounding lipid chains. This interaction also increases the mechanical rigidity of fluid bilayers and decreases their lateral diffusion. In contrast, the addition of cholesterol to gel phase bilayers disrupts local packing orders and increases the diffusion coefficient and decreases the elastic modulus.

See the original Q&A on Quara:

http://www.quora.com/What-is-the-effect-of-the-phase-transition-temperature-of-the-lipid-on-the-liposome-formulation

Celsion Receives European Orphan Drug Designation for ThermoDox to Treat Primary Liver Cancer

2011-03-05T09:43:00.005-06:00

COLUMBIA, MD -- (MARKET WIRE) -- 03/02/11 -- Celsion Corporation (NASDAQ: CLSN), a leading oncology drug development company, today announced that the European Commission (EC) has granted orphan drug designation for the Company's lead compound, ThermoDox®, a proprietary heat-activated liposomal encapsulation of doxorubicin, for the treatment of hepatocellular carcinoma (HCC), commonly referred to as primary liver cancer. ThermoDox®, which also holds orphan drug designation in the U.S., is currently being evaluated under a Special Protocol Assessment with the U.S. Food and Drug Administration in a 600 patient pivotal Phase III trial, the HEAT study, in patients with non-resectable primary liver cancer.

"EC's orphan drug designation for ThermoDox® recognizes the urgent need for new therapies in addressing primary liver cancer, a rapidly progressing disease and for which few effective treatment options exist," stated Mr. Michael H. Tardugno, Celsion's President and Chief Executive Officer. "As the HEAT study nears enrollment completion and we look to its outcome, this designation, along with U.S. orphan drug status, FDA Fast Track designation and regulatory agency support in 11 countries worldwide for our Phase III HEAT study, provides us with speed and strategic flexibility both through the registration process and in the commercial setting. We look forward to the potential of delivering ThermoDox® to patients in need and to establishing a new standard of care in HCC."

As established by the European Medicine Agency (EMA), orphan designation for a medicinal product provides for scientific advice and regulatory assistance from the EMA during the product development phase, direct access to centralized marketing authorization, and certain financial incentives for companies developing product candidates intended for the treatment of a life-threatening or chronically debilitating condition affecting no more than five in 10,000 persons in the European Union. The designation also provides 10 years of marketing exclusivity subsequent to product approval. Orphan drugs are eligible for a reduction of fees associated with pre-authorization inspections, as well as, marketing authorization application fees and certain other fees for qualifying companies.

About Primary Liver Cancer

Primary liver cancer is one of the most deadly forms of cancer and ranks as the fifth most common solid tumor cancer. The incidence of primary liver cancer is approximately 20,000 cases per year in the United States, approximately 40,000 cases per year in Europe and is rapidly growing worldwide at approximately 750,000 cases per year, due to the high prevalence of Hepatitis B and C in developing countries. The standard first line treatment for liver cancer is surgical resection of the tumor; however, 90% of patients are ineligible for surgery. Radio frequency ablation (RFA) has increasingly become the standard of care for non-resectable liver tumors, but the treatment becomes less effective for larger tumors. There are few non-surgical therapeutic treatment options available as radiation therapy and chemotherapy are largely ineffective in the treatment of primary liver cancer.

About ThermoDox® and the Phase III HEAT Study

ThermoDox® is a proprietary heat-activated liposomal encapsulation of doxorubicin, an approved and frequently used oncology drug for the treatment of a wide range of cancers. In the HEAT study, ThermoDox® is administered intravenously in combination with RFA. Localized mild hyperthermia (39.5 - 42 degrees Celsius) created by the RFA releases the entrapped doxorubicin from the liposome. This delivery technology enables high concentrations of doxorubicin to be deposited preferentially in a targeted tumor.

For primary liver cancer, ThermoDox® is being evaluated in a 600 patient global Phase III study at 76 clinical sites under an FDA Special Protocol Assessment. The study is designed to evaluate the efficacy of ThermoDox® in combination with Radio Frequency Ablation (RFA) when compared to patients who receive RFA alone as the control. The primary endpoint for the study is progression-free survival (PFS) with a secondary confirmatory endpoint of overall survival. A pre-planned, unblinded interim efficacy analysis will be performed by the independent Data Monitoring Committee when enrollment in the HEAT Study is complete and 190 PFS events are realized in the study population. Additional information on the Company's ThermoDox® clinical studies may be found at http://www.clinicaltrials.gov.

See here for original URL: http://www.celsion.com/releasedetail.cfm?ReleaseID=553789

How many liposome based drugs are in the market?

2011-03-05T02:20:00.004-06:00

There are several liposome formulations that have been commercialize and there are many liposome formulations that are in various stages of clinical trials.

These are several of the commercialized and phase III formulations:

1) Myocet (Liposomal doxorubicin)- This is a non PEGylated formulation of liposomal doxorubicin. The liposomes are composed of egg PC (EPC): cholesterol (55:45 molar ratio). It is used in combinational therapy for treatment of recurrent breast cancer.

2) Doxil, Caelyx (Liposomal doxorubicin)- This is a PEGylated formulation of liposomal doxorubicin. The liposomes are composed of hydrogenated soy PC (HSPC): cholesterol: PEG 2000-DSPE (56:39:5 molar ratio). It is used for treatment of refractory Kaposi's sarcoma, recurrent breast cancer and ovarian cancer.

3) LipoDox (Liposomal doxorubicin)- This is a PEGylated formulation of liposomal doxorubicin. The liposomes are composed of DSPC: cholesterol: PEG 2000-DSPE (56:39:5 molar ratio). It is used for treatment of refractory Kaposi's sarcoma, recurrent breast cancer and ovarian cancer.

4) Thermodox (Liposomal doxorubicin)- This is a PEGylated formulation of liposomal doxorubicin. Thermodox is a triggered release formulation. The liposomes will release their content upon heat. The tumor is heated up using radio frequency ablation (RFA). The liposomes release their content inside the tumor upon heat. The liposomes are composed of DPPC, mono steroyl PC (MSPC) and PEG2000-DSPE. It is used for treatment of primary liver cancer (Hepatocellular carcinoma) and also recurrent chest wall breast cancer. Thermodox is in phase III of clinical trial.

5) DaunoXome (Liposomal Daunorubicin)- This is a non PEGylated formulation of liposomal Daunorubicin. The liposomes are composed of DSPC and cholesterol. It is used for treatment of Kaposi's sarcoma.

6) Ambisome (Liposomal Amphoteracin B)- This is a non PEGylated formulation of liposomal Amphoteracin B. The liposomes are composed of HSPC, DSPG, cholesterol and amphoteracin B in 2:0.8:1:0.4 molar ratio. It is used for treatment of fungal infection.

7) Marqibo (Liposomal vincristine)- This is a non PEGyated formulation of liposomal vincristine. The liposomes are composed of egg sphingomylin and cholesterol. It is used for the treatment of metastatic malignant uveal melanoma. Marqibo is in phase III of clinical trial.

8) Visudyne (Liposomal verteporfin)- This is a non PEGylated formulation of liposomal verteporfin (BPD-MA). The liposomes are composed of BPD-MA:EPG:DMPC in 1:05:3:5 molar ratio. It is used for treatment of age-related macular degeneration, pathologic myopia and ocular histoplasmosis.

9) DepoCyt (Liposomal cytarabine)- This is a non PEGylated formulation of liposomal cytarabine. The Depo-Foam platform is used in DepoCyt. Depo-Foam is a spherical 20 micron multi-lamellar liposome matrix comprised of Cholesterol: Triolein: Dioleoylphosphatidylcholine (DOPC): Dipalmitoylphosphatidylglycerol (DPPG) in 11:1:7:1 molar ratio. The drug is used by intrathecal administration for treatment of neoplastic meningitis and lymphomatous meningitis.

10) DepoDur (Liposomal morphine sulfate)- This is a non PEGylated formulation of liposomal cytarabine. The Depo-Foam platform is used in DepoCyt. Depo-Foam is a spherical 20 micron multi-lamellar liposome matrix comprised of Cholesterol: Triolein: Dioleoylphosphatidylcholine (DOPC): Dipalmitoylphosphatidylglycerol (DPPG) in 11:1:7:1 molar ratio. The drug is used by epidural administration for treatment of postoperative pain following major surgery.

11) Arikace (Liposomal amikacin)- This is a non PEGylated formulation of liposomal amikacin. The liposomes are composed of DPPC and cholesterol. The size of the liposomes is between 200-300 nm. It is used for treatment of lung infections due to susceptible pathogens. Arikace is used in nebulized form and it is inhaled by the patients. The drug is in phase III of clinical trial.

12) Lipoplatin (Liposomal cisplatin)- This is a PEGylated formulation of liposomal
cisplatin. The liposomes are composed of DPPG, Soy PC, cholesterol and PEG2000-DSPE. It is used for treatment of epithelial malignancies such as lung, head and neck, ovarian, bladder and testicular cancers.

13) LEP-ETU (Liposomal Paclitaxel)- This is a non PEGylated formulation of liposomal Paclitaxel. The liposomes are composed of DOPE, cholesterol and cardiolipin. Its is used for treatment of ovarian, breast and lung cancer. LEP-ETU is completing phase II of clinical trials.

14) Epaxal (Hepatitis A vaccine)- Liposomes have been used as a vaccine adjuvant in this formulation. Inactivated vaccines usually contain an adjuvant which potentiates the immune response to the antigen. During the last 70 years aluminium salts have been the only adjuvant licensed for human. The adjuvanting activity is based on their serving as an antigen depot and inducing a localized inflammatory response. These liposomes also known as immunopotentiating reconstituted influenza virosomes (IRIV) are composed of DOPC/DOPE in 75:25 molar ratio. The liposomes are sized to 150 nm.

15) Inflexal V (Influenza vaccine)-Liposomes have been used as a vaccine adjuvant in this formulation. The liposomes are composed of DOPC/DOPE in 75:25 molar ratio. The liposomes are sized to 150 nm.

See the original Q&A here: http://www.quora.com/How-many-liposome-based-drugs-are-in-the-market

New MIT nanoparticles could lead to powerful vaccines for HIV and other diseases

2011-02-26T16:12:00.003-06:00

MIT engineers have designed a new type of nanoparticle that could safely and effectively deliver vaccines for diseases such as HIV and malaria.

The new particles, described in the Feb. 20 issue of Nature Materials, consist of concentric fatty spheres that can carry synthetic versions of proteins normally produced by viruses. These synthetic particles elicit a strong immune response — comparable to that produced by live virus vaccines — but should be much safer, says Darrell Irvine, author of the paper and an associate professor of materials science and engineering and biological engineering.

Such particles could help scientists develop vaccines against cancer as well as infectious diseases. In collaboration with scientists at the Walter Reed Army Institute of Research, Irvine and his students are now testing the nanoparticles’ ability to deliver an experimental malaria vaccine in mice.

Vaccines protect the body by exposing it to an infectious agent that primes the immune system to respond quickly when it encounters the pathogen again. In many cases, such as with the polio and smallpox vaccines, a dead or disabled form of the virus is used. Other vaccines, such as the diphtheria vaccine, consist of a synthetic version of a protein or other molecule normally made by the pathogen.

When designing a vaccine, scientists try to provoke at least one of the human body’s two major players in the immune response: T cells, which attack body cells that have been infected with a pathogen; or B cells, which secrete antibodies that target viruses or bacteria present in the blood and other body fluids.

For diseases in which the pathogen tends to stay inside cells, such as HIV, a strong response from a type of T cell known as “killer” T cell is required. The best way to provoke these cells into action is to use a killed or disabled virus, but that cannot be done with HIV because it’s difficult to render the virus harmless.

To get around the danger of using live viruses, scientists are working on synthetic vaccines for HIV and other viral infections such as hepatitis B. However, these vaccines, while safer, do not elicit a very strong T cell response. Recently, scientists have tried encasing the vaccines in fatty droplets called liposomes, which could help promote T cell responses by packaging the protein in a virus-like particle. However, these liposomes have poor stability in blood and body fluids.

Irvine, who is a member of MIT’s David H. Koch Institute for Integrative Cancer Research, decided to build on the liposome approach by packaging many of the droplets together in concentric spheres. Once the liposomes are fused together, adjacent liposome walls are chemically “stapled” to each other, making the structure more stable and less likely to break down too quickly following injection. However, once the nanoparticles are absorbed by a cell, they degrade quickly, releasing the vaccine and provoking a T cell response.

In tests with mice, Irvine, postdoctoral associate James Moon (lead author of the paper) and their colleagues used the nanoparticles to deliver a protein called ovalbumin, an egg-white protein commonly used in immunology studies because biochemical tools are available to track the immune response to this molecule. They found that three immunizations of low doses of the vaccine produced a strong T cell response — after immunization, up to 30 percent of all killer T cells in the mice were specific to the vaccine protein.

That is one of the strongest T cell responses generated by a protein vaccine, and comparable to strong viral vaccines, but without the safety concerns of live viruses, says Irvine. Importantly, the particles also elicit a strong antibody response. Niren Murthy, associate professor at Georgia Institute of Technology, says the new particles represent “a fairly large advance,” though he says that more experiments are needed to show that they can elicit an immune response against human disease, in human subjects. “There’s definitely enough potential to be worth exploring it with more sophisticated and expensive experiments,” he says.

In addition to the malaria studies with scientists at Walter Reed, Irvine is also working on developing the nanoparticles to deliver cancer vaccines and HIV vaccines. Translation of this approach to HIV is being done in collaboration with colleagues at the Ragon Institute of MIT, Harvard and Massachusetts General Hospital. The institute, which funded this study along with the Gates Foundation, Department of Defense and National Institutes of Health, was established in 2009 with the goal of developing an HIV vaccine.

Source: http://web.mit.edu/newsoffice/2011/nano-sized-vaccines-0222.html

I have a membrane protein and I would like to incorporate the protein into liposomes. What methods can I use?

2011-02-21T12:52:00.001-06:00

Incorporation of membrane proteins into liposomes is an art more than science. It requires quite a bit of patience and you need to test various methods and see which one will work better for your particular protein. Not all methods can be used for all proteins.

There are essentially four presently known mechanisms for incorporating or reconstituting of membrane proteins into liposomes.

1) Methods involving the use of an organic solvent.
2) Methods involving the use of the mechanical means.
3) Methods involving the use of detergents.
4) Direct incorporation of the protein into the preformed liposomes.

These techniques will be described briefly in the following sections.

Organic solvent -medicated reconstitution

Organic solvents have been used widely to prepare large liposomes in procedures including ethanol injection, ether infusion and reverse phase evaporation. However, the usefulness of these techniques is limited because of exposure of membrane proteins to the organic solvents, which often denatures them.

The only suitable method reported to date for organic solvent medicated reconstitution of membrane proteins is the reverse phase evaporation. Szoka and Papahadjopoulos developed a technique for incorporation of membrane proteins into large unilamellar liposomes after extraction into a hydrocarbon solvent together with phospholipids. The membrane protein was added to a
suspension of lipids in a suitable buffer then a solvent e.g. hexane, pentane,
diisopropyl ether, and diethyl ether was added and the mixture was sonicated for a few minutes under argon. After the removal of the organic solvent in a rotary evaporator, the large unilamellar liposomes of about 1 micrometer are formed. Critical aspects of this technique are the selection of the organic solvent as well as the volume ratio of aqueous to non-aqueous phase.

Reconstitution by Mechanical Means

Second method uses mechanical means to produce large and small unilamellar vesicles (ULV) from multilamellar vesicle (MLV) by swelling of the dry phospholipid films in excess buffer.Such mechanical means include sonication of MLVs and forcing multilamellar lipid vesicles through a French press. Sonication and freeze thawing of a mixed suspension of lipids and isolated proteins has been widely used in earlier stages of membrane protein reconstitution to demonstrate the function of these purified proteins for which detergent dialysis was insufficient.

The incorpration efficiencies are usually low using this technique. The protein ca also get denatured during the process.

Reconstitution by using detergents

Of the several different methods used to remove detergent from mixture of detergent, phospholipid and protein, the dialysis procedure has proved highly successful. In such a method, the protein and phospholipids are co-solubilized in a detergent to form micelles. The detergent is then removed, resulting in the spontaneous formation of bilayer vesicles with the protein incorporated therein. The detergent is incorporated into liposomes as well as the protein and thus, these methods require removal of the detergent by methods such as dialysis, gel exclusion chromatography or absorption on hydrophobic resins. The method that use detergent are very slow because detergent removal must be as complete as possible. Also a phase change that take place during this process slows detergent removal even further. Another disadvantage is that one can not control the orientation of protein incorporated into the liposomes using detergent methods.

The critical aspect of this technique is the selection of a detergent with the proper critical micelle concentration (CMC). It is advised to use a detergent with high CMC such as Octyl-b-D-glucopyranoside which has a CMC of 20-26 mM.
Detergents with high CMC can easily be removed from the mixture by dialysis.

Direct incorporation of proteins into preformed liposome

The forth process involves the direct incorporation of protein into preformed liposomes. One of the key features for successful incorporation of the delipidated proteins into preformed liposomes appear to be the organization of the bilayer. Bilayer conductive to spontaneous incorporation of large membrane proteins are achieved by incorporatingimpurities such as fatty acids, lysophospholipid, cholesterol, detergent and membrane bound proteins. The putative effect of impurities is the formation of organizational defects that act as sites for fusion of vesicles with the proteins.

See the whole question and answer on Quora page:

http://www.quora.com/Zahra-Mirafzali/answers

Do you have any liposome related questions? Ask me about it on Quora.

Insmed Acquires Transave for Late-Stage Inhaled Lung Infection Therapy

2011-01-30T21:41:00.001-06:00

Biopharmaceutical firm Insmed has taken over inhaled drugs company Transave through a cash and shares-based deal that gives Insmed the acquired firm's late-stage lung infection candidate, Arikace. The takeover deal, through which Insmed is also paying off Transave's $7.8 million in debt, will shuffle around about 25.9 million shares of Insmed common stock, 91.7 million shares of Insmed Series B conditional convertible preferred stock, and $561,280 in cash. The merger effectively gives former Transave shareholders a 46.7% equity stake in the combined entity on an as-converted, fully diluted basis.

Transave’s lead candidate, Arikace™ (liposomal amikacin for inhalation), is based on the firm’s liposomal technology for the delivery of drugs to the lung. The drug is poised to start in Phase III trials for the treatment of pseudomonas lung infections in cystic fibrosis patients, and lung infections due to non-TB mycobacteria (NTM). Insmed says it hopes to start parallel Phase III development of Arikace for both indications during the second half of 2011.

The firm admits it has been on the hunt for a late-stage, high-value product candidate. “The drug’s previously completed phase II and earlier stage clinical studies highlighted the potential of Arikace to become a leading treatment in two high-growth orphan indications with significant unmet medical needs,” comments Melvin Sharoky, Ph.D., Insmed board member and previous chairman.

Transave claims the liposomal technology on which Arikace is based may be key in allowing the antibiotic to overcome the physical barriers presented by mucus in the lungs of cystic fibrosis patients and by bacterial pseudomonas biofilm. The firm says in vitro experiments have previously shown Arikace liposomes penetrate both human cystic fibrosis sputum and the biofilm of pseudomonas macrocolonies.

In clinical trials Arikace has been delivered locally via inhalation using Pari Pharma’s eFlow nebulizer system. The drug has been granted Orphan Drug status in both the U.S. and EU for the treatment of pseudomonas lung infections in cystic fibrosis. Transave had in addition been planning to file for orphan drug designation covering the use of Arikace for the treatment of non-tuberculosis mycobacterial lung infections. Arikace has separately been granted orphan drug designation in the U.S. for a third indication, the the treatment of bronchiectasis in patients with infections due to pseudomonas or other susceptible pathogens.

Pacira licenses DepoFoam to Novo Nordisk, plans IPO

2011-01-30T21:38:00.003-06:00

Parsippany, NJ-based Pacira Pharmaceuticals, which makes controlled-release injectable products, has licensed its DepoFoam drug delivery technology to Danish drugmaker Novo Nordisk. The deal could be worth more than $45.5 million, according to The Associated Press.

What puts Pacira on the drug-delivery map is its DepoFoam technology, which consists of multi-vesicular liposome particles that contain internal chambers for encapsulated drugs. According to the company, DepoFoam is capable of sustained release of a drug over a period of between one and 30 days. It works with narrow gauge needles and pen systems and offers flexible delivery that can be designed to for an immediate release dose followed by sustained delivery.

Pacira is also planning an IPO. According to Dan Primack, writing in Fortune's Term Sheet newsletter, Pacira has set its IPO terms to 4.25 million common shares being offered at between $14 and $16 per share. The company has raised VC funding from MPM Capital, HBM BioVentures, ORbiMed Advisors and Sanderling Ventures.

Original URL: http://www.fiercedrugdelivery.com/story/pacira-licenses-depofoam-novo-nordisk-plans-ipo/2011-01-25

Celator Pharmaceuticals and Cephalon, Inc. Agree to Extend Research Agreement into Next Phase of Development

2011-01-25T07:37:00.000-06:00

Celator Pharmaceuticals announced that it has agreed with Cephalon, Inc. to extend an existing research agreement into the next phase of development

PRINCETON, NJ, USA | January 10, 2011 | Celator Pharmaceuticals today announced that it has agreed with Cephalon, Inc. to extend an existing research agreement into the next phase of development. The research agreement provides for the utilization of Celator's proprietary technology in an ongoing drug development and life-cycle management program at Cephalon.

"We are pleased that our progress to date allows Cephalon and Celator to advance this promising work," said Scott Jackson, chief executive officer, Celator Pharmaceuticals. "It is rewarding to have a company of Cephalon’s stature demonstrate the potential of our technology in its portfolio and make the ongoing financial commitment to continue this research."

Terms and conditions of the research agreement are confidential.

Celator is advancing a number of its own programs based on the Company's proprietary nano-scale delivery platforms. The Company has announced positive results from its Phase 2 study of CPX-351 (Cytarabine:Daunorubicin) Liposome Injection versus conventional cytarabine and daunorubicin therapy (known as the “7+3” regimen) in patients with newly diagnosed acute myeloid leukemia. The study showed that patients treated with CPX-351 demonstrated a higher aplasia rate, a higher remission rate (including complete remissions [CR] and complete remissions with incomplete neutrophil/platelet recovery [CRi]), lower induction mortality, improved median event-free survival (EFS), and improved median overall survival (OS). Even more noteworthy were the improvements seen in high risk patients. In particular, patients with secondary AML treated with CPX-351 experienced a statistically significant improvement in survival (p=0.01) with median overall survival of 12.1 months versus 6.1 months.

CPX-351 is one of a pipeline of investigational cancer therapies developed using Celator's CombiPlex® drug-ratio technology. Celator also has an agreement with the National Cancer Institute's Nanotechnology Characterization Laboratory (NCL) whereby the NCL selected the Company's hydrophobic docetaxel prodrug nanoparticle formulation for intensive physical characterization, in-vitro studies, and in-vivo pharmacology and toxicology protocols to support an eventual investigational new drug (IND) filing with the U.S. Food and Drug Administration.

About Cephalon, Inc.

Cephalon is a global biopharmaceutical company dedicated to discovering, developing and bringing to market medications to improve the quality of life of individuals around the world. Since its inception in 1987, Cephalon has brought first-in-class and best-in-class medicines to patients in several therapeutic areas. Cephalon has the distinction of being one of the world's fastest-growing biopharmaceutical companies, now among the Fortune 1000 and a member of the S&P 500 Index, employing approximately 4,000 people worldwide. The company sells numerous branded and generic products around the world. In total, Cephalon sells more than 150 products in nearly 100 countries. More information on Cephalon and its products is available at http://www.cephalon.com/.

About Celator Pharmaceuticals, Inc.

Celator Pharmaceuticals, Inc., with locations in Princeton, NJ, and Vancouver, BC, is a privately held pharmaceutical company developing new and more effective therapies to treat cancer. CombiPlex®, the company's proprietary drug ratio technology platform, represents a novel approach that identifies molar ratios of drugs that will deliver a synergistic benefit, and locks the desired ratio in a nano-scale drug delivery vehicle that maintains the ratio in patients with the goal of improving clinical outcomes. The company pipeline includes two Phase 2 products; CPX-351 (a liposomal formulation of cytarabine:daunorubicin) for the treatment of acute myeloid leukemia and CPX-1 (a liposomal formulation of irinotecan:floxuridine) for the treatment of colorectal cancer; a preclinical stage compound, CPX-571 (a liposomal formulation of irinotecan:cisplatin); and multiple research programs, including the hydrophobic docetaxel prodrug nanoparticle (HDPN) formulation being studied by the National Cancer Institute's Nanotechnology Characterization Laboratory. Based on the applications of CombiPlex and the proprietary nanoparticle prodrug delivery platform, Celator is positioned to advance a broad pipeline of cancer therapies involving both previously approved and novel drug agents. For more information, please visit the company's website at www.celatorpharma.com. Information on ongoing trials is available at www.clinicaltrials.gov.

http://www.pipelinereview.com/index.php/2011011039638/Small-Molecules/Celator-Pharmaceuticals-and-Cephalon-Inc.-Agree-to-Extend-Research-Agreement-into-Next-Phase-of-Development.html

Celsion to Obtain $5M in Financing and Fast-Track Milestone Payments from Japanese Partner

2011-01-25T07:28:00.002-06:00

Celsion entered into a registered direct offering in which it expects to receive gross proceeds of about $5 million. The company is leveraging heat-activated liposome drug delivery technology to deliver high concentrations of known chemotherapeutics to the lesion site.

Celsion also amended its development, product supply, and commercialization agreement with Yakult Honsha covering lead candidate Thermodox®. The candidate is a formulation of doxorubicin and is being investigated as a treatment for hepatocellular carcinoma (HCC) and recurrent chest wall (RCW) breast cancer.

Yakult Honsha will provide up to $4 million in an accelerated partial payment to Celsion of a future drug approval milestone. Celsion will receive $2 million immediately and the rest once Celsion is allowed to resume enrollment of Japanese patients in its Phase III trial, which is being conducted in the Japan. In consideration of these accelerated milestone payments from Yakult, Celsion agreed to reduce future drug approval milestone payments by approximately 40%.

In September 2010, Celsion reported that after reviewing data from 401 patients enrolled in its Phase III study for primary liver cancer, the Data Monitoring Committee (DMC) unanimously recommended that the trial continue to enroll patients in trials being conducted in Canada, China, Hong Kong, Italy, Korea, U.S., and Taiwan. DMC continues to independently assess safety in patients randomized at Japanese sites.

Radio frequency ablation (RFA) has become an increasingly common method for treating liver cancer. However, while RFA uses extremely high temperatures (80º to 100ºC) to ablate tumors, it may fail to treat the outer margins of larger tumors since temperatures in the periphery are not high enough to destroy the cancer cells.

Celsion’s ThermoDox treatment approach is designed to deliver high concentrations of doxorubicin directly to those cancer cells that survive RFA. In conjunction with ablating the center of the tumor, RFA simultaneously activates ThermoDox to release its encapsulated doxorubicin, killing the remaining viable cancer cells throughout the heated region including the tumor margins.

Celsion notes that its lysolipid thermally sensitive liposome technology is different from other liposomal technologies because it has a low heat-activated release of encapsulated chemotherapeutic agents right at the cancer site.

Commenting on the $5 million registered direct offer, Michael H. Tardugno, Celsion’s president and CEO, says, “We believe that we now have the financial runway sufficient to complete enrollment in our Phase III primary liver cancer trial, the HEAT study, as well as other related clinical and CMC milestones in 2011.”

According to securities purchase agreement, Celsion is selling 5,000 shares of 8% redeemable convertible preferred stock with a stated value of $1,000 and warrants to purchase up to 2,083,333 shares of common stock in a registered direct offering.

The convertible preferred stock and warrants will be sold in units, with each unit consisting of one share of convertible preferred stock and a warrant to purchase up to 416.6666 shares of common stock at an exercise price of $3.25 per share of common stock. The units are being offered and sold at a purchase price of $1,000 per unit. Each share of preferred stock is convertible into shares of common stock at an initial conversion price of $2.40 per share.

See here for original URL:

http://www.genengnews.com/gen-news-highlights/celsion-to-obtain-5m-in-financing-and-fast-track-milestone-payments-from-japanese-partner/81244532/

Celator® Pharmaceuticals Presents Positive Data from Phase 2 Study of CPX-351 at the American Society of Hematology Annual Meeting

2010-12-07T08:08:00.007-06:00

PRINCETON, N.J.--(BUSINESS WIRE)--

Celator Pharmaceuticals today announced positive clinical data in elderly patients with newly-diagnosed acute myeloid leukemia (AML) treated with CPX-351 (Cytarabine:Daunorubicin) Liposome Injection. Data were presented from the podium at the 52nd American Society of Hematology Annual Meeting in Orlando, Florida. The results were based on 12 months of follow-up in a randomized, Phase 2 trial that compared CPX-351 to conventional cytarabine and daunorubicin (the “7+3” regimen), the current standard of care (ASH Abstract #655).

“Our findings suggest that CPX-351 may provide a long-sought opportunity to improve clinical outcomes over “7+3” in previously untreated AML,” said Jeffrey E. Lancet, M.D., associate professor, H. Lee Moffitt Cancer Center, who gave the presentation. “Importantly, these improvements were seen in the overall patient population but were greater in patients with high-risk AML, especially those with secondary AML.”

The randomized, open-label Phase 2 study enrolled 126 patients between the ages of 60-75 years with newly-diagnosed AML at 18 sites in the United States and Canada. Patients were stratified as high risk (age 70 or older, secondary AML, or ≥3 chromosomal abnormalities) or standard risk (all other patients). Following randomization, 85 patients received CPX-351 and 41 received the “7+3” regimen.

Compared to patients in the “7+3” arm, patients treated with CPX-351 demonstrated a higher aplasia rate, a higher remission rate (including complete remissions [CR] and complete remissions with incomplete neutrophil/platelet recovery [CRi]), lower induction mortality, improved median event-free survival, and improved median overall survival (OS).


	CPX-351	“7+3”
Aplasia Rate	87.7%	71.1%
Remission Rate (CR+CRi)	66.7%	51.2%
Induction Mortality, Day 60	4.7%	14.6%
Median EFS	5.4 months	2.0 months
Median OS	14.7 months	12.9 months

Follow-up is continuing for another 12 months. Larger improvements over “7+3” were seen in high-risk patients, where median event free survival was 4.5 months versus 1.8 months and median overall survival was 11.0 months versus 7.5 months. Even more noteworthy were the improvements seen in patients with secondary AML treated with CPX-351 who experienced a median event free survival of 3.8 months versus 1.4 months for “7+3” and a statistically significant improvement in median overall survival of 12.1 months versus 6.1 months (p=0.01).

Treatment with CPX-351 was associated with well-characterized and manageable adverse events that were generally comparable to conventional therapy. The myelosuppressive effect of CPX-351 was 7 days and 10 days longer for neutrophil and platelet recovery respectively, than those treated with “7+3,” effects consistent with the pharmacokinetics of CPX-351. The frequency and severity of cardiac events was low and similar in both groups.

“These encouraging results provide multiple paths for further development of CPX-351, with some patient populations representing opportunities for rapid clinical development and potential product registration,” said Scott Jackson, chief executive officer of Celator Pharmaceuticals. “As we plan for Phase 3, we will continue to collect data from this study until completion of the 24 month follow-up period and look forward to presenting initial results from our second Phase 2 study of CPX-351 in patients with AML in first relapse next year.”

Other CPX-351 presentations from the American Society of Hematology Annual Meeting
CPX-351 in Sequential Therapy for Refractory Leukemia
Results of a Phase 1 trial of sequential treatment for relapsed or primary refractory acute leukemia demonstrated that CPX-351 can be utilized as salvage chemotherapy prior to a reduced intensity conditioning regimen and allogeneic stem cell transplantation (ASH Abstract #1334). Further research is planned to define a maximum tolerated dose of CPX-351 and to shorten the interval between the administration of CPX-351 and the stem cell transplant.

Activity of CPX-351 Against Human Leukemias

A laboratory evaluation of the cytotoxicity of CPX-351 in 35 patient specimens suggested that CPX-351 possesses potent anti-leukemic activity against a wide range of human leukemia cell types (ASH Abstract #2886). The data establish a rationale for clinical testing of CPX-351 in other leukemia diagnoses besides AML and provide a model for in vitro screening that could help identify specific patient populations most likely to benefit from CPX-351.

About CPX-351

CPX-351 (Cytarabine:Daunorubicin) Liposome Injection represents a new approach to developing combinations of drugs in which drug molar ratios with synergistic anti-tumor activity are encapsulated in a drug delivery vehicle in order to maintain the desired ratio following administration. CPX-351 has been granted orphan drug status by the U.S. Food & Drug Administration (FDA) for the treatment of acute myeloid leukemia (AML). CPX-351 is currently in phase 2 clinical development for the treatment of AML. Celator has completed a successful randomized, phase 2 study comparing CPX-351 to the standard "7+3" regimen of cytarabine:daunorubicin in patients 60 years of age up to and including 75 years of age with newly diagnosed AML and has completed enrollment in a randomized, phase 2 study of CPX-351 versus intensive salvage therapy in patients 18 years of age up to and including 65 years of age with AML in first relapse. The second study is supported by The Leukemia & Lymphoma Society.

About Celator Pharmaceuticals, Inc.

ASH Abstracts on CPX-351:

Abstract #655 – Podium Presentation
Jeffrey E. Lancet, MD, Jorge E. Cortes, MD, Donna E. Hogge, MD, PhD, Martin Tallman, MD, Tibor Kovacsovics, MD, Lloyd E. Damon, MD, Ellen Ritchie, MD, Rami S. Komrokji, MD, Arthur C. Louie, MD and Eric J. Feldman, MD. Phase 2B randomized study of CPX-351 vs. Cytarabine + Daunorubicin (7+3 regimen) in newly diagnosed AML patients aged 60-75.

Abstract #1334 – Poster
Usama Gergis, MD, Ellen Ritchie, MD, Gail J. Roboz, MD, Joseph M. Scandura, MD, PhD, Sebastian Mayer, MD, Tomer M. Mark, MD, MSc, Tsiporah B. Shore, MD, Usama Wissa, MD and Eric J. Feldman, MD. A novel sequential treatment utilizing CPX-351 as salvage chemotherapy followed by a reduced intensity conditioning allogeneic stem-cell transplantation for patients with refractory leukemia.

Abstract #2886 – Poster
Jeffrey W. Tyner, PhD, Paul Tardi, PhD, Lawrence Mayer, PhD, Luke B. Fletcher, Stephen Spurgeon, MD, Tibor Kovacsovics, MD and Marc M. Loriaux, MD, PhD. Evaluation of CPX-351 (Cytarabine:Daunorubicin) Liposome Injection anti-leukemic activity against primary patient leukemia ceRead more: http://www.benzinga.com/press-releases/10/12/b674359/celator%C2%AE-pharmaceuticals-presents-positive-data-from-phase-2-study-of-c#ixzz17R2DubY9

Celator® Pharmaceuticals Announces Data to be Presented at the American Society of Hematology Annual Meeting

2010-11-30T10:57:00.002-06:00

PRINCETON, N.J., Nov. 29, 2010 -- /PRNewswire/ -- Celator Pharmaceuticals today announced that three abstracts on its lead program, CPX-351 (Cytarabine:Daunorubicin) Liposome Injection, will be presented at the 52nd American Society of Hematology (ASH) Annual Meeting in Orlando, Florida, including a podium presentation on results from a randomized, Phase 2 trial in elderly patients with newly diagnosed acute myeloid leukemia (AML) that compared treatment with CPX-351 to conventional cytarabine and daunorubicin (the "7+3" regimen), the current standard of care.

ASH Abstracts on CPX-351:

Abstract #655 – Podium Presentation

Jeffrey E. Lancet, MD, Jorge E. Cortes, MD, Donna E. Hogge, MD, PhD, Martin Tallman, MD, Tibor Kovacsovics, MD, Lloyd E. Damon, MD, Ellen Ritchie, MD, Rami S. Komrokji, MD, Arthur C. Louie, MD and Eric J Feldman, MD

Phase 2B randomized study of CPX-351 vs. Cytarabine (CYT) + Daunorubicin (DNR) (7+3 regimen) in newly diagnosed AML patients aged 60-75.

Session Name:	Acute Myeloid Leukemia - Therapy, excluding Transplantation: Novel Therapeutics
Session Date:	Monday, December 6, 2010
Session Time:	4:30 PM - 6:00 PM
Presentation Time:	4:30 PM
Room:	Orange County Convention Center, 311 ABCD

Abstract #1334 – Poster

Usama Gergis, MD, Ellen Ritchie, MD, Gail J. Roboz, MD, Joseph M. Scandura, MD, PhD, Sebastian Mayer, MD, Tomer M Mark, MD, MSc, Tsiporah B. Shore, MD, Usama Wissa, MD and Eric J Feldman, MD

A novel sequential treatment utilizing CPX-351 as salvage chemotherapy followed by a reduced intensity conditioning allogeneic stem-cell transplantation for patients with refractory leukemia.

Session Name:	Clinical Care - Transplantation Regimen Toxicities and Engraftment: Poster I
Date:	Saturday, December 4, 2010
Presentation Time:	5:30 PM - 7:30 PM
Location:	Orange County Convention Center, Hall A3/A4
Poster Board no.:	I-314

Abstract #2886 – Poster

Jeffrey W Tyner, PhD, Paul Tardi, PhD, Lawrence Mayer, PhD, Luke B Fletcher, Stephen Spurgeon, MD, Tibor Kovacsovics, MD and Marc M Loriaux, MD, PhD

Evaluation of CPX-351 (Cytarabine:Daunorubicin) Liposome Injection anti-leukemic activity against primary patient leukemia cells.

Session Name:	Molecular Pharmacology, Drug Resistance: Poster II
Date:	Sunday, December 5, 2010
Presentation Time:	6:00 PM - 8:00 PM
Location:	Orange County Convention Center, Hall A3/A4
Poster Board no.:	II-766

About CPX-351

CPX-351 (Cytarabine:Daunorubicin) Liposome Injection represents a new approach to developing combinations of drugs in which drug molar ratios with synergistic anti-tumor activity are encapsulated in a drug delivery vehicle in order to maintain the desired ratio following administration. CPX-351 has been granted orphan drug status by the U.S. Food & Drug Administration (FDA) for the treatment of acute myeloid leukemia (AML). CPX-351 is currently in phase 2 clinical development for the treatment of AML. Celator has completed a successful randomized, phase 2 study comparing CPX-351 to the standard "7+3" regimen of cytarabine:daunorubicin in patients 60 years of age up to and including 75 years of age with newly diagnosed AML and has completed enrollment in a randomized, phase 2 study of CPX-351 versus intensive salvage therapy in patients up to 65 years of age with AML in first relapse. The second study is supported by The Leukemia & Lymphoma Society.

About Celator Pharmaceuticals, Inc.

Original URL: http://www.sacbee.com/2010/11/29/3218455/celator-pharmaceuticals-announces.html#ixzz16mmQfEsW