WO1994017789A1

WO1994017789A1 - Polyalkylcyanoacrylate nanocapsules

Info

Publication number: WO1994017789A1
Application number: PCT/IE1993/000005
Authority: WO
Inventors: Valery Alexandrovich Dyatlov; Georgy Arkadievich Katz
Original assignee: Shield Research Limited
Priority date: 1993-02-15
Filing date: 1993-02-15
Publication date: 1994-08-18
Also published as: AU3462193A; EP0683667A1

Abstract

Nanocapsules typically having a diameter in the range 150-400 nm are provided and consist of a phase enclosed in a polymeric shell formed of a network of polymers. The nanocapsules, can be used to encapsulate a wide range of water soluble active agents in high yield for delivery to a target system or locus, for example, drugs for use in therapy or prophylaxis. The nanocapsules can be formed by interfacial polymerisation of alkyl-2-cyanoacrylates in a two phase system consisting of immiscible water soluble polymers under polymerisation initiating conditions, resulting in encapsulation of the active agent within a polyalkylcyanoacrylate shell.

Description

Description PO LYALKY LC YANOACRY LATE NANOC APSU LES

Technical Field

This invention relates to nanocapsules, a process for their preparation and their use in the delivery of a wide range of active agents to humans and other animals and other systems.

Background Art

Nanocapsules are examples of nanoparticles which are used inter alia as drug carrier systems. Nanoparticles are either small solid spheres (nanospheres) or small capsules (nanocapsules) formed of a central cavity surrounded by a shell or wall. The latter include polybutylcyanoacrylate nanocapsules (Al Khouri Fallouh, N. (1984); Pharm. Ph.D., No. 207, Paris XI) and polyisobutylcyanoacrylate nanocapsules (Al Khouri Fallouh, N. (1986); International Journal of Pharmaceutics 28, 125-132). The latter paper describes a process for the formation of nanocapsules with an average diameter of 200-300 nm by a mechanism which is described as being probably that of interfacial polymerisation resulting from the dispersion of an alcoholic solution of isobutylcyanoacrylate and oil in water. This process involves the use of two immiscible phases and the nanocapsules so formed are oil-filled and can be used to entrap lipophilic substances.

Damge, C. et al. (Diabetes (1988) 37, page 246) describe polyalkylcyanoacrylate nanocapsules as a drug carrier for insulin. The rate of encapsulation of insulin was found to be 54.9%. The nanocapsules were prepared by the method of Al Khouri Fallouh N. (1984) supra and as such the insulin was encapsulated in a lipophilic phase. There is a need for stable, aqueous-filled nanocapsules so as to extend the range of active substances that can be delivered by means of such nanocapsules.

Other examples of microcapsular drug carrier systems include liposomes which are small phospholipid based vesicles having an aqueous core. Liposomes have lipoidic walls structurally related to those of biological membranes and as such have a shell defined by a molecular bi-layer. However, liposomes are difficult to manufacture on an industrial scale and are of limited stability. Moreover, the entrapment levels are low (see Al Khouri Fallouh, N. (1986) supra).

Nanoparticles are used inter alia to administer labile active agents or toxic anti-tumour agents to a subject. Conventionally, nanoparticles are administered by the intramuscular or intravenous route and are transported into the epithelial cells, blood cells and liver cells by phagocytosis. Alternatively, the nanoparticles are degraded by chemical and/or enzymatic processes in the blood.

Nanoparticles, such as polyethylcyanoacrylate particles, are broken down by the Kupffer cells of the liver resulting in release of the active agent.

EP-A 0 274 961 describes the preparation of nanocapsules from dispersible colloidal systems. It is indicated that a wide range of substances (B) which are soluble or dispersible in a given solvent can be encapsulated by the process described. However, the process as described will result in a core of an organic solvent, an oily phase or a particulate substance. This will limit the nature of the active agent that can be encapsulated. For example, many of the organic solvents or solvent systems described would affect the stability of biologically active agents, such as peptides and proteins, and would be likely to lead to denaturation thereof and loss of pharmacological activity. The document does not describe the formulation of aqueous-filled nanocapsules. Ways of improving drug delivery, so as to achieve better bioavailability and pharmacokinetics are constantly being sought, especially for active agents which are subjected to rapid degradation following administration.

Oral administration is one of the modes of administering drugs which has the greatest degree of patient compliance and thus ways are constantly being sought of formulating active agents for administration by the oral route which it has not hitherto been possible to administer by that route.

Disclosure of invention

The invention provides nanocapsules comprising an aqueous phase enclosed in a polymeric shell, said polymeric shell being formed of a network of polymers.

The nanocapsules according to the invention are stable and can be used to entrap effective amounts of an active agent. It is possible to achieve a degree of encapsulation of 75% or higher with the nanocapsules according to the invention.

By polymer herein as regards the polymeric shell is meant any suitable polymer according to the I.U.P.A.C. definition of polymer.

The polymeric shell of the nanocapsules according to the invention is made up of a network of polymer chains typically of the order of 10 or more monomer units. The network formed is distinguishable from the ordered arrangement of the membrane of a liposome by having an essentially disordered arrangement of polymers defining said network and being generally thicker than the micellar, bi- molecular layer typical of a liposome.

Preferably, the nanocapsules in accordance with the invention have a diameter in the range 150-400 nm. The size of the nanocapsules is determined by the method of preparation as hereinafter described.

The nanocapsules according to the invention are primarily intended for use in the delivery of active agents to the human or animal body, including delivery for the purposes of medical diagnosis involving imaging. However, the nanocapsules according to the invention are not limited to such use and will also find application in agriculture, in cosmetics for delivery of a wide variety of active agents including fragrances, the food industry and other areas of technology to which their properties are adapted to provide a desired effect. For example, the nanocapsules according to the invention are ideally suited for the encapsulation and subsequent delivery of systemic fungicides, herbicides and pesticides and plant growth controlling agents to plants.

Thus, typically an active agent is contained in the aqueous phase.

An especially preferred polymeric material for the nanocapsular shell is a poly alkylcyanoacry late material, more especially a poly(Cι- C i o)-alky 1-2-cy anoacry late material.

According to a preferred method, the nanocapsules are formed by interfacial polymerisation in a two phase aqueous polymeric emulsion as hereinafter described.

The active agent encapsulated in the nanocapsules according to the invention is any water soluble active agent, including naturally occurring substances and synthetic analogues thereof.

Given that the active agent is dissolved or dispersed in an aqueous phase in the core of the nanocapsule, the stability thereof is maximised.

Preferred active agents include amino acids, peptides and polypeptides. Such active agents include hormones, hormone release factors, cytokines, encephalins, blood factors and products including enzymes and antibodies, and other active agents which are susceptible to degradation and/or modification by proteolytic and other enzymes before exerting their effect, especially if administered by the oral route. The latter type of active agents also includes anti-tumour agents, antibiotics, opiates such as apomorphine, dopamine, serotonin and other agents active on the central nervous system, and steroid hormones such as progesterone and testosterone.

When the active agent is an antibody, the antibody can be a monoclonal or polyclonal antibody.

The nanocapsules according to the invention are also especially suitable for the encapsulation and subsequent delivery of immunomodulating agents, for example, cyclosporin.

The nanocapsules according to the invention can also be used to encapsulate various vaccines.

It will be appreciated that the nanocapsules according to the invention can increase the bioavailability and efficacy of a wide range of water soluble active agents by protecting said agents from premature degradation in the gastrointestinal tract and the blood and allowing for a sustained release thereof.

The invention also provides a drug delivery system comprising nanocapsules as hereinbefore described.

The nanocapsules according to the invention are stable and release their contents on degradation following administration to the target system or locus.

The nanocapsules according to the invention when intended to deliver an active agent for use in therapy or prophylaxis may be administered orally, parenterally or topically to the human or animal body. Following oral administration the nanocapsules traverse the gut wall and are taken up into the blood stream and the product is released on degradation of the nanocapsule shell or wall. The nanocapsules are useful in delivering active agents to the blood stream by the oral route that are not normally suitable for administration by this route in traditional conventional pharmaceutical formulations. The encapsulated product is protected from the harsh conditions of the gut, such that a significantly greater proportion of active agent is delivered to the blood stream than would be possible by simple oral administration of the non-encapsulated active agent. For example, insulin, a protein, is normally given by intramuscular injection. If given orally it is normally degraded by the normal digestive processes of the gut and only a very small and variable proportion finds its way into the bloodstream. Insulin encapsulated by the method according to the invention can be given orally with minimal loss of pharmacological effect. Thus, there are major benefits for the patient both in terms of reducing stress and increasing convenience.

Suitable formulations of the nanocapsules according to the invention for administration by the oral route include capsules, dragees, elixirs, granules, lozenges, pellets, powders, suspensions and tablets. In the case of tablets care should be taken that the tabletting technique does not lead to any disruption of the nanocapsules and alteration of their release properties. Such tablets can be formulated for rapid disintegration in the gastric and/or intestinal juices, if required or, alternatively, coated so as to further delay the release of the active agent.

The nanocapsules can also be formulated as solutions or suspensions for injection intramuscularly, intravenously and subcutaneously. It is also possible to formulate the nanocapsules according to the invention in liquid form for administration by perfusion.

Further types of formulations according to the invention include nasal formulations, ocular agents, including slow release implants containing the nanocapsules, pessaries, suppositories, lozenges coated on one surface with a bioadhesive for use in the buccal cavity or formulations for administering an active agent sublingually. The nanocapsules will generally be formulated in unit dosage form for administration or application in an amount and for a time prescribed by an attending physician.

The preferred method for preparing the nanocapsules according to the invention comprises interfacial polymerisation of an alkyl-2- cyanoacrylate under polymerisation initiating conditions at the interface of a two phase aqueous system. The initiating conditions preferably involve the use of an initiator of polymerisation located within droplets of a discrete phase, while a continuous phase is or contains an inhibitor of polymerisation.

Generally, the continuous phase will be present in a large excess relative to the discrete phase, for example in a ratio of 100:1-50:1.

The aqueous system preferably comprises an aqueous solution of two or more water soluble immiscible polymers. Such water soluble immiscible polymers are known (see for example, Alberdsson, P. A. "Partition of cell particles and macromolecules", Wiley, International Scientific N.Y. (1971 ) pp 30-37).

The polymers are selected primarily on the basis of their compatibility and density. As regards the former criterion, there should be little or no affinity between the polymers, such that they do not form aggregates or interact unfavourably in solution.

The following are examples of suitable combinations of polymers:

Dextran sulphate and methylcellulose Dextran sulphate and polyethyleneglycol

Dextran sulphate and polyvinylalcohol

Diethylaminoethyldextran and polyethyleneglycol

Dextran and polyvinylalcohol

Dextran and methylcellulose Dextran and polyethyleneglycol Dextran and Ficoll (Ficoll is a Trade Mark) Dextran and oxypropyldextran

Dextran and a mixed polymer of ethylene oxide and propylene glycol such as Pluronic (Pluronic is a Trade Mark) Dextran and polypropyleneglycol

Oxypropyldextran and polyethyleneglycol

Sodium carboxymethyldextran and polyvinylpyrrolidone

Dextran and chitosan

Dextran and dextran sodium sulphate

The formation of a stable two phase system also depends on the concentrations of the respective polymers in the solution. If the concentration of the polymers is below a critical level, then the two aqueous polymers will not separate into layers. The behaviour and characteristics of different polymers in combination must be determined empirically (see Alberdsson, P. A. (1971) supra).

As described above, even when concentrations of polymers which allow separation of the two polymers are used, this separation is not absolute. Thus, small amounts of the less dense polymer in solution become entrapped within the more dense one and vice versa. The amount of each polymer which becomes entrapped is also dependent on the concentration of polymers used in the system. The amount of each polymer entrapped in the other can be calculated over a range of concentrations which permit stable two phase system formation.

According to a further aspect of the invention, there is provided a process for the manufacture of nanocapsules comprising an aqueous phase enclosed in a polymeric shell, said process comprising the following steps:

1 ) forming a two phase aqueous system from a solution of two or more immiscible water soluble polymers, said polymers being equilibrated in the respective phases of the two phase system; 2) selecting a large excess of one phase formed in step 1 and adding it to a minor amount of the second phase formed in step 1 and forming an emulsion therefrom, said emulsion as formed containing discrete droplets of the minor phase dispersed in a continuum of the phase present in large excess; and

3) adding a predetermined amount of a monomer capable of polymerising by interfacial polymerisation at the surface of the droplets formed in step 2 and any material entrained therein, so as to form nanocapsules.

The equilibration in step 1 ensures that the respective phases are saturated by the respective polymers prior to the emulsification step and allows one to ensure that a given amount of droplets of one phase is encapsulated by the added monomer when said monomer is added in a predetermined amount.

If required, the pH of either phase can be altered so as to promote interfacial polymerisation. For example, an acid, such as citric acid, can be added prior to emulsification so as to inhibit polymerisation in the continuous phase and ensure that interfacial polymerisation occurs at the surface of the droplets defining the discrete phase as hereinafter described.

If it is desired to encapsulate an active agent, this is added prior to emulsification. One or more auxiliary agents may be added with the active agent to stabilise or otherwise to ensure optimal encapsulation of said active agent. Inclusion of an initiator of polymerisation may also be appropriate at this stage as hereinafter described.

The uptake of active agent in droplets of the discrete phase can be promoted by adding, prior to emulsification, one or more substances which cause said active agent to be expelled by the continuous phase as hereinafter described. Alternatively, the uptake of active agent in droplets of the discrete phase can be promoted by adding, prior to emulsification, one or more substances which cause the active agent to be attracted by said discrete phase as hereinafter described.

The principal stages in the encapsulation process are as follows:

Stage 1 :

A two phase aqueous system is formed containing an aqueous solution of two or more immiscible water soluble polymers capable of forming such a two phase system as follows. The polymers selected are dissolved in water, whereupon they settle into two layers, following equilibration of the respective polymers as hereinabove described. This separation occurs primarily because the polymers are of different densities. If required, a further polymer which partitions selectively into one or other of the layers may be added during this stage, said further polymer having the capability to selectively concentrate a target active agent to be added in stage 2 and which it is desired to encapsulate in either one or the other phase of the two phase system.

The rate or time required for the separation of the layers depends on the choice of the individual polymers. Left to gravity alone, the separation can take from several hours to several days. Separation can be accelerated by centrifugation.

After separation, the two aqueous solutions, upper and lower, are decanted into separate flasks. At this point, two stable systems have been created in which the upper phase is equilibrated with lower phase and vice versa. Both phases are used in the creation of an emulsion in Stage 2.

Stage 2:

In this stage, an emulsion is formed between the two separated phases described above. Typically, the emulsion formed contains the two phases in a ratio of the order of 100:1. On emulsification, small droplets of the minor component form a discrete phase dispersed within a bulk or continuous phase of the major component which is present in excess. As both components in the emulsion have been mutually equilibrated in Stage 1 , the droplets are relatively stable in the mixture and thus an emulsion can be formed.

The emulsion is formed by vigorous agitation such as that achieved by means of sonication or vortex mixing. The size of the droplets forming the discrete phase is primarily controlled by the degree and rate of agitation.

Apart from the degree of agitation a number of other factors determine the size of the nanocapsules according to the invention. In general, the more rapid the rate of polymerisation, the lesser the degree of control over polymerisation and the greater the size of the nanocapsules formed.

Furthermore, the rate of polymerisation is inversely proportional to the size of the alkyl group in the alkylcyanoacrylate monomer, so that the larger the alkyl group, the slower the polymerisation and hence the smaller the nanocapsules formed.

Also the lower the pH of the aqueous solution, the slower the rate of polymerisation and the smaller the size of the nanocapsules formed.

The rate of polymerisation is primarily controlled by pH and the size of the ester (alkyl) groups.

The active agent to be encapsulated is included in the emulsification process. The choice of upper or lower phase (created in Stage 1 ) to form the minor component in the emulsion is determined by the physical and chemical properties of the active agent. Indeed, such properties also influence the choice of polymers used in Stage 1. The droplets within the emulsion become encapsulated by the alkyl-2- cyanoacrylate added in the next stage - Stage 3. Accordingly, it is desirable to selectively concentrate the active agent inside the droplets. Thus the choice of upper or lower phase to form the droplets in the emulsion is primarily determined by the affinity of the active agent for the respective phases.

The method also allows for an initiator of polymerisation to be concentrated inside the droplets. In some instances, this may be the active agent itself. If this is not an initiator, however, this must also be added at this stage.

Stage 3:

In this stage, an alkyl-2-cyanoacrylate is added and the droplets are encapsulated following polymerisation at the droplet surface. Alkyl-2-cyanoacrylates polymerise on contact with an initiator of polymerisation. Nucleophilic chemical groups are good initiators of polymerisation. Conversely, acids, particularly strong acids, inhibit the polymerisation process. In the emulsion created in Stage 2, polymerisation in the bulk phase or continuous phase is inhibited by a low pH. This effect is reversed when the monomers encounter the initiator at the surface of the droplet, resulting in encapsulation by polymerisation of the droplet which includes the active agent.

Concentration of active agent in droplets

For optimum yield, it is desirable to concentrate the drug or other substance constituting the active agent in the dispersed droplets which are in low concentration relative to the surrounding polymeric solution. Thus, by assessing the characteristics and properties of the target drug or other substance, one can determine its affinity for the respective phases.

However, it is possible that a given active agent will not have a strong affinity for either phase in the system and will be evenly distributed between them. The efficiency of encapsulation in this case will be low and the yield obtained poor. The method according to the invention provides a means for selectively concentrating the drug in the droplets by ionic, hydrophobic or other interaction and, thereby, increasing the yield.

In the case of poor affinity of the active agent for one phase or the other a third polymer may be added to the system. This third polymer has characteristics such that it is compatible with one of the phases and does not partition into a third phase. The role of the third polymer is to either attract or repel the active agent by hydrophobic, ionic or other interaction. For example, in the dextran/ polyethyleneglycol system described below dextran sulphate can be added which concentrates in the dextran phase of the two phase system. At the appropriate pH, the negatively charged dextran sulphate will attract positively charged species such as peptides, concentrating them in the dextran phase. Alternatively, chitosan may be added to the dextran phase. This polymer at the appropriate pH is positively charged and will attract negatively charged species or repel positively charged ones. Examples of these interactions are given below.

The nanocapsules prepared in accordance with the invention can be collected in a manner known per se, for example by filtration. Alternatively, they can be dried, for example by lyophilization and later rehydrated prior to use. Of course, drying may not be acceptable in the case of certain active agents which might be denatured in this way, for example polypeptides.

In Examples 1 -4 of the following Examples the active agents are all peptides and at the pH indicated are positively charged. They therefore function as initiators of alkyl 2-cyanoacrylate polymerisation. However, in some instances as in the case of Example 5 the active agent may not itself act as an initiator. In these instances an initiator must be added which will partition into the dispersed droplet phase. Again, this partitioning may be manipulated by adding a third polymer to the appropriate phase as discussed above. Suitable initiators are ammonium salts, amino acids, peptides and proteins or other substances compatible with the final use of the nanocapsules. Best modes for carrying out the invention

The invention will be further illustrated by the following Examples.

In the following Examples the method according to the invention is exemplified by the use of the dextran/polyethylene glycol (PEG) two phase system to encapsulate therapeutic peptides normally administered to man by injection.

Example 1

Insulin in a Two Component Two Phase System

In this Example, the method according to the invention is used to encapsulate insulin (porcine 25.9 IU/mg; 80.9% purity Kaynas Plant of Endocrine Preparations, Kaynas, Lietuva), and thus the product naturally concentrates in the PEG phase. Insulin, at the pH used in this Example concentrates in the PEG phase, in a ratio of 5.6:1 i.e. the concentration in the PEG phase is 5.6 times higher than that in the dextran phase.

Preparation of Two Phase System:

10 g of dextran (dextran as sold under the Trade Mark Polyglykin by Krasnoyarsky Plant of Medical Preparations Krasnoyarsk, Russia) and 1.14g of PEG (supplied by Schuchardt,

Munich, Federal Republic of Germany) are dissolved in 50 ml of water by mixing and heating to 80°C. After cooling, 50 mg of citric acid (citric acid monohydrate obtained from Belgorodsky Plant of Citric Acid, Belgorod, Russia) and 0.2 ml of H3PO4 are added. If required, further H3PO4 may be added to adjust the pH to 2.5-3. The mixture is then allowed to stand and the layers formed separated in a separatory funnel. Preparation of Emulsion and Polymerisation:

0.2 ml of the upper phase (primarily PEG) are added to 20 ml of the lower phase (primarily dextran) along with 40 mg of insulin and 1 mg of cysteine. Insulin contains disulphide bridges which are essential for its structure and the cysteine is added only as a stabiliser of these disulphide bridges. The mixture is next placed in a cooled sonication reactor vessel and is sonicated for 2 min. with continuous cooling. 0.4 ml of ethylcyanoacrylate is then added and sonication with continuous cooling in an ice bath continued for a further 10 min. After this time the suspension is transferred to a magnetic stirrer and stirred for 6 hours. The pH is then adjusted to 7.2-7.4 by the addition of IN NaOH with continuous stirring.

The nanocapsules produced are sized by a Coulter Counter. In the remaining Examples, the nanocapsules produced are sized in the same way. The mean diameter of nanocapsules produced in this

Example are 274 nm. The concentration of insulin not associated with the nanocapsules is 0.41 mg/ml, corresponding to a yield of encapsulation of 79.5%.

Example 2

Dalargin Encapsulation in a Three Component Two Phase System

In this Example the active agent to be encapsulated is the peptide Dalargin (Dalargin is the trivial name for a peptide with the following structure: Tyr-D.Ala-Gly-Phe-Leu-Arg obtained from the Cardiological Scientific Centre of the Academy of Medical Sciences of Russia), and the product distributes evenly between the phases. A third polymer, dextran sulphate is added to concentrate the peptide in the dextran phase.

The distribution of Dalargin between the two phases is approximately equal. Dextran sulphate (dextran sodium sulphate obtained from Ioba Feinchemia, Austranal, A-2401 Fishhamend, Austria) is added as a third component in the two phase system. On addition of dextran sulphate the distribution is skewed to the dextran phase with a concentration ratio of 5.7:1 in the dextran phase/PEG phase.

Preparation of Two Phase System:

Preparation of the two phase system is carried out in the manner as described in Example 1.

Preparation of Emulsion and Polymerisation:

0.5 ml of the lower phase, 50 mg of Dalargin, 50 mg of sodium dextran sulphate and 0.1 ml of H3PO4 are added to 50 ml of the upper phase of the above preparation. After the components are dissolved, the mixture is placed in a sonication reaction vessel and sonicated with continuous cooling for 2 min. After this, 1 ml of ethylcyanoacrylate is added and the emulsion sonicated for a further 10 min. with continuous cooling. The suspension is then adjusted to pH 7.2-7.4 by the addition of IN NaOH with continuous stirring.

The mean diameter of the nanocapsules so produced is 295 nm. The concentration of Dalargin not associated with the nanocapsules is 0.1 mg/ml, corresponding to a yield of encapsulation of 90%.

Example 3

Surfagon Encapsulation in a Three Component Two Phase System

This Example shows an alternative method of using an ionic interaction to that illustrated in Example 2. In this case, a positively charged substance, chitosan, is added to the two phase system and this stays in the dextran phase. This forces the positively charged peptide used in the Example, Surfagon (Surfagon is the trivial name for a peptide with the following structure: Glu-His-Trp-Ser-Tyr-D.Ala-Leu- Arg-Pro obtained from the Cardiological Scientific Centre of the Academy of Medical Sciences of Russia), into the PEG phase. The distribution of Surfagon between the two phases is approximately equal, as for Dalargin in the case of Example 2. However, in this Example the addition of the third component, chitosan, results in the peptide concentrating in the PEG phase, with a concentration ratio (PEG/dextran) of 7.4:1.

Preparation of Two Phase, Three Component System:

0.25 g of chitosan is added to 50 ml of 5% acetic acid solution containing 0.2 ml of H3PO4. After the components have dissolved, the pH is adjusted if necessary to 1.5-2.0 by addition of IN NaOH. 10 g of dextran and 1.14 g of PEG are added to the chitosan solution. After mixing by stirring, the layers are allowed to separate in a separatory funnel and the phases separated.

Preparation of Emulsion and Polymerisation:

0.05 ml of the upper phase is added to 5 ml of the lower phase and the mixture placed in a sonication vessel. 5 mg of Surfagon is added and the mixture sonicated for 3 min. with continuous cooling. 0.1 ml of ethylcyanoacrylate is added to the emulsion and sonication continued for a further 10 min. with cooling. After this period, the suspension is transferred to a conical flask and stirred continuously for 6 hours. After this time, the pH is adjusted to 7.2-7.4 by addition of IN NaOH.

The mean diameter of the nanocapsules so produced is 310 nm. The concentration of Surfagon not associated with the nanocapsules is routinely 0.1 mg/ml, corresponding to an encapsulation yield of 90%. Example 4

Insulin Encapsulation in a Three Component Two Phase System

In Example 1 above, it was demonstrated that insulin may be encapsulated efficiently in the dextran/PEG system since it concentrates preferentially in the PEG phase. However, this system may be further manipulated by the addition of a third component such that the product further concentrates in the upper or PEG phase. In this Example polyvinyl alcohol is added to the system and the hydrophobic interaction between this substance and insulin is such that the concentration ratio (PEG/dextran) becomes 9.3:1 , as compared to 5.6:1 in Example 1.

Preparation of a Three Component, Two Phase System:

10 g of dextran, 1.14 g of PEG and 0.75 g of polyvinyl alcohol (polyvinyl alcohol as sold under the Trade Mark "Polydez" by the Polyvinylacetate Plant, Erevan, Armenia) are dissolved in 50 ml of water by stirring and heating to 80°C. After cooling, 50 mg of citric acid is added and the pH adjusted to 2.5-3 by addition of H3PO4 if required. After separation of the two layers, the phases are decanted into separate containers.

Preparation of Emulsion and Polymerisation:

0.2 ml of the upper phase is added to 20 ml of the lower phase and then 1 mg of cysteine, 18 mg of citric acid and 40 mg of insulin are added and the mixture stirred. The mixture is then placed in a sonication vessel and sonicated for 2 min. with continuous stirring. Next, 0.4 ml of ethylcyanoacrylate monomer is added and sonication continued for a further 10 min. After this time, the suspension is stirred for 6 hours and finally the pH adjusted to 7.2-7.4 by addition of IN NaOH. The mean diameter of the nanocapsules produced by this method is 310 nm. The concentration of insulin not associated with the nanocapsules is routinely 0.35 mg/ml, corresponding to a yield of encapulation 82.5%.

Example 5

Encapsulation of Apomorphine

In this Example, the drug apomorphine does not function as an initiator of polymerisation. Accordingly, an initiator is added to the system and in the reaction conditions described below this partitions in the dextran phase. The anticoagulant drug heparin is used as the initiator since this is a biologically compatible substance without significant pharmacological effect at the concentrations used. Structurally heparin is an aminosulphopolysaccharide and therefore functions efficiently as an initiator.

Preparation of Two Phase System:

Preparation of the two phase system is carried out in the same manner as described in Example 1.

Preparation of Emulsion and Polymerisation:

2.3 mg of cysteine are added to 60 ml of the top phase (primarily PEG). 50 μl of an aqueous heparin solution, containing 2.5 mg of heparin (Sigma Chemical Company) are added to 1.5 ml of the bottom phase prepared above (primarily dextran) and then 25 mg of apomorphine (as apomorphine hydrochloride obtained from Sigma Chemical Company) are added.

After mixing, the pH is adjusted to 2-2.5 if required by addition of H3PO4. The mixture is then placed in a sonication reaction vessel and sonicated with continuous cooling for 2 min. 1.8 ml of butyl-2- cyanoacrylate are added to the emulsion and sonication continued with continuous cooling for a further 30 min. The reaction mixture is then transferred to a magnetic stirrer at room temperature and the reaction continued for a further 6 hours. After this time 150 mg of ascorbic acid are added to prevent oxidation of the drug during storage. Immediately before use the mixture is titrated to pH 7.2-7.4. The product is stored at 4°C prior to use.

The mean diameter of the nanocapsules produced by this method is 320 nm. The concentration of apomorphine not associated with the nanocapsules is routinely 0.03 mg/ml corresponding to a yield of encapsulation of 92%.

In vivo studies

The pharmacological effect of drug-loaded nanocapsules fed orally was examined in animal models. The results of studies with Surfagon and Dalargin nanocapsules prepared as described in Example 3 and 2, respectively are given below.

Surfagon

Surfagon is a proprietary luteinising hormone releasing hormone analogue and acts by controlling the release of luteinising hormone (LH) in males and females. It is used clinically to treat endometriosis and prostate cancer and is currently given as a s.c. depot injection.

The encapsulated Surfagon used in these experiments was prepared by the method in Example 3 above and the concentration of the nanocapsules was adjusted such that the concentration of Surfagon contained in the nanocapsule preparation was 0.8 mg/ml.

LH Release

This study involved a total of 51 male rats, divided into 3 experimental groups. LH levels were examined in blood samples taken following sacrifice of animals at 120, 180 and 240 minutes after administration of the test sample. LH (ng/ml) was measured by standard radioimmunoassay.

The experimental rationale was as follows:

Group 1 : Control group, 15 animals, 0.2 ml of physiological saline introduced by intra-abdominal injection.

Group 2: Native Surfagon, injected intra-abdominally at a rate of 2 μg/Kg (18 animals)

Group 3: Surfagon nanocapsules, introduced orally by gastric tube at a rate of 20 μg/Kg in 0.5 ml of pasteurised bovine milk (18 animals).

In groups 2 and 3, 6 animals were sacrificed and assayed at each time point. In group 1 , 5 animals were similarly treated at each time point.

The results are presented in Table 1; the means and standard deviations are shown.

Table 1

Concentration of LH in Serum (ng/ml) following Surfagon administration

Group Sample Point (Min.)

120 180 240

1 0.55 + 0.065 0.25 + 0.100 0.30 + 0.030

2 0.52 + 0.100 0.78 + 0.070 0.30 + 0.100

3 0.45 + 0.065 0.91 + 0.071 0.35 + 0.240 It is known that the uptake of free Surfagon across the gut wall is negligible. The results indicated in Table 1 show that encapsulated Surfagon given orally exerts a significant pharmacological effect.

Testosterone Release

In this study the effect of Surfagon nanocapsules on testosterone production over a period of 30 days was examined. On day 32, the animals were sacrificed and the appropriate organs analysed. In the study, 25 rats divided into three groups were used. The experimental rationale was as follows with 10 animals in groups 2 and 3 and 5 animals in group 1 :

Group 1 : Control, no injection.

Group 2: Intramuscular injection of free Surfagon at a rate of 10 μg/Kg in 0.2 ml of physiological saline every day for the duration of the experiment.

Group 3: Intramuscular injection of Surfagon nanocapsules at a rate of 100 μg/Kg once every 10 days for the duration of the experiment (3 times in total).

The results are presented in Table 2; the means and standard deviations are shown.

Table 2

Testicle and prostate mass in Surfagon treated rats

Group Testicle Mass (g) Prostate Mass (mg)

1 2.10 ± 0.18 51.0 ± 4.0

2 1.40 ± 0.09 31.0 ± 0.1

3 1.40 ± 0.09 39.2 ± 2.6

The results indicated in Table 2 show that encapsulated Surfagon given intramuscularly exerts a significant pharmacological effect.

Dalargin

Dalargin is a proprietary encephalin-like peptide with activity in wound healing, prevention and treatment of anaphylactic shock and analgesia. Dalargin nanocapsules were prepared as described in Example 2 and assessed in two separate animal model systems.

1. Anaphylactic Shock.

Anaphylactic shock was induced in male guinea-pigs with body weight of between 250-300 g by the sensization method described by Shatemikoo, B.A. et al. (Soviet Academy of Sciences (1982) 2, pp 27- 31), which is outlined below.

Chicken ovalbumin was used as an allergen and was fed in 50 mg doses orally through a gastric tube daily for 14 days. After this time, anaphylaxis was induced by injection of ovalbumin into the heart. The degree of response was determined by the death rate and also by the number with convulsive syndrome and by the anaphylactic index (Weigle, W. et al. J. Immunol. (1960) 55, pp 469-477). The concentration of Dalargin was 0.8 mg/ml of nanocapsule preparation. On days 11 to 13 of the experiment the test substances were administered to the different experimental groups each of which consisted of 18 animals.

Group 1 Controls, intraperitoneal injection of physiological saline (0.5 ml).

Group 2 Intraperitoneal injection of free Dalargin at a rate of 50μg/Kg in 0.5 ml of physiological saline.

Group 3 Intraperitoneal injection of Dalargin nanocapsule preparation at a rate of 150 μg/Kg of Dalargin, given on the 11th day only.

Group 4 Oral administration of Dalargin nanocapsules given via a gastric tube at a rate of 50 μg/Kg in 1 ml pasteurised bovine milk.

The results are presented in Table 3.

Table 3

Effect of encapsulated Dalargin in experimentally induced anaphylaxis

Group Severity of Response Death Convulsion Anaphylactic Index

1 27.8% 66.7% 2.9

2 16.7% 38.9% 2.06

3 22.2% 33.3% 1.58

4 16.7% 27.8% 1.89 These results show that encapsulated Dalargin given orally and intraperitoneally is effective in reducing the degree of anaphylaxis.

2. Wound Healing.

The wound healing effect of nanoencapsulated Dalargin was assessed in a rat model, with an individual animal body weight between 120-130 g.

First, the animals were anaesthesised with nembutane (35 mg/Kg, intraperitoneally) and the hair shaved from the back of the animal to an area of approximately 10-12 cm². The shaved area was surface sterilized with an alcohol/iodine solution and then identical outlines for the wound site accurately drawn on each animal using a template. The template was positioned at approximately 50 mm from the distal angle of the eyes. The diameter of the outline was 17 mm. Then full depth wounds were made in the area with a sharp edged scissors, the surface of the wound dried with sterile gauze and then lightly dusted with a topical antibiotic to prevent opportunistic infection.

The test substances were introduced immediately after wound formation and administration continued for 12 days. The Dalargin nanocapsule preparation used was as described in the previous experiment. 50 rats divided into groups of 12 or 13 animals were used. Measurement of the wound area was carried out every 4 days. This measurement was based on the degree of granulation and wound healing. The experimental rationale was as follows.

Group 1 Controls, 0.1 ml physiological saline was administered intraperitoneally by injection daily for 12 days.

Group 2 Free Dalargin administered by intraperitoneal injection at a rate of 10 μg/Kg in 1 ml of physiological saline daily for 12 days. Group 3 Dalargin in nanocapsules administered intraperitoneally at a rate of 50 μg/Kg of active agent in 0.1 ml of physiological saline every 5 days.

Group 4 Dalargin in nanocapsules administered orally by gastric tube at a rate of 50 μg/Kg in 0.5 ml of pasteurized bovine milk daily for 12 days.

The results are presented in Table 4; the units shown are percentage of the test area with full wound healing.

Table 4

The effect of Dalargin on wound healing

Group Time (days) 4 8 12

1 28.2 61.6 82.4

2 58.9 82.9 99.5

3 43.6 69.5 91.7

4 60.4 78.4 97.3

The effect of Dalargin administered orally was negligible (data not shown). The results indicated in Table 4 clearly show that Dalargin is delivered to the appropriate site at a significant pharmacological concentration following oral administration in nanocapsules.

Apomoφhine

The pharmacological effects and bioavailability of encapsulated apomoφhine were investigated in an alcoholic rat model. The basis of this model is that apomoφhine reduces the urge for alcoholic rats to take ethanol in a situation where the animals have free access to both water and ethanol. Opiates and other mood altering drugs can be assessed in this system for efficacy or bioavailability by monitoring the 27

relative consumption of water and ethanol. The encapsulated apomoφhine was prepared in the manner described in Example 5.

Alcoholic Female Rat Model.

Second stage alcoholic Wistar rats were used in which free-will consumption of ethanol had been established for 4.5 months. 20 animals were included in each experimental group, as described below, and all animals had free access to 96% ethanol, water and feed. The experimental rationale was as follows.

Group 1: 0.2 ml of physiological saline administered s.c. daily for 14 days, per animal.

Group 2: Free apomoφhine administered s.c. once daily for 14 days at a rate of 1 mg/Kg, per animal.

Group 3: Encapsulated apomoφhine administered s.c. once on days 1, 3, 6, 9 and 12 at a rate of 1 mg/Kg, per animal.

The results are presented in Table 5 and 6; the means and standard deviations are shown.

Table 5

Free consumption of water

Table 6

Free consumption of Ethanol

The results indicated in Tables 5 and 6 clearly show that encapsulated apomoφhine is active and is bioavailable when administered by s.c. injection. Furthermore, the pharmacokinetics of the encapsulated product are more favourable, allowing a more sustained period of reduced alcohol consumption than the free product.

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Claims

1. Nanocapsules comprising an aqueous phase enclosed in a polymeric shell, said polymeric shell being formed of a network of polymers.

2. Nanocapsules according to Claim 1, having a diameter in the range 150-400 nm.

3. Nanocapsules according to Claim 1 or 2, wherein an active agent is contained in the aqueous phase.

4. Nanocapsules according to any preceding claim, wherein the polymeric shell is comprised of a polyalkylcyanoacrylate material.

5. Nanocapsules according to Claim 4, which are formed by interfacial polymerisation in a two phase aqueous polymeric emulsion.

6. Nanocapsules according to Claim 5, wherein the respective phases are present as a discrete phase and a continuous phase

7. Nanocapsules according to Claim 5 or 6, wherein the uptake of active agent in droplets of the discrete phase is promoted by adding one or more substances which cause the active agent to be expelled by the continuous phase.

8. Nanocapsules according to Claim 5 or 6, wherein the uptake of active agent in droplets of the discrete phase is promoted by adding one or more substances which cause the active agent to be attracted by said discrete phase.

9. Nanocapsules according to Claim 7 or 8, wherein the or each substance is a charged polymer.

10. Nanocapsules according to any one Claims 3-9, wherein the active agent is a peptide or polypeptide.

11. An active agent delivery system comprising nanocapsules according to any one of Claims 1-10.