CA2275717C - Chitosan-gelatin a microparticles - Google Patents

Abstract

There is provided a pharmaceutical composition for use in the improved up--take of therapeutic agents across mucosal surfaces which comprises a mixture of chi-tosan and a type A, cationic, gelatin, together with a therapeutic agent. The composition is preferably in the form of microparticles, such as microspheres.

Description

CHITOSAN-GELATIN A MICROPARTICLES

This invention relates to novel drug delivery conlpositions which provide for the improved uptake of therapeutic agents across mucosal surfaces.
Polar drugs, including high molecular weiglit peptides, proteins and polysaccharides, are typically not effectively absorbed across mucosal membranes, such as the gastrointestinal tract, the eye, the vagina, the jo nasal cavity or the rectum. Such molecules are thus normallv onlv given bv injection, which inevitably gives rise to well known problems associated widi patient compliance, the cost of treatment, as well as the potentially harmful effects, such as phlebitis and pain, of the injection.

It is well known in the literature that the absorption of polar molecules across niucosal membranes may be greatlv improved if they are administered in combination with so-called "absorption enhancers".
L=xamplcs of absorption enhancers whicli have been described in the literature include non-ionic surfactants, cvclodextrins, phospholipids and bile salts. (For a review see Davis et aI (eds.), Delivery Systems for Peptide Dru.gs, Plenum Press, New York, 1987; and Lee (ed.), Peptide and Protein Delivcry, Marcel Dekker lnc., New York, 1991.) EP-A-023 359 and EP-A-122 023 describe powdery pharmaceutical compositions for application to the nasal niucosa, as well as metliods for the administration of suc11 compositions. The pllarmaceutical compositions allow polypeptides and derivatives thereof to be effectively absorbed through the nasal mucosa. Similarly, US 4,226,849 describes a metliod for administering a powdery medicanlent to the nasal nlucosa, in whicli the preferred composition lias niucoadhesive properties.

Formulations based on microspheres for mucosal delivery have been described in WO 88/09163. Tlie forniulations contain certain enhancers to aid effective penetration of the mucosa bv the drug. WO 89/03207 describes microsphere forniulations which do not require an enhancer.

Chitosan is a derivative of chitin or poly-N-acet_yl-D-glucosamine in which io the greater proportion of the N-acetyl groups have been removed through hvdrolysis. It is available from several suppliers including Pronova, Drammen, Norway, and, depending on the grade selected, is soluble in water and/or aqueous acid up to pH values of between 6.0 and 7Ø

Cliitosan lias previously been used to precipitate proteinaceous material and to make surgical sutures. It has also been employed previously in oral drug formulations in order to improvc the dissolution of poorly solublc drui-,s (sec Sawavanagi et al, Cheni. Pharni. Bull., 31 (1983) 2062-2068) or for thc sustained release of drugs by a process of slow erosion from a hvdrated compressed matrix (Nagai et al, Proc. Jt. US Jpn. Semin. Adv.
Chitin Chitosan Relat. Enzvmcs, 21-39, Zikakis J.P. (ed.), Academic Press, Orlando, 1984).

WO 90/09780 describes a composition comprising a drug and a polycationic substance (e.g. chitosan) that proniotes the transport of the drug across mucosal membranes. The composition may also comprise microspheres of the polvcationic substance.

T 1 i WO 96/05810 describes a conlposltlOn comprising a pharmacologically active compound and particles, preferably powders or microspheres, of chitosan or a chitosan derivative or salt, where the particles are either solidified or partially cross-linked such that they have a zeta-potential of betwecn +0.5 and +50 mV. Solidified particles are made by treating particles niade from a water soluble chitosan salt with an alkaline agent, such as sodium hydroxide, in non-acid contalnlng water to render them insoluble.

io Cliitosan niicrospheres liave also been produced for use in enhanced chromatouaphic separation (Li Q. et al, Biomater. Artif. Cells Immobilization Bioteclinology, 21 (1993) 391-398), for the topical delivcrv of drups (Macllida Y., Yakugaku Zasshl., 113 (1993) 356-368), for drug targeting after injection (Ohya Y et al, J. Microcncap., 10 (1993) 1-9), as an implantable controlled release delivery system (Jameela and Jayakrishnan. Biomaterials, 16 (1995) 769-775) and for the controlled release of'drugs (see Bodmeier R. et al, Pharm. Res., 6 (1989) 413-417 and Chithanlbara et al, J. Pharni. Pharniacol.. 44, 1992. 283-286).

EP 454044 and EP 486959 describe polvelectrolvte microparticlcs or polysaccharide niicrospheres, including chitosan niicrospheres, for use in the ccmtrolled release of drugs. Chitosan microspheres crosslinked with olutaraldehydc have also been described in JP 539149.

Gelatin is a purified protein obtained eitlier by partial acid hydrolysis (type A) or by partial alkaline hydrolvsis (type B) of animal collagen. Type A
gelatin is cationic with an isoelectric point between pH values of 7 and 9, whereas type B gelatin is anionic witll an isoelectric point between pH
values of 4.7 and 5. Gelatin is known to swell and soften when inunersed in cold water, cventually absorbing between 5 and 10 tinles its own weight in water. It is soluble in liot water, forming a gel on cooling. Gelatin is used as a liaemostatic in surgical procedures as an absorbable film or sponge, which can absorb many times its own weight in blood. It is also employed as a plasma substitute, and may be used in the preparation of pastes, pastilles, suppositories, tablets and hard and soft capsule shells for oral formulations.

The production of gelatin microspheres has been widely described in the Ic~ literature. Gelatin microspheres liave been produced bv an emulsification method involviny crosslinking with -lutaraldehyde, producing microspheres of less than 2 m in diameter (Tabata and Ikada, Pharm.
Res. 6 (1989) 422-427). Cortesi er al (Int. J. Pharm. 105 (1994) 181-186), Natruzzi et al (J. Microencapsulation, 11 (1994) 294-260) and Esposito el al (Int. J. Pharm., 117 (1995) 151-158) have reported the production of microspheres of a mean diameter of 22 m using a coacervation emulsification method. Microspheres as produced by the latter processes were not crosslinked. Microspheres of a smaller size have been produced accordinp to a similar niethod by Esposito el al (Pharm.

2n Sci. Conunun. 4 (1994) 239-246). The type of gelatin (A or B) used in these studies was not spccified.

The production of microspheres by complexation, between a negatively charged nlaterial such as alginate and a positively cliarged chitosan has been described in the literature. For example. Polk et al, J. Pharm. Sci., 83 (1994) 178-185) describes the production of chitosan-alginate microspheres by the addition of an alginate solution to a solution of chitosan and calcium ions. The highest concentration of chitosan used in the microsphere fornlulations was 5.2% w/w. Similarlv, the formation of T..__...._.T__.
T

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complex coacervates between oppositely cliarged poiyions, namely a positively cllarged chitosan and a negatively charged type B gelatin has been described by Remunan-Lopez and Bodmeier (Int. J. Pharni. 135 (1996) 63-72). These workers found the optimum chitosan:gelatin ratio to be in the range 1: 10 to 1:20. The coacervate was obtained in a dry form by decanting the supernatant after centrifugation and drying at 60 C.

We have now found, surprisingly, that microparticles, produced from a combination of a chitosan and a cationic type A gelatin, possess particularly advantageous properties, which enable the improved transport of therapeutic agents, including polar drugs, across mucosal surfaces such as the nasal cavity.

Thus, according to a first aspect of the invention there is provided a composition comprising a mixture of chitosan and type A. cationic, gelatin, togetlier with a therapeutic agent (hereinafter referred to as "the compositions according to the invention").

By "mixture of chitosan and type A gelatin" we include any composition comprising a chitosaii, as defined hereinafter, and a type A gelatin, as defined hereinafter, whether a physical and/or chemical association between these two coiistituents exists or not.

Tlie term "chitosan" will be understood by those skilled in the art to include all derivatives of cliitin, or poly-N-acetyl-D-glucosamine ( including all polyglucosamine and oligomers of glucosamine niaterials of different molecular weights), in which the greater proportion of the N-acetvl groups llave been removed througli hydrolysis. We prefer that the chitosan has a positive charge.

Chitosan, cllitosan derivatives or salts (e.g. nitrate, phosphate, sulphate, hydrochloride, glutamate, lactate or acetate salts) of chitosan may be used. We use the term chitosan derivatives to include ester, ether or other derivatives formed by bonding of acyl and/or alkyl groups with OH
groups, but not the NH2 groups, of chitosan. Examples are 0-alkyl ethers of chitosan and 0-acyl esters of chitosan. Modified chitosans, particularly those conjugated to polyethylene glycol, are included in this definition.
Low and medium viscosity chitosans (for example CL113, G210 and io CLI10) nzay be obtained from various sources, including Pronova Biopolymer, Ltd., UK; Seigagaku America Inc., MD, USA; Meron (India) Pvt, Ltd., India; Vanson Ltd, VA, USA; and AMS Biotechnology Ltd., UK. Suitable derivatives include those which are disciosed in Roberts, Chitui Clremistry, MacMillan Press Ltd., London (1992).

The chitosan or chitosan derivative or salt used preferably has a molecular weight of 4,000 Dalton or more, preferably in the range 25,000 to

2,000,000 Dahon, and most preferably about 50,000 to 300,000 Dalton.
Chitosans of different low molecular weights can be prepared by enzymatic degradation of chitosan using chitosanase or by the addition of nitrous acid. Both procedures are well known to those skilled in the art and are described in recent publications (Li et al, (1995) Plant Physiol.
Biocheni. 33, 599-603; Allan and Peyron, (1995) Carbohydrate Research 277, 257-272; Damard and Cartier, (1989) Int. J. Biol. Macromol. 11, 297-302).

Preferably, the cliitosan is water-soluble and may be produced from cliitin by deacetylation to a degree of greater than 40%, preferably between 50%
and 98%. and more preferably between 70% and 90%. Particular T.......... 17--deacetylated chitosans which may be nlentioned include the "Sea Cure "
series of chitosan glutamates available from Protan Biopolymer A/S, Dranunen, Norway.

The term "type A gelatin" includes all cationic proteins wliich are, or may be, obtained by partial acid liydrolysis of animal collagen, and excludes type B gelatins.

Although the compositions according to the invention may be prepared in to a varietv of physical forms using techniques which will be well known to the skilled persoii, we prefer that the compositions are in the form of microparticles. The term "microparticles" includes microspheres, microcapsules and powders. However, we prefer that the microparticles are microspheres.

We have found, surprisingly, that when the compositions according to the invetition are provided in the form of microparticles, such microparticles retain a positive charge and may provide for the improved transport of polar drugs across, or for the improved presentation of vaccines to, rniucosal surfaces, such as the nasal cavity, to such an extent that the effect is superior to that obtained for a chitosan solution, or microparticles produced froni chitosan or type A gelatin alone (e.(,. soluble (spray dried) cliitosan microspheres and gelatin microspheres). The effect is also similar to that obtained for partially aldehyde crosslinked chitosan inicrospheres, yet the compositions according to the invention are sufficiently hard/solid not to require crosslinking. We have further found that the flow properties of these cliitosan/type A gelatin microparticles are superior to those of spray dried chitosan microsplieres and crosslinked chitosan microspheres.

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The microparticles may be prepared by spray drying, emulsification, solvent evaporation, precipitation or other methods known to a person skilled in the art. The therapeutic agent can be incorporated into the microparticles during their production or sorbed onto the microparticles after their production.

When the compositions according to the invention are in the form of microspheres, they may be prepared using for example either io emulsification or spray drying techniques.

When microspheres are prepared by spray drying, a warm mixture of cliitosan and type A gelatin is spray dried with instant cooling of the resultant microspheres. The therapeutic agent may be incorporated by 1s adsorbing onto the surface of the microspheres by freeze drying or spray drying a suspension of the microspheres with the therapeutic agent, or by plivsicallv or mechanically mixing the dried niicrospheres with the therapeutic a~~ent.

20 However, xve have found that microspheres may advantageously be prepared by warming a solution of a chitosan niixed with type A gelatin, whicii is then einulsified and gelated by cooling. We have found tllat, in particular, nlicrospheres prepared in accordance with this technique exhibit the advantageous properties referred to hereinbefore.

In the emulsification technique, the chitosan may be dissolved in water and niixed with type A gelatin under heating to 40 C causing the gelatin to melt. This mixture may be emulsified, at a temperature above the melting point of the gelatin, in an organic medium (e.~.:. a vegetable oil, . . ......... ... TI

such as sunflower oil, soya oil, cotton seed oil or coconut oil), in the presence of an emulsifier with a low hydrophilic-lipophilic balance (HLB) value. Such emulsifiers, which are useful for stabilising water-in-oil emulsions, are known to those skilled in the art (e.g. SpanTM 80). The microspheres may then be solidified by decreasing the temperature of the emulsion to below 10 C with stirring. The microspheres may then be harvested using conventional techniques, for example by adding a pharmaceutically acceptable organic solvent, e.g. chilled acetone or petroleum ether, to the emulsion, centrifugation, washing and drying. The therapeutic agent may be incorporated into the microspheres by adding it to the chitosan/gelatin mixture before emulsification. Alternatively, the therapeutic agent may be adsorbed onto the surface of the microspheres by freeze drying or by spray drying a suspension of the microspheres with the therapeutic agent, or by physically or mechanically mixing the dried microspheres with the therapeutic agent.

Thus, according to a further aspect of the invention there is provided a drug delivery composition in a form suitable for administration to a mucosa comprising a therapeutic agent and microparticles made from a mixture of chitosan and type A gelatin and where the agent is either incorporated into the particles during production or is adsorbed to the surface of the particles, or is present as an admixture.

Microcapsules and powders may be made by modifying the process as defined herein in accordance with techniques which are well known to those skilled in the art, or may be prepared in accordance with other techniques which will be well known to those skilled in the art, including double emulsification processes.

According to a furtlier aspect of the invention there is provided a process for the preparation of a composition according to the invention, which process comprises preparation of type A gelatin/chitosan mi.croparticles (i.e.
microparticles comprising a mixture of type A gelatin and cllitosan) by a 5 process of spray drying or by emulsification, which emulsification may comprise warming a solution of a chitosan mixed with type A gelatin, emulsification and gelation by cooling.

The flow properties of" the niicroparticles can be measured by methods io known to those skilled in the art. One possible method involves the measurcment of the Hausner Ratio where a known weight of material is poured into a nieasuring cylinder and the volume recorded. The c_ylinder is then tapped against a surface a specified number of times and the volume again recorded. The poured and tapped densities are then determined and the I-Iausner Ratio = tapped density/poured density calculated. A ratio of < 1.25 indicates a free flowing material while a ratio of > 1.5 indicates a poor flowing (cohesive) niaterial. Another possible niethod involves the measurement the Angle of Repose by pouring niaterial througlt a funnel held at a fixed height onto a piece of graph paper until a cone is formed. The height (H) and the radius (R) of the cone is determined and the angle calculated (tan 0 = H/R). An Angle of Repose 0 < 300 indicatcs good flow properties while an Angle of Repose 0 > 400 indicates very poor flow properties (James I. Wells, Pharmaceutical Preformuiation, Ellis Horwood Series in Pharmaceutical Technology, 1988).

The size of the microparticles. which includes microcapsules and especially microspheres, is preferably in the range I to 200 m, more II
preferably 1 to 100 gtn, as measur-ed by e.g. liglit nlicroscopy or sieve fractionation.

The microparticles will consist of preferably between 50 and 95%, more preferably between 70 and 90% and niost preferably between 75 and 85 %
of type A gelatin, and correspondingly between 50 aiid 5%, preferably between 30 and 10% and most preferably between 25 and 15% of cliitosan, as measured in relation to the total arnount of gelatin and chitosan in the final composition (i.e. excluding therapeutic agent and Io other ingredients which nlay be included).

The term "therapeutic agent" includes drugs, genes (DNA) or gene constructs, vaccines and components thereof (for example isolated antigens or parts thereof) and monoclonal antibodies. For applications employing such materials as genes, gene constructs, vaccines and monoclonal antibodies, the microparticles can be used to enhance the delivery of the therapeutic agent into the mucosal tissue for enhanced therapeutic effect, for example presentation of an antigen to the underlying lymphoid tissue, and/or transfection of the cells in the mucosal lining.

Preferably the tlierapeutic agent is a polar drug. By "polar drugs" we mean niolecules witli a partition coeff icient (octanol - water system) of less than 50.

The conipositions may be used with therapeutic agents selected from the following non-exclusive list: insulin, PTH (parathyroid hormone), PTH
analogues, PTHrP (human parathyroid hormone peptide), calcitonins (for example porcine, liuman, salmon, cliicken or eel) and synthetic modifications tliereof', enkephalins, LHRH (luteinising hornione releasing llormone) and analogues (nafarelin, buserelin. leuprolide, goserelin), glucagon, TRH (tliyrotropine releasing hormone), vasopressin, desmopressiil, growtll hormone, lleparins, GHRH (growth hormone releasing hormone), CCK (cholecystokinin), THF (thymic humoral factor), CGRP (calcitonin gene related peptide). atrial natriuretic peptide, nifedipine, metoclopramide, ergotamine, pizotizin, pentamidine and vaccines (particularly but not limited to AIDS vaccines, measles vaccines, rhinovirus Type 13 and respiratory syncytial virus vaccines, influenza vaccines, pertussis vaccines, meningococcal vaccines, tetanus vaccines, io diphtheria vaccines, cholera vaccines and DNA vaccines (e.g. one containing a plasmid DNA coding for a suitable antigen)).

Further therapeutic agents include but are not limited to: antibiotics and antimicrobial agents, such as tetracycline hydrochloride, leucomycin, ts penicillin, penicillin derivatives, erythromycin, sulphathiazole and nitrofurazone; anti-migraine compounds, such as naratriptan, sumatriptan, alnitidan or other 5-HT1 agonists; vasoconstrictors, such as phenvlephedrine hydrnchloride, tetrahydrozoline hvdrochloride, naphazoline nitrate, oxvmetazoline hvdrochloride and tramazoline 20 livdrochloride; cardiotonics, such as digitalis and digoxin; vasodilators, such as nitroglycerine and papaverine hvdrochloride; bone metabolism controlling agents, such as vitamin D and active vitamin D3; sex horniones; hypotensives; anti-tumour agents; steroidal anti-inflammatory agents, such as llydrocortisone, prednisone, fluticasone, prednisolone, 25 triamcinolone, triamcinolone acetonide, dexamethasone, betamethasone, beclomethasone and beclometliasone dipropionate; non-steroidal anti-inflanunatory agents, such as acetaniinophen, aspirin, aminopyrine, phenylbutazone, mefanic acid, ibuprofen, diclofenac sodium, indonlethacin, colchicine and probenecid; enzymatic anti-inflammatory T -T..-agents, such as chymotrypsin and bromelain seratiopeptidase; anti-liistaminic agents, such as dephenhydranline llydrochloride, chloropheniramine maleate and clemastine; anti-tussive-expectorants, such as codeine phosphate and isoproterenol hydrochloride; analgesics, such as s opioids (like dianlorphine, morphine and its polar metabolites, such as niorphine-6-glucuronides and niorphine-3-sulphate); anti-emetics, such as nletoclopramide, ondansetron, chlorpromazine; drugs for treatment of epilepsy, such as clonazepam; drugs for treatment of sleeping disorders, such as nlelatonin; drugs for treatment of asthma, such as salbutamol.

Conibinations of the abovenientioned therapeutic agents may be employed.
The compositions according to the invention may be administered orally, nasally, vaginally, buccally, rectally, via the eye, or via the pulmonary route, in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art. For example, compositions may be adtninistered via the nasal route as a powder using a nasal powder device.
-ra the pulmonarv route usin~, a powder inhaler or nietered dose inlialer, i-ia the vaginal route as a powder usiiig a powder device, formulated into a 211 vagina suppository or pessary or vaginal tablet or vaginal gel, via the huccal route formulated into a tablet or a buccal pateh, via the rectal route founiulated into suppositories; via the eye in the forni of a powder or a dry ointnient; and via the oral route in the form of a tablet_ a capsule or a pellet (which conipositions nlay administer agent via the stomach, the small intestine or the colon), all of which may be formulated in accordance with techniques which are well known to those skilled in the art. The compositions may gei on the nlucosa at least to some extent and this may facilitate retention of the composition on the niucosa.

The preferred route of administration is nasal. Devices which may be used to deliver the compositions according to the invention nasally include the Direct Haler , the BespakO powder device, the Monopoudre (Valois) and the Insufflator (Teijin).

Compositions according to the invention which may be administered orally may be adapted to deliver thcrapeutic agent to the small intestine or the colonic, especially the proximal colonic, region of the gastrointestinal tract.

io Preferably, a means is provided to prevent release of therapeutic agent until the formulation reaches the snlall intestine or colon. Means which may be employed in order to prevent release until the small intestine is reached are well known to those skilled in the art (see for example dosage forms coated with so-called enteric polymers that do not dissolve in the acidic conditions which exist in the stomach, but dissolve in the more alkaline conditions found in the small intestine of a mammal. Suitable enteric coating materials include modified cellulose polymers and acrylic polymers, and in particular tltosr sold uiider the trademark EudragitGy.) Means which may be employed in order to prevent release until the colon is reaclled are wcll known to those skilled in the art. Such materials include cellulose acetate trimellitate (CAT). hvdroxvpropylmethyl cellulose phthalate (HPMCP), polyvinvl acetate phthalate (PVAP), cellulose acetate phthalate (CAP) and shellac, as described by Healy in his article "Enteric Coatings and Delayed Releasc", Chapter 7 in Drug Delivery to the Gastrointestinal Tract, eds. Hardy et al, Ellis Horwood, Chichester, 1989). Especially preferred niaterials are nlethylmetliacrylates or copolymers of methacrylic acid and methylmethacrylate. Such materials are available as Eudragit enteric polymers (Rohni Pharma, Darmstadt, Germany). Such a coating may also suitably comprise a material whicli is redox-sensitive (e.g. azopolymers which may, for example, consist of a random copolymer of styrene and hydroxyethyl methacrylate, cross-linked with divinylazobenzene synthesised by free radical polymerisation, or disulphide polymers (see International Patent Application WO 91/11175 and Van den Mooter, Int. J.

5 Pharm. 87, 37 (1992)). See also International Patent Application WO
97/05903.

It will be appreciated by those skilled in the art that the site of delivery may also be selectively controlled by varying the thickness of certain of the 10 above-mentioned polymer coatings, It will be well understood by those skilled in the art that further excipients may be employed in formulations comprising the compositions according to the invention. For example, in solid dosing forms, further excipients 15 which may be employed include diluents such as microcrystalline cellulose (e.g. Avicel , FMC), lactose, dicalcium phosphate and starch(es);
disintegrants such as microcrystalline cellulose, starch(es) and cross-linked carboxymethylcellulose; lubricants such as magnesium stearate and stearic acid; granulating agents such as povidone; and release modifiers such as hydroxypropyl methylcellulose and hydroxypropyl cellulose. Suitable quantities of such excipients will depend upon the identity of the active ingredient(s) and the particular dosing form which is used.

If desired, other materials may be included in the composition, for example absorption enhancers. Suitable absorption enhancers include non-ionic surfactants, cyclodextrins, bile salts and, preferably, phospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol and generally those mentioned in WO 88/09163.

According to a further aspect of the invention, there is provided a pharmaceutical formulation in a form suitable for administratioii to a mucosal surface which comprises a composition according to the invention in a pharmaceutically acceptable dosage form.

Compositions according to the invention have been found to have the advantage that they provide improved transport of polar drugs across mucosal surfaces, such as the nasal cavity, have improved flow properties when compared to prior art compositions, and avoid the need for the use of chemical crosslinking agents.

According to a further aspect of the invention there is thus provided a method for the improved transport of therapeutic agents across (or into) mucosal surfaces (whicii includes the presentation of vaccines to mucosal surfaces) in tnammals, and a method of treating a human or other mammal, which methods comprise administering a composition, as described above, preferably to a mucosal surface of that human or other mammal, for exaniple the vagina, buccal cavity, rectum, lungs, eye, colon, small intestinc, stoniach or nasal cavity.

The amount of therapeutic agent which may be emploved in the contpositions according to the invention will depend upon the agent which is used. Iiowever, it will be clear to the skilled person that suitable doses of therapeutic agents can he readily determined non-inventively. Suitable doses arc in the range 1 g to 1 g depending upon the therapeutic agent(s) which is/are employed and the route of administration.

The invention is illustrated, but in no way limited, by the following examples with reference to the figures in whicli:

. _.. .t ..___ ,...... _ Figure 1 shows the mean plasma glucose/time curves atter administration to sheep of 2 IU/kg insulin in gelatin microspheres and in gelatin/chitosan microspheres containing either 9.6% or 19.28% G210 chitosan glutamate.

Figure 2 shows the mean plasma insulin/time curves after administration to sheep of 2 IU/kg insulin in gelatin microspheres and in gelatin/chitosan microsplieres containing either 9.6% or 19.28% G210 chitosan glutamate.

Figure 3 shows the mean plasma calcium/time curves after administration to sheep of 20 IU/kg salmon calcitonin in gelatin microspheres and in gelatiu/chitosan microspheres containing either 39.9% G110 or 19.9%
G210 chitosan glutamate.

is Figure 4 shows the mean plasma insulin/time curves after administration to slieep of 2 IU/kg insulin in 0.5% chitosan solution (G210) and in gclatin/chitosan microspheres containing 19.28 T. G210 chitosan -lutamate.

Figure 5 shows the mean plasma insulin/time curves after administration to slicep of 2 IU/kg insulin with chitosan powder (G210) and in gelatin/chitosan microsplteres containing 19.28 % G210 chitosan glutamate.

Figure 6 shows tlre mean changes in plasma PTH concentration for a PTH/gelatin/chitosan microsphere formulation (PTH CHI/GER) as compared to a formulation comprising PTH (alone) in saline (PTH sol) and PTH witli chitosan glutamate (PTH CHI Sol).

Figure 7 shows the effect on plasma glucose level of gelatin/chitosan microspheres comprising different amounts of insulin.

Figure 8 shows the effect of repeated administration of gelatin/chitosan microspheres on plasma insulin level.

Example 1 Preparation of Microspheres Containing 3.6% w/w lnsulin 86.7% w/w Gelatin A and 9.6% w/w Chitosan Glutamate (Sea Cure G210) 193 mg cliitosan glutamate was weighed into a 50 mL beaker and 15 mL
of water was added and stirred until dissolution occurred. 1735 mg of gelatin A (Sigma) was added to the cllitosan solution and stirred at 40 C
until dissolution occurred. The pH of the solution was adjusted to 4 by adding an appropriate amount of 1 M HC1. 72 mg of human zinc insulin (1.8 mL of a 40 mg/mL insulin stock solution) was added to the gelatin/chitosan solution, which was transferred to a 20 mL volumetric flask and water added up to volume.

2 g of Span 80 was weighed into a metal beaker, 200 n1L of sunt7ower oil was added and the mixture warmed to 40 C. The 40 C
insulin/gelatin/chitosan solution was added and emulsified at 1000 rpm for 5 minutes using a Heidolph stirrer fitted wit11 a four blade stirrer arm maintaining the temperature at 40 C. The beaker was transferred to an ice bath and stirring continued at 1000 rpm until the temperature had dropped to below 10 C. The stirring speed was reduced to 500 rpm, 150 mL of chilled acetone was added to the emulsion at 5 mL/min, and the mixture was then centrifuged at 2500 rpm in centrifuge tubes for 10 min.
The supernatant was discarded and the pellet resuspended in 50 mL
acetone. The microspheres were recovered by vacuum filtration and 1 I i washing with further 50 mL of chilled acetone. The filter cake was allowed to dry and the microspheres placed in 50 mL of acetone in a screw capped bottle containing a magnetic stirrer and stirred overnight.
The microspheres were vacuum filtered and dried in a desiccator.

Example 2 Preparation of Microspheres Containing 3.6% w/w Insulin. 77.12% w/w gelatin A and 19.28% w/w Chitosan Glutamate (Sea Cure G210) 386 mg of chitosan glutamate was weighed into a 50 mL beaker, 15 mL of to water was added and the resultant stirred until dissolution occurred. 1542 mg of gelatin A was added to the chitosan solution, which was then stirred at 40 C until dissolution occurred. The pH of the solution was adjusted to 4 by adding an appropriate amount of 1M HCI. 72 mg of human zinc insulin (1.8 niL of a 40 mg/mL insulin stock solution) was added to the gelatin/chitosan solution, which was then transferred to a 20 mL
volumetric ilask, and water was added up to volume.

2v uf Span 80 was weighed into a metal beaker, 200 mL of sunf7ower oil was added and the mixture warmed to 40 C. The 40 C
insulin/eelatin/chitosan solution was added and emulsified at 1000 rpm for 5 niinutes using a Heidolph stirrer fitted witli a four blade stirrer arm maintaining the temperature at 40 C. The beaker was transferred to an ice bath and stirring continued at 1000 rpm until the temperature had dropped to below 10 C. The stirring speed was reduced to 500 rpm and 2 5 150 niL of chilled acetone was added to the emulsion at 5 mL/min which was then centrifuged at 2500 rpm in centrifuge tubes for 10 min. The supernatant was discarded and the pellet resuspended in 50 mL acetone.
The microspheres were recovered by vacuum filtration and washed with further 50 mL of chilled acetone. The filter cake was allowed to dry, the microspheres placed in 50 mL of acetone in a screw capped bottle containing a magnetic stirrer and stirred overnight. The microspheres were vacuum filtered and dried in a desiccator.

5 Example 3 Preparation of Microspheres Containing 0.2% w/w Salmon Calcitonin (SCT). 59.9% w/w Gelatin A and 39.9% w/w Cliitosan Glutamate(Sea Cure GI 10) 798 mg chitosan glutamate (G110) was weighed into a 50 mL beaker, 15 to mL of water was added and the resultant stirred until dissolution occurred.
1198 mg of gelatin A was added to the chitosan solution, which was then stirred at 40 C until dissolution occurred. The pH of the solution was adjusted to 4 by adding an appropriate amount of 1 M HC1. 20,000 IU of SCT (0.91 mL. of a 4 mg/mL SCT stock solution) was added to the 15 gelatin/chitosan solution which was transferred to a 20 mL volumetric flask, and water was added up to volume.

2 g of Span 80 was weighed into a nietal beaker, 200 mL of'sunflowcr oil was added and the mixture warmed to 40 C. The 40 C
20 SCT/gelatin/chitosan solution was added and emulsified at 1000 rpm for 5 minutes using a Heidoiph stirrer fitted with a four blade stirrer arm maintaining the temperature at 40 C. The beaker was transfcrred to an ice bath and stirring continued at 1000 rpm until the temperature had dropped to below 10 C. The stirring speed was reduced to 500 rpm, 150 mL of chilled acetone was added to the emulsion at 5 mL/min which was tllen centrifuged at 2500 rpm in centrifuge tubes for 10 min. The supernatant was discarded and the pellet resuspended in 50 mL acetone.
The microspheres were recovered by vacuum filtration and washed with a further 50 niL of chilled acetone. The filter cake was allowed to dry and r 1 the microspheres placed in 50 mL of acetone in a screw capped bottle containing a niagnetic stirrer and stirred overnight. The microspheres were vacuum filtered and dried in a desiccator.

Example 4.

Preparation of Microspheres Containing 0.2% w/w SC'T 79.9% w/w Gelatin A and 19.9% w/w Chitosan Glutamate (Sea Cure G210) 398 mg chitosan glutamate (G210) was weighed into a 50 mL beaker, 15 mL of water was added and the resultant mixture stirred until dissolution lo occurred. 1598 mg of'gelatin A was added to the chitosan solution, which was stirred at 40 C until dissolution occurred. The pH of the solution was adjusted to 4 by adding an appropriate amount of 1M HCI. 20,000 ItJ of SCT (0.91 mL of a 4 mg/niL SCT stock solution) was added to the gelatin/chitosan solution, which was then transferred to a 20 mL
volumetric flask and water was added up to volume.

2g of Span 80 was weighed into a nietal beaker, 200 niL of sunflower oil was added and the mixture was warmed to 40 C. The 40 C
SCT/gclatin/chitosan solution was added aiid emulsified at 1000 rpm for 5 minutes using a Heidolph stirrer fitted with a four blade stirrer arm, maintaining the temperature at 40 C. The beaker was transferred to an ice bath and stirring continued at 1000 rpni until the temperature had dropped to below 10 C. The stirring speed was reduced to 500 rpm, 150 mL of chilled acetone was added to the emulsion at 5 mL/min which was then centrifuged at 2500 rpm in centrifuge tubes for 10 min. The supernatant was discarded and the pellet resuspended in 50 mL acetone.
The nlicrospheres were recovered by vacuum filtration and waslied with a fitrther 50 mL of chilled acetone. Tlie filter cake was allowed to dry and the microspheres placed in 50 mL of acetone in a screw capped bottle containing a magnetic stirrer and stirred overnigl2t. The microspheres were vacuum filtered and dried in a desiccator.

Example 5 The insulin-chitosan/gelatin microsphere formulations from Examples 1 and 2 were administered nasally to sheep and the effect of the formulations was compared to the effect of administering insulin in gelatin A
microspheres.

to The insulin - gelatin niicrosplieres were prepared in the following wav:
1928 mg gelatin A was added to 14 niL of water in a 50 mL beaker and heated under stirring at 40 C until the gelatin had dissolved. The pH of the gelatin solution was adjusted to 4 using IM HC1 and an equivalent of 72 mg of human zinc insulin (1.8 mL of 40 mg/mL insulin stock solution) was added to the solution. The solution was transferred to a 20 mL
volumetric flask and made up to volume. 2 g of Span 80 was weighed into a metal beaker, 200 niL of sunflower oil was added and the mixture warmed to 40 C. The 40 C insulin/gelatin solution was added and etnulsified at 1000 rpm for 5 minutes using a Heidolph stirrer fitted with a four blade stirrer arm, with the temperature maintained at 40 C. The beaker was transi"erred to an ice bath and stirring continued at 1000 rpm until the teniperature had dropped to below 10 C. The stirring speed was reduced to 500 rpm and 150 niL of chilled acetone was added to the emulsion at 5 mL/min. The emulsion was centrifuged at 2500 rpm in centrifuge tubes for 10 min., the supernatant discarded and the pellet resuspended in 50 mL acetone. The microspheres were recovered by vacuum filtration and waslied with furtlier 50 mL of chilled acetone. The filter cake was allowed to dry and the microspheres were placed in 50 mL
of acetone in a screw capped bottle containing a magnetic stirrer and stirred overnight. The niicrospheres were vacuum filtered and dried in a desiccator.

Each of the microsphere formulations were administered nasally to groups of five sheep using blueline siliconised tubes at an insulin dose of 2 IU/kg and a microsphere dose of 2.0 mg/kg. Biood saniples were collected at specified time points from the cannulated external jugular veins and plasma glucose and insulin concentrations measured. The mean clianges in plasma glucose concentration with time for the three formulations are io sllown in Figure 1. It can be seen that insulin given nasally in combination with gelatin microspheres did not result in any significant lowering of the plasnia glucose levels (CR,,õ = 95.9%) wllereas the formulations containing 9.6% chitosan and 19.28% chitosan gave glucose lowering effects of Cm;n = 74.6% and Cmin = 53.8% of basal level, respectively. The corresponding plasma insulin levels for the three formulations are shown in Figure 2. It can be seen that C11õx for both the chitosan/gelatin microsphere formulations (131.6 mU/L and 439.7 mU/L
for the 9.6/86.7% and 19.28/77.12% chitosan/gelatin niicrosphere, respectively ) were sit*nificantly higher than the C,,,dx, seen for the gelatin microsplieres (53.5 mU/L).

Example Thc calcitonin-chitosan/gelatin microsphere fornwlations described in Example 3 and 4 were administered nasall_y to sheep and the effect of the calcitonin in gelatin formulations compared to the effect of administerinp microspheres.
The calcitonin - gelatin niicrospheres were prepared in the following way:
1996 n-ig gelatin A was added to 15 mL of water in a 50 mL beaker and heated under stirring at 40 C until the gelatin had dissolved. The pH of the gelatin solution was adjusted to 4 using IM HCI and an equivalent of 20,000 IU of salmon calcitonin (0.91 mL of 4 mg/mL SCT stock solution) was added to the solution. The solution was transferred to a 20 mL

volumetric flask and made up to volume. 2 g of Span 80 was weighed into a metal beaker, 200 mL of sunflower oil was added and the mixture warmed to 40 C.

The 40 C insulin/gelatin solution was added and emulsified at 1000 rpm for 5 minutes using a I-leidolph stirrer fitted with a four blade stirrer arm, with the temperature maintained at 40 C. The beaker was transferred to an ice bath and stirrini! continued at 1000 rpm until the temperature had dropped to below 10 C. The stirring speed was reduced to 500 rpm and 150 mL of chilled acetone was added to the emulsion at 5 mL/min. The emulsion was centrifuged at 2500 rpm in centrifuge tubes for 10 min., the supernatant was discarded and the pellet resuspended in 50 mL acetone.
The nlicrospheres were recovered by vacuum filtration and washed with further 50 niL of chilled acetone. The filter cake was allowed to dry and the microspheres placed in 50 niL of acetone in a screw capped bottle containing a inagnetic stirrer and stirred overnight. The microspheres were vacuum filtered and dried in a desiccator.

Each of tlie microsphere formulations were administered nasally to groups of five sheep using blueline siliconised tubes at an SCT dose of 20 IU/kg and a microsphere dose of 2.004 mg/kg. Blood samples were collected at specified time points froni the cannulated external jugular veins and plasma calcium concentrations measured. The mean changes in plasma calcium concentration with time for the three formulations are shown in Figure 3. It can be seen that SCT given nasally in combination with _ ._ ...__._ j ge.latin microsplieres only resulted in a minimal lowering of the plasma calcium levels (Cmjn = 91.1 %) wliereas tlle formulations containing 39.9% G110 chitosan and 19.9% G210 chitosan gave calcium lowering effects of C,,,;n = 73.6% and C;n = 74.7% of basal level, respectively.

There was no significant difference between the effects obtained for the 39.9% G110 and 19.9% G210 chitosan levels in the gelatin microspheres.
Example 7 The insulin-chitosan/gelatin microsphere formulation described in jo Example 2 was administered nasally to sheep and the effect of the formulation compared to the effect of administering insulin in a simple chitosan solution.

The microsphere formulation was administered nasally to a group of five sheep using blueline siliconised tubes at an insulin dose of 2 IU/kg and a microsphere dose of 2.0 mg/kg. As a comparison, a solution of 200 IU/niL insulin in 5 mg/mL G210 chitosan glutamate solution was adniinistercd nasally at 2 IU/k(; to a group of four sheep. Blood samples werc: collected at specified tinle points froni the cannulated external jugular veins and plasma insulin concentrations measured. The mean chanues in plasma insulin concentration with time for the two formulations are shown in Figure 4. It can be seen that the plasma insulin level is significantly liigher for the gclatin/chitosan microsphere forniulation (Cmax = 450 mU/L) as compared to the cllitosan solution fornlulation (C,nax =
100 mU/L).

Example 8 The insulin-chitosan/gelatin nlicrosphere formulation described in Example 2 was administered nasally to slieep and the effect of the formulation compared to the effect of administering insulin with a chitosan powder fornlulation.

The gelatin/chitosan microsphere formulation was administered nasally to ~ a group of five slieep using blueline siliconised tubes at an insulin dose of 2 IU/kg and a niicrosphere dose of 2.0 mg/kg. As a comparison, a mixture of 640 IU insulin with 800 mg G210 chitosan glutamate was administered nasally at 2 IU/kg to a group of four sheep at 2 IU/kg.
Blood samples were collected at specified time points from the cannulated Ic~ external jugular veins and plasma insulin concentrations nieasured. The mean changes in plasma iiisulin concentration with time for the two formulations are shown in Figure 5. It can be seen that the plasma insulin level is significantly higher for the gelatin/chitosan niicrosphere formulation (Cmax = 450 mU/L) as compared to the chitosan powder 15 forniulation (Cm,x = 250 mU/L). It should also be noted that the amount of chitosan administercd in the two formulations is much higher for the chitosan pnwder formulation than for the gelatin/chitosan microsphere fOrmulation.

2() ECaniple y Preparation of Microspheres Containing 0.4% w/w PTH. 19.92 m, w/w Chitosan Glutamate (Sea Cure 210) and 79.68% w/w Gelatin A

9.28 mg of PTf1 was added to 20 mL of a solution containing 400 mg cliitosan glutamate and 1.6 g of gelatin A and maintained at 50 - 60 C_ 2 25 g of Span 80 was weighed into a beaker and 200 mL of soya oil was added. The resultant was mixed and heated to 40 C. The PTH/
chitosan/gelatin solution was added and emulsified at 1000 rpm for 10 min. using a Heidolph stirrer fitted witli a four blade stirrer arm, maintaining the temperature at 40 C. The beaker was transferred to an i ~ ~

ice bath and stirring continued at 100 rpm until the temperature had dropped to below 10 C. The stirring speed was reduced to 500 rpm and 150 niL of chilled acetone was added to the emulsion at 5 mL/min, followed by centrifugation at 3000 rpm for 10 min. The supernatant was discarded and the pellet resuspended in acetone. The microspheres were recovered by vacuum filtration and washed with 50 mL of cllilled acetone.
The filter cake was allowed to dry, the microspheres placed in 50 niL of acetone in a screw capped bottle containing a magnetic stirrer and stirred overnight. The microspheres were vacuum filtered and dried in a I d issector.

Slieep study The PTH gelatin/chitosan microsphere formulation was administered nasally to a group of 6 sheep using blueline siliconised tubes at a PTH
dose of 4pg/kg. As a comparison, the same group of sheep was also administercd the same dose of PTH in saline and in saline containing 0.5 % chitosan glutamate. Blood samples were collected at specified time points fronl the cannulatcd external jugular veins and plasma PTI-I
concentrations measured. The mean changes in plasma PTH

concentration with tinie for the three formulations are shown in Figure 6.
It can be: seen that the plasnla PTH is significantly higher for the gelatin in chitosan niicrosphere formulation (C,,,, = 2.5 ng/mL) as compared to the chitosan solution formulation (Cm~x = 0.25 ng/niL) and the control PTH
solution (Cm,x = 0 ng/mL).

Example 10 Deterrnination of Hausner Ratio for Cliitosan/Gelatin A Microspheres and for Spray Dried Chitosan Microspheres (Sea Cure G210) A known weight (see below) of chitosan/gelatin microsplieres, prepared as in Example 2, was carefully poured into a 10 niL measuring cylinder and the volume recorded (poured volume). The measuring cylinder was tapped (onto the bench) 50 times and the volume of the chitosan/gelatin microspheres again recorded (tapped volume). The measurement was carried out in triplicate.

Weight Poured Poured Tapped Tapped Hausner Vol.(cm') Den.(g/cm') Vol.(cm3) Den. (g/cm') Ratio 2.2481 7.3 0.3079 5.8 0.3876 1.26 2.3680 7.6 0.3116 6.0 0.3947 1.27 2.3220 7.5 0.3096 6.1 0.3807 1.23 Haustier Ratio (chitosan/gelatin microspheres) = 1.25 (good flow properties) A known wcight (see below) of spray dried chitosan niicroparticles (Sea Cure G210; Pronova) was carefully poured into a 10 niL nieasuring cylinder and the volunle recorded (poured volume). The measuring cylinder was tapped (onto the bench) 50 tinies and the volume of the chitosan again recorded (tapped volume). The nieasurement was carried out in triplicate.

Weiglit Poured Poured Tapped Tapped Hausner Vol.(cm3) Den.(g/cm3) Vol.(cm3) Den. (g/cm3) Ratio 1.5609 9.0 0.1734 4.1 0.3807 2.20 1.4728 8.5 0.1733 3.8 0.3876 2.24 1.4004 8.0 0.1751 3.6 0.3890 2.22 Hausner Ratio (chitosan) = 2.22 (very poor flow properties) Example 11 io Deterniination of Angle of Repose for Chimsan/Gelatin A Microspheres and for Spray Dried Cliitosan Microspheres (Sea Cure G210) The Angle of Repose (0) was determined by pouring about 3 g of chitosan/gelatin microspheres, prepared as in Example 2. through a funnel (held at a fixed height) onto a piece of graph paper until a cone was ls formed. The height (1-1) and the radius (R) of the cone were determined and the Angle calculated (tan 0 = H/R). The measurement was carried out in triplicate.

Mean Heiizht = 10 mm 210 Mean Radius = 18 mm Angle of Repose (chitosan/gelatin microspheres) = 29 (good flow properties) The Angle of Repose (0) was determined by pouring about 3 g of spray 25 dried chitosan microparticles (Sea Cure G210; Pronova) through a funnel (held at a fixed height) onto a piece of graph paper until a cone was formed. The heiglit (H) and the radius (R) of the cone were deternlined and the Angle calculated (tan 0 = H/R). The measurement was carried out in triplicate.

Mean Height = 25 nun Mean Radius = 24 mm Angle of Repose (chitosan) = 46 (very poor flow properties).

Example 12 Determination of the Effect of Dose of Microspheres on the Absorption of Insulin Microspheres were prepared as in Example 2 with the final concentration io of insulin in the microspheres being 2.0%, 4.0%, 5.3%, 7.7% and 14.4%
w/w.

The microspheres were administered nasally to groups of 4 sheep with a fixed dose of 1 IU insulin/kg and 2.0, 1.0, 0.75, 0.5 and 0.25 mg/kg of i s gelatin/chitosan microspheres as described in Example 8. The mean changes in plasma glucose level expressed as AUC are given in Figure 7.
It can be seen that the effect of the gelatin/chitosan microspheres on the AUC is no different whether 2.0 nie/kg or down to 0.25 mg/kg of microspheres are adnlinistered with a constant dose of insulin.

Example 13 Effect of Repeated Administration of Gelatin A/Chitosan Microsphcres on Plasma Insulin Level Gelatin/chitosan/insulin microspheres were prepared as in Example 2.
The microspheres and a chitosan solution formulation, prepared as in Example 8, were administered nasally to groups of 4 sheep once daily for 5 consecutive days. A SC injection of insulin solution was given to the third group of sheep for five consecutive days. Plasma insulin levels expressed as AUC are given in Figure 8 . It can be seen that the AUC

_ t- - T T

obtained for each day is consistently higher for the gelatin/chitosan microspllere formulation as compared to the cilitosaii solution formulation.
It can also be seen that the AUCs obtained on the five consecutive days are similar for the nasal formulation, thus showing a consistent and reproducible effect, whereas a certain accumulative effect can be seen for the SC repeated injection.

Claims (50)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition comprising a mixture of chitosan and type A, cationic, gelatin together with a therapeutic agent.

2. A composition as claimed in Claim 1, wherein the composition is in the form of microparticles.

3. A composition as claimed in Claim 1 or Claim 2, wherein the therapeutic agent is incorporated into the particles during production, adsorbed to the surface of the particles, or is present as an admixture.

4. A composition as claimed in Claim 2 or Claim 3, wherein the microparticles are microspheres.

5. A composition as claimed in any one of claims 1 to 4, wherein the composition is suitable for delivery of a therapeutic agent across a mucosal membrane into the systemic circulation.

6. A composition as claimed in any one of claims 1 to 5, wherein the chitosan has a molecular weight greater than 4000 Dalton.

7. A composition as claimed in Claim 6, wherein the chitosan has a molecular weight in the range 25,000 to 2,000,000 Dalton.

8. A composition as claimed in Claim 7, wherein the chitosan has a molecular weight in the range 50,000 to 300,000 Dalton.

9. A composition as claimed in any one of Claims 1 to 8, wherein the chitosan is a derivative, which derivative is formed by bonding of acyl or alkyl groups with the hydroxyl moieties of the chitosan.

10. A composition as claimed in any one Claims 1 to 8, wherein the chitosan is in the form of a salt selected from the group nitrates, phosphates, sulphates, hydrochloride, glutamates, lactate or acetate.

11. A composition as claimed in any one of Claims 2 to 10, wherein the microparticles are produced by spray drying, emulsification, solvent evaporation or precipitation.

12. A composition as claimed in any one of Claims 1 to 11, wherein the chitosan has a degree of deacetylation of greater than 40%.

13. A composition as claimed in Claim 12, wherein the degree of deacetylation is between 50 and 98%.

14. A composition as claimed in Claim 13, wherein the degree of deacetylation is between 70 and 90%.

15.A composition as claimed in any one of Claims 2 to 14, wherein the microparticles have a diameter of between 1 to 200 µm.

16. A composition as claimed in Claim 15, wherein the diameter is between 1 to µm.

17. A composition as claimed in any one of Claims 1 to 16, wherein the composition comprises between 50 and 95% of type A gelatin.

18.A composition as claimed in Claim 17, wherein the composition comprises between 75 and 85% of type A gelatin.

19. A composition as claimed in any one of Claims 1 to 18, wherein the therapeutic agent is a polar drug.

20. A composition as claimed in any one of Claims 1 to 19, wherein the therapeutic agent is a polypeptide.

21. A composition as claimed in Claim 20, wherein the therapeutic agent is selected from the group insulin, calcitonin, luteinising hormone releasing hormone, growth hormone or a growth hormone releasing factor.

22. A composition as claimed in any one of Claims 1 to 19, wherein the therapeutic agent is an analgesic agent or a drug for the treatment of migraine.

23. A composition as claimed in any one of Claims 1 to 19, wherein the therapeutic agent is an antigen intended for mucosal immunisation.

24. A composition as claimed in any one of Claims 1 to 19, wherein the therapeutic agent is a gene or gene construct (DNA) intended for the transfection of cells in the mucosal surface.

25. A composition as claimed in any one of Claims 1 to 24, which further comprises an absorption enhancing agent.

26. A composition as claimed in Claim 25 where the absorption enhancing agent is a phospholipid.

27. A pharmaceutical formulation in a form suitable for administration to a mucosal surface which comprises a composition as defined in any one of claims 1 to 26, in a pharmaceutically acceptable dosage form.

28. A formulation as claimed in Claim 27, wherein the mucosal surface is in the nose.

29. A formulation as claimed in Claim 27, wherein the mucosal surface is in the buccal cavity.

30. A formulation as claimed in Claim 27, wherein the mucosal surface is in the vagina.

31. A formulation as claimed in Claim 27, wherein the mucosal surface is in the gastrointestinal tract and the formulation is for oral delivery.

32. A formulation as claimed in Claim 27, wherein the mucosal surface is in the rectum.

33. A formulation as claimed in Claim 27, wherein the mucosal surface is in the eye.

34. A formulation as claimed in Claim 27, wherein the mucosal surface is in the lung.

35. The use of a composition, as defined in any one of Claims 1 to 26, or a formulation as defined in any one of Claims 27 to 34, for the transport of a therapeutic agent across a mucosal surface in a mammal.

36. The use of a composition, as defined in any one of Claims 1 to 26, or a formulation as defined in any one of Claims 27 to 34 in the manufacture of a medicament for a method of treating a mammalian patient.

37. The use as claimed in Claim 36, wherein the composition or formulation is for delivery to a mucosal surface.

38. The use as claimed in Claim 35 or Claim 37, wherein the composition or formulation is adapted to deliver a therapeutic agent across a mucosal membrane into the systemic circulation.

39. The preparation of type A gelatin-chitosan microparticles by a process of spray drying.

40. The preparation as claimed in Claim 39, characterised in that the microparticles are prepared by spray drying a warm mixture of chitosan and gelatin with instant cooling of the resultant microparticles.

41. The preparation of type A gelatin-chitosan microparticles by emulsification.

42. The preparation as claimed in Claim 41, characterised in that the microparticles are prepared by warming a solution of a chitosan mixed with type A gelatin, emulsification and gelation by cooling.

43. The preparation as claimed in Claim 42, characterised in that the chitosan is dissolved in water and mixed with the gelatin under heating to 40°C.

44. The preparation as claimed in Claim 42 or Claim 43, characterised in that the mixture is emulsified at a temperature above the melting point of the gelatin in an organic medium in the presence of an emulsifier.

45. The preparation as claimed in any one of Claims 42 to 44, characterised in that the microparticles arc solidified by decreasing the temperature of the emulsion below 10°C.

46. The preparation as claimed in any one of Claims 42 to 45, characterised in that the microparticles are harvested by adding chilled acetone to the emulsion, centrifugation, washing and drying.

47. The preparation as claimed in any one of Claims 41 to 46, characterised in that the microparticles further comprise a therapeutic agent, which agent is incorporated into the microparticles by adding it to the mixture before emulsification.

48. The preparation as claimed in any one of Claims 39 to 46 characterised in that the microparticles further comprise a therapeutic agent, which agent is adsorbed onto the surface of the microparticles by freeze drying or spray drying a suspension of microparticles with the therapeutic agent.

49. The preparation as claimed in any one of Claims 39 to 46, characterised in that the microparticles further comprise a therapeutic agent, which agent is adsorbed onto the surface of the microparticles by physically or mechanically mixing the dried microparticles with the therapeutic agent.

50. The use of a composition, as defined in any one of Claims 1 to 26, or a formulation as defined in any one of Claims 27 to 34, in the manufacture of a medicament for use in the improved transport of therapeutic agents across mucosal surfaces in mammals.