Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
  • A61K47/40—Cyclodextrins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/10—Drugs for disorders of the urinary system of the bladder
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• compositions comprising imidazoquinolin(amines) and derivatives thereof suitable for local administration
• the present invention relates in general to the field of modulators of the innate immune system, particularly to pharmaceutical compositions comprising imidazoquinolin(amines) and derivatives thereof, preferably suitable for local administration, such as, intravesical administration.
• the present invention concerns the use of imidazoquinolin(amines) and derivatives thereof for intravesical treatment of bladder diseases, such as, for example, bladder cancer and cystitis.
• the present invention furthermore comprises methods of treatment for these diseases as well as methods of administration of the inventive pharmaceutical compositions.
• pattern recognition receptors typically recognize conserved molecular patterns that distinguish foreign organism, like viruses, bacteria, fungi and parasites from cells of their hosts.
• pattern recognition receptors include, inter alia, so called members of the Toll-like receptor (TLR) family, the first family of pattern recognition receptors studied in detail.
• TLR Toll-like receptor
• TLRs are transmembrane proteins which recognize ligands of the extracellular milieu or of the lumen of endosomes. Following ligand-binding they transduce the signal via cytoplasmic adaptor proteins which leads to triggering of a host-defence response and entailing production of antimicrobial peptides, proinflammatory chemokines and cytokines, antiviral cytokines etc. To date, at least 10 members of Toll-like receptors (TLRs 1 -10) have been identified in human and 13 (TLRs 1 -13) in mice.
• TLRs Toll-like receptors in human include TLR1 -TLR2, which recognize Triacyl lipopeptides; Toll-like receptors TLR1 - TLR6, which recognize diacyl lipopeptide; Toll-like receptor TLR2, which recognize peptidoglycans; Toll-like receptor TLR3, which is known to recognize dsRNA, a viral product; Toll-like receptor TLR4, which has LPS (lipopolysachharide) of Gram-negative bacteria as a known ligand; Toll-like receptor TLR5, which recognizes bacterial flagellin(s); Toll-like receptors TLR7/8, which known ligands comprise imidazoquinolines, guanosine analogs and ssRNA; Toll-like receptor TLR9, which recognizes unmethylated CpG motifs frequently found in the genome of bacteria, viruses and protozoans, but not in vertebrates; TLR9 furthermore recognizes malaria pigment hemozoin, a digestion product of haem
• TLRs After recognition of microbial pathogens, these TLRs typically trigger intracellular signalling pathways that result in induction of inflammatory cytokines (e.g. TNF-alpha, IL-6, IL-1 -beta and IL-12), type I interferon (IFN- beta and multiple IFN-alpha) and chemokines (Kawai, T. and S. Akira (2006). "TLR signalling.” Cell Death Differ 13(5): 816-25).
• inflammatory cytokines e.g. TNF-alpha, IL-6, IL-1 -beta and IL-12
• IFN- beta and multiple IFN-alpha type I interferon
• chemokines Yamanes, T. and S. Akira (2006). "TLR signalling.” Cell Death Differ 13(5): 816-25).
• TLR3, TLR7 and TLR9 are of major importance.
• TLR7 recognizes small synthetic immune modifiers including imiquimod, R-848, loxoribine, and bropirimine, all of which are already applied or promising for clinical use against viral infections and cancers.
• plasmacytoid dendritic cells express TLR7 and TLR9, and respond to TLR7 and TLR9 ligands by producing a large amount of interferon (IFN- alpha).
• IFN- alpha interferon
• imiquimod belongs to the class of imidazoquinolin(amine) immune modifiers.
• the immunomodulatory molecule imiquimod (1 -isobutyl-1 Mimidazo[4,5-c
• the mechanism for the immunostimulatory activity of imiquimod is thought to be due in substantial part to enhancement of the immune response by induction of various important cytokines (e.g., interferons, interleukins, tumor necrosis factor, etc.).
• TLR7 toll-like receptor 7
• IFN- ⁇ interferon- ⁇
• IL-6 interleukin-6
• TNF- ⁇ tumor necrosis factor- ⁇
• immune modifiers such as imiquimod
• the ability to provide therapeutic benefit via local administration of these immune modifiers for treatment of a particular condition at a particular location may be hindered by a variety of factors. These factors include insolubility and/or degradation of these immune modifiers in the formulation prior, during or even subsequent to administration, but also physical instability of the formulation, including factors such as separation of components, thickening, precipitation/agglomeration of active ingredients, and the like, as well as poor permeation of the immune modifier(s) into the surrrounding tissue or cells upon administration.
• solubility of the immune modifier imiquimod is critical, and its use in pharmaceutical compositions, in particular, in liquid or semi-liquid compositions is limited due to its hydrophobic properties.
• imiquimod is soluble in low concentrations in polar organic solvents such as DMSO, dimethyl formide, and N-methyl-2- pyrrolidone, such solvents do not allow for administration of imiquimod at or in the human body due to their various toxic effects.
• Non-toxic solvents such as water or ethanol solubilise member of the imidazoquinolin(amin)es, such as imiquimod, only slightly and only allow for administration of imiquimod at or in the human body in low (subtherapeutic) concentrations.
• a pharmaceutical composition particularly as a liquid or semi-liquid formulation, which allows to solubilise imidazoquinolin(amin)es, such as imiquimod, in an appreciably manner and therefore allows for higher effective concentrations of immune modifiers such as imiquimod, when administered to the subject in need thereof.
• a pharmaceutical composition particularly as a liquid or semi-liquid formulation, which allows to solubilise imidazoquinolin(amin)es, such as imiquimod, in an appreciably manner and therefore allows for higher effective concentrations of immune modifiers such as imiquimod, when administered to the subject in need thereof.
• immune modifiers particularly of members of the class of imidazoquinolin(amin)es, such as imiquimod
• imiquimod is reported to cause, for example, agitation, anemia, angioedema, arrhythmias, capillary leak syndrome, cardiac failure, cardiomyopathy, cerebrovascular accident, depression, dyspnea, erythema multiforme, exfoliative dermatitis, Henoch-Sch ⁇ nlein purpura syndrome, idiopathic thrombocytopenia purpura, insomnia, ischemia, leukopenia, liver function abnormal, lymphoma, multiple sclerosis aggravated, paresis, proteinuria, pulmonary edema, seizure, syncope, thrombocytopenia, and thyroiditis.
• routes for systemic administration in general include, for example, transdermal, oral, or parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes.
• transdermal, oral, or parenteral routes including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes.
• Such systemic administration typically leads to an overall distribution of the immune modifiers through the humans body and therefore significantly increases the risk of side effects.
• routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections, wherein such administration typically occurs at the site of affliction and allows for a direct action of the drug while systemic side effects are significantly reduced, which are envisaged to occur upon systemic administration.
• imidazoquinolin(amin)es such as imiquimod or its derivatives
• the specific requirement of local administration due to the above-mentioned physico-chemical properties of these compounds and side effects upon systemic administration limits their therapeutical application and the number of diseases to be treated therewith.
• the majority of diseases, which may be treated with imiquimod or its derivatives are specific diseases of the skin, including skin cancers, such as, basal cell carcinoma, Bowen's disease, superficial squamous cell carcinoma as well as genital warts (Condylomata acuminata).
• a further prominent disease, which may be treated with immune modifiers, in particular imidazoquinolin(amin)es, such as imiquimod includes bladder diseases, in particular bladder cancer and cystitis.
• bladder cancer refers to any of several types of (malignant or non-malignant) neoplastic diseases of the urinary bladder. It is one of the fastest growing cancers worldwide due to the rapidly aging populations of most countries. Every year in the United States more than 60,000 people are newly diagnosed with bladder cancer, 80 % of these have noninvasive bladder cancer. Since the mortality rate of bladder cancer is relatively low, the total number of patients in the US and in Europe is above 400,000. So far the majority of noninvasive (superficial) bladder cancer patients are treated with so called “bacillus Calmette- Guerin (BCG) solutions", which are administered via the intravesical route. However, such BCG solutions are uncharacterized products composed by an attenuated form of the bacterium Mycobacterium tuberculosis, and therefore, exhibiting a poor safety profile.
• BCG Bacillus Calmette- Guerin
• cystitis typically comprises an inflammation of the urinary bladder and occurs when the normally sterile lower urinary tract (urethra and bladder) is infected by bacteria and becomes irritated and inflamed. Because of the risk of the infection spreading to the kidneys and due to the high complication rate in the elderly population and in diabetics, prompt treatment is almost always recommended. In order to control the bacterial infection cystitis is usually treated with antibiotics. Commonly used antibiotics for the treatment include, for example, nitrofurantoin, trimethoprim-sulfamethoxazole, amoxicillin, cephalosporins, ciprofloxacin or levofloxacin, and doxycycline.
• antibiotic therapies often disrupt the normal balance of the intestinal flora causing diarrhea.
• an antibiotic-induced disruption of the population of the bacteria normally present as constituents of the normal vaginal flora may also occur, and may lead to overgrowth of yeast species of the genus Candida in the vulvo-vaginal area.
• imidazoquinolin(amines) in particular the hydrophobicity profile of this compound class, have to be taken into account, which (in view of the approaches described in the art) typically lead to an insufficient in vivo concentration of the administered immune modifier compound acting as TLR-7 ligand.
• imiquimod is an active agent efficient for the treatment of oncological, viral, and inflammatory diseases.
• imidazoquinolin(amin)es such as imiquimod and its derivatives
• the use of imidazoquinolin(amin)es, such as imiquimod and its derivatives, in manufacturing a medicament is strictly limited by its solubility characteristics.
• various side effects are caused by imidazoquinolin(amin)es, such as imiquimod, if administered systemically. Accordingly, administration of this agent in a formulation which allows the agent to be specifically delivered to the target organ by using an appropriate pharmaceutical composition is of utmost importance.
• imidazoquinolin(amin)es such as imiquimod
• imiquimod in sufficiently solubilised amounts to allow efficient treatment of diseases mentioned herein.
• imidazoquinolin(amin)es such as imiquimod and derivatives thereof, were formulated in a suitable formulation to be administered locally, in particular, intravesical Iy, in order to combat bladder diseases, thereby significantly reducing the risk of severe systemic side effects.
• a pharmaceutical composition suitable to comprise immune modifiers in particular imidazoquinolin(amin)es, such as imiquimod, in therapeutically effective amounts.
• the object(s) of the present invention is (are) solved by the attached claims.
• the object(s) of the present invention is (are) solved by a pharmaceutical composition comprising (a) an imidazoquinolin(amine) or a derivative thereof, e.g. imiquimod or a derivative thereof, and (b) at least one organic acid selected from acetic acid and/or lactic acid or a mixture thereof.
• the inventive pharmaceutical composition comprises as a first component at least one organic acid selected from acetic acid and/or lactic acid or a mixture thereof.
• organic acids “acetic acid” and “lactic acid” were already known to a skilled person for formulating pharmaceutical compositions in general, the inventors of the present invention surprisingly found that specifically these short chain carboxylic acids are suitable to efficiently solubilise imidazoquinolin(amin) or derivatives thereof, a finding which has not yet been published or discussed in the art.
• acetic acid and lactic acid exhibit solubilisation properties clearly superior to any other (carboxylic) acid.
• acetic acid CH 3 COOH
• lactic acid (2-hydroxy propionic acid)
• solubilise imidazoquinolin(amine) 3 to 100 fold better than other short chain (carboxylic) acids, namely, phosphoric acid, succinic acid and citric acid.
• imidazoquinolin(amin)es refers both to the generic class of imidazoquinolins and, more specifically in a preferred embodiment, also to the subclass of imidazoquinolinamines.
• imidazoquinolin(amines) or their derivatives as used herein have a basic functional group, particularly an amine moiety
• imidazoquinolin(amines) can be solubilised in any acidic solution at pH values below the pK a of compounds of that class, e.g. in case of imiquimod, i.e. of about 4.
• solubility of imidazoquinolin(amines) or their derivatives as used herein does not merely depend on the pH of the solution.
• the inventive pharmaceutical composition as defined above comprises an organic acid selected from acetic acid and/or lactic acid or a mixture thereof in a concentration of about 0.025 M to about 0.200 M, preferably in a concentration of about 0.025 M to about 0.100 M or in a concentration of about 0.100 M to about 0.200 M, e.g.
• solubilised imidazoquinolin(amines) or their derivatives as used herein in solution is directly related to the acid concentration, i.e. to the concentration of acetic acid and lactic acid. Accordingly, higher concentrations of acetic acid and/or lactic acid or a mixture of both may be preferred for solubilising, preferably within the above defined ranges.
• the inventive pharmaceutical composition as defined above comprises a mixture of (two organic acids selected from) acetic acid and lactic acid, both together preferably exhibiting a "common concentration" of organic acids as defined above for the inventive pharmaceutical composition in general of about 0.025 M to about 0.200 M or any further concentration as defined above.
• common concentration of organic acids means that molarities of both organic acids acetic acid and lactic acid used for such a mixture, lead to a concentration as defined above.
• both components of a mixture of (two organic acids selected from) acetic acid and lactic acid as defined above comprises a ratio of acetic acid : lactic acid of about 1 :30 to about 30:1 , e.g. of about 2:30, of about 3:30, of about 4:30, of about 5:30, of about 3:1, of about 3:1, of about 7:30, of about 8:30, of about 9:30, of about 10:30, of about 1 1 :30, of about 12:30, of about 13:30, of about 14:30, of about 15:30, of about 16:30, of about 17:30, of about 18:30, of about 19:30, of about 20:30, of about 21 :30, of about 22:30, of about 23:30, of about 24:30, of about 25:30, of about 26:30, of about 27:30, of about 28:30, of about 29:30, of about 30:29, of about 30:28, of about 30:27, of about 30:26, of about 30:25, of about 30:24, of about 30:23, of about
• Ranges of acetic acid lactic acid are disclosed herein, which may be formed on the basis of any of the above values, e.g. 1 :30 to 30:24, 1 :30 to 23:30, 30:12 to 30:1 , 30:8 to 30:1 etc.
• the inventive pharmaceutical composition as defined above comprises exclusively either acetic acid or lactic acid as acid components.
• specific embodiments relate to compositions of the invention as described above which do not encompass any other acid, be it an organic or an inorganic acid, except for acetic acid and/or lactic acid.
• the pharmaceutical composition comprises exclusively acetic acid (and no lactic acid) as (organic) acid component and, more preferably, apart from acetic acid no other acid at all, be it organic or inorganic.
• the pharmaceutical composition may (in another specific embodiment) comprise exclusively lactic acid (and no acetic acid) as (organic) acid and, more preferably, apart from lactic acid no other acid at all, be it organic or inorganic.
• the pharmaceutical composition may comprise just acetic and or lactic acid as organic acid components, however, there may be at least one additional inorganic acid be included in the pharmaceutical composition of the invention, e.g. phosphoric acid, HCI etc.
• the inventive pharmaceutical composition as defined herein comprises exclusively lactic acid as acid component, preferably exhibiting a concentration of lactic acid as defined above for the inventive pharmaceutical composition in general of about 0.025 M to about 0.200 M or any further concentration as defined above.
• the inventive pharmaceutical composition as described above does not encompass any other acid than lactic acid, be it an organic or an inorganic acid.
• the inventors of the present invention surprisingly found that lactic acid is even more efficient in solubilising imidazoquinolin(amines) or their derivatives as used herein than acetic acid.
• lactic acid is twice as efficient in solubilising imidazoquinolin(amines) or their derivatives as used herein as acetic acid. It was also found, that the solubility of imidazoquinolin(amines) or their derivatives as used herein in lactic acid is not enhanced by the addition of surfactants, such as Tween or Pluronic, which usually function as solubility enhancers in pharmaceutical compositions. This finding suggests that imidazoquinolin(amines) or their derivatives as used herein are not entrapped in micelles formed by surfactants and, therefore, supports the assumption that these imidazoquinolin(amines) interact with lactic acid by forming specifically structured adducts.
• surfactants such as Tween or Pluronic
• the inventive pharmaceutical composition as defined herein typically comprises an pH-value of about 3 to about 8, preferably of about 3 to about 7, more preferably of about 3 to about 6, even more preferably of about 3 to about 5, and most preferably a pH-value of about 3.5 to about 4, including a pH-value in a range of about 3.5 to about 4.9, of about 3.5 to about 4.8, of about 3.6 to about 4.7, of about 3.6 to about 4.6, of about 3.7 to about 4.5, of about 3.7 to about 4.4, of about 3.8 to about 4.3, of about 3.8 to about 4.2, or of about 3.9 to about 4.1.
• the inventive pharmaceutical composition may be prepared and administered in a pH- value as defined above. If necessary, the pH-value may be further adjusted for the specific treatment and administration requirements, e.g. to a more neutral pH-value of about 5, 6, or 7 (pH 5 to 7), e.g. using buffers and additives as disclosed herein.
• the inventive pharmaceutical composition comprises as a further component an imidazoquinolin(amine) compound, such as imiquimod or a derivative thereof, as a biologically (therapeutically) active agent.
• imidazoquinolin(amines) or their derivatives as used herein preferably refers to imidazoquinolin(amines) as defined in the following.
• the inventive pharmaceutical composition comprises imidazoquinolin(amines) selected from following formula (I):
• R 1 , R 2 , and R 3 are each independently selected from hydrogen; cyclic alkyl of three, four, or five carbon atoms; straight chain or branched chain alkyl containing one to about ten carbon atoms and substituted straight chain or branched chain alkyl containing one to about ten carbon atoms, wherein the substituent is selected from the group consisting of cycloalkyl containing three to about six carbon atoms and cycloalkyl containing three to about six carbon atoms substituted by straight chain or branched chain alkyl containing one to about four carbon atoms; fluoro- or chloroalkyl containing from one to about ten carbon atoms and one or more fluorine or chlorine atoms; straight chain or branched chain alkenyl containing two to about ten carbon atoms and substituted straight chain or branched chain alkenyl containing two to about ten carbon atoms, wherein the substituent is selected from the group consisting of cycloalkyl
• R y is hydrogen or a carbon-carbon bond
• R x is alkoxy of one to about four carbon atoms, hydroxyalkoxy of one to about four carbon atoms, 1 -alkynyl of two to about ten carbon atoms, tetrahydropyranyl, alkoxyalkyl wherein the alkoxy moiety contains one to about four carbon atoms and the alkyl moiety contains one to about four carbon atoms, 2-, 3-, or 4-pyridyl, and with the further proviso that when R y is a carbon-carbon bond R y and R x together form a tetrahydrofuranyl group optionally substituted with one or more substituents independently selected from the group consisting of hydroxy or hydroxyalkyl of one to about four carbon atoms; straight chain or branched chain alkyl containing one to about eight carbon atoms, straight chain or branched
• R a and R b are each independently hydrogen, (C,-C 6 )alkyl, hydroxy(C,-C 6 ) alkyl, amino(C 1 -C 6 )alkyl, aminosulfonyl, (C,-C 6 )alkanoyl, aryl, or benzyl, all of them optionally being substitued by one or more amino groups; or R a and R b together with the nitrogen to which they are attached form a pyrrolidino, piperidino, or morpholino group; the dashed lines in the five membered ring of formula (I) above denote an optional bond that connects a nitrogen of the five membered ring to the carbon that is between the two nitrogens ofthe five membered ring, and when the bond is present, either R 1 or R 3 is absent; provided, that R a and R b together allow formation of a quarternary ammonium ion either at the nitrogen of the central stuctural element N(R a
• R b or a pharmaceutically acceptable salt thereof.
• a pharmaceutically acceptable salt in the meaning of the present invention typically refers to an acetic or lactic acid salt.
• the inventive pharmaceutical composition comprises as imidazoquinoline or its derivative an imidazoquinolinamine such as 1 H- imidazo[4,5-c]quinolin-4-amines, typically selected from one of following formulas (II) to (Vl):
• R 11 is selected from the group consisting of alkyl of one to about ten carbon atoms, hydroxyalkyl of one to about six carbon atoms, acyloxyalkyl wherein the acyloxy moiety is alkanoyloxy of two to about four carbon atoms or benzoyloxy, and the alkyl moiety contains one to about six carbon atoms, benzyl, (phenyl)ethyl and phenyl, said benzyl, (phenyl)ethyl or phenyl substituent being optionally substituted on the benzene ring by one or two moieties independently selected from the group consisting of alkyl of one to about four carbon atoms, alkoxy of one to about four carbon atoms and halogen, with the proviso that if said benzene ring is substituted by two of said moieties, then said moieties together contain no more than six carbon atoms;
• R 21 is selected from the group consisting of hydrogen, alkyl of one to about eight carbon atoms, benzyl, (phenyl)ethyl and phenyl, the benzyl, (phenyl)ethyl or phenyl substituent being optionally substituted on the benzene ring by one or two moieties independently selected from the group consisting of alkyl of one to about four carbon atoms, alkoxy of one to about four carbon atoms and halogen, with the proviso that when the benzene ring is substituted by two of said moieties, then the moieties together contain no more than six carbon atoms; and each R 1 is independently selected from the group consisting of alkoxy of one to about four carbon atoms, halogen, and alkyl of one to about four carbon atoms, and n is an integer from 0 to 2, with the proviso that if n is 2, then said R 1 groups together contain no more than six carbon atoms;
• R 12 is selected from the group consisting of straight chain or branched chain alkenyl containing two to about ten carbon atoms and substituted straight chain or branched chain alkenyl containing two to about ten carbon atoms, wherein the substituent is selected from the group consisting of straight chain or branched chain alky I containing one to about four carbon atoms and cycloalkyl containing three to about six carbon atoms; and cycloalkyl containing three to about six carbon atoms substituted by straight chain or branched chain alkyl containing one to about four carbon atoms; and
• R 22 is selected from the group consisting of hydrogen, straight chain or branched chain alkyl containing one to about eight carbon atoms, benzyl, (phenyl)ethyl and phenyl, the benzyl, (phenyl)ethyl or phenyl substituent being optionally substituted on the benzene ring by one or two moieties independently selected from the group consisting of straight chain or branched chain alkyl containing one to about four carbon atoms, straight chain or branched chain alkoxy containing one to about four carbon atoms, and halogen, with the proviso that when the benzene ring is substituted by two such moieties, then the moieties together contain no more than six carbon atoms; and each
• R 2 is independently selected from the group consisting of straight chain or branched chain alkoxy containing one to about four carbon atoms, halogen, and straight chain or branched chain alkyl containing one to about four carbon atoms, and n is an integer from zero to 2, with the proviso that if n is 2, then said R 2 groups together contain no more than six carbon atoms;
• R 23 is selected from the group consisting of hydrogen, straight chain or branched chain alkyl of one to about eight carbon atoms, benzyl, (phenyl)ethyl and phenyl, the benzyl, (phenyl)ethyl or phenyl substituent being optionally substituted on the benzene ring by one or two moieties independently selected from the group consisting of straight chain or branched chain alkyl of one to about four carbon atoms, straight chain or branched chain alkoxy of one to about four carbon atoms, and halogen, with the proviso that when the benzene ring is substituted by two such moieties, then the moieties together contain no more than six carbon atoms; and each
• R 3 is independently selected from the group consisting of straight chain or branched chain alkoxy of one to about four carbon atoms, halogen, and straight chain or branched chain alkyl of one to about four carbon atoms, and n is an integer from zero to 2, with the proviso that if n is 2, then said R 3 groups together contain no more than six carbon atoms; or a pharmaceutically acceptable salt of any of the foregoing.
• R 14 is -CHR x R y wherein R y is hydrogen or a carbon-carbon bond, with the proviso that when R y is hydrogen R x is alkoxy of one to about four carbon atoms, hydroxyalkoxy of one to about four carbon atoms, 1 -alkynyl of two to about ten carbon atoms, tetrahydropyranyl, alkoxyalkyl wherein the alkoxy moiety contains one to about four carbon atoms and the alkyl moiety contains one to about four carbon atoms, 2-, 3-, or 4-pyridyl, and with the further proviso that when R y is a carbon-carbon bond R y and R x together form a tetrahydrofuranyl group optionally substituted with one or more substituents independently selected from the group consisting of hydroxy and hydroxyalkyl of one to about four carbon atoms;
• R 24 is selected from the group consisting of hydrogen, alkyl of one to about four carbon atoms, phenyl, and substituted phenyl wherein the substituent is selected from the group consisting of alkyl of one to about four carbon atoms, alkoxy of one to about four carbon atoms, and halogen; and is selected from the group consisting of hydrogen, straight chain or branched chain alkoxy containing one to about four carbon atoms, halogen, and straight chain or branched chain alkyl containing one to about four carbon atoms;
• R 15 is selected from the group consisting of: hydrogen; straight chain or branched chain alkyl containing one to about ten carbon atoms and substituted straight chain or branched chain alkyl containing one to about ten carbon atoms, wherein the substituent is selected from the group consisting of cycloalkyl containing three to about six carbon atoms and cycloalkyl containing three to about six carbon atoms substituted by straight chain or branched chain alkyl containing one to about four carbon atoms; straight chain or branched chain alkenyl containing two to about ten carbon atoms and substituted straight chain or branched chain alkenyl containing two to about ten carbon atoms, wherein the substituent is selected from the group consisting of cycloalkyl containing three to about six carbon atoms and cycloalkyl containing three to about six carbon atoms substituted by straight chain or branched chain alkyl containing one to about four carbon atoms; hydroxyalkyl of one
• R s and R ⁇ are independently selected from the group consisting of hydrogen, alkyl of one to about four carbon atoms, phenyl, and substituted phenyl wherein the substituent is selected from the group consisting of alkyl of one to about four carbon atoms, alkoxy of one to about four carbon atoms, and halogen;
• X is selected from the group consisting of alkoxy containing one to about four carbon atoms, alkoxyalkyl wherein the alkoxy moiety contains one to about four carbon atoms and the alkyl moiety contains one to about four carbon atoms, hydroxyalkyl of one to about four carbon atoms, haloalkyl of one to about four carbon atoms, alkylamido wherein the alkyl group contains one to about four carbon atoms, amino, substituted amino wherein the substituent is alkyl or hydroxyalkyl of one to about four carbon atoms, azido, chloro, hydroxy, 1 -morpholino, 1
• the inventive pharmaceutical composition comprises as imidazoquinoline or its derivative imidazoquinolin(amines) from following formula (VII):
• n 1 ,2, or 3;
• R 16 is selected from the group consisting of hydrogen; cyclic alkyl of three, four, or five carbon atoms; straight chain or branched chain alkyl containing one to about ten carbon atoms and substituted straight chain or branched chain alkyl containing one to about ten carbon atoms, wherein the substituent is selected from the group consisting of cycloalkyl containing three to about six carbon atoms and cycloalkyl containing three to about six carbon atoms substituted by straight chain or branched chain alkyl containing one to about four carbon atoms; fluoro- or chloroalkyl containing from one to about ten carbon atoms and one or more fluorine or chlorine atoms; straight chain or branched chain alkenyl containing two to about ten carbon atoms and substituted straight chain or branched chain alkenyl containing two to about ten carbon atoms, wherein the substituent is selected from the group consisting of cycloalkyl containing three to about six
• R y is hydrogen or a carbon-carbon bond
• R x is alkoxy of one to about four carbon atoms, hydroxyalkoxy of one to about four carbon atoms, l-alkynyl oftwo to about ten carbon atoms, tetrahydropyranyl, alkoxyalkyl wherein the alkoxy moiety contains one to about four carbon atoms and the alkyl moiety containsone to about four carbon atoms, 2-, 3-, or 4-pyridyl, and with the further proviso that when R y is a carbon-carbon bond R y and R x together form a tetrahydrofuranyl group optionally substituted with one or more substituents independently selected from the group consisting ofhydroxy and hydroxyalkyl of one to about four carbon atoms, is selected from the group consisting of hydrogen, straight chain or branched chain alkyl containing one to about eight carbon
• R is selected from the group consisting ofhydrogen, fluoro, chloro, straight-chain or branched chain alkyl containing one to about four carbon atoms, and straight chain or branched chain fluoro- or chloroalkyl containing one to about four carbon atoms and at least one fluorine or chlorine atom;
• the inventive pharmaceutical composition comprises as imidazoquinoline or its derivative imidazoquinolin(amines) from following formula (VIII):
• R 27 is selected from the group consisting of hydrogen, straight chain or branched chain alkyl containing one to about eight carbon atoms, straight chain or branched chain hydroxyalkyl containing one to about six carbon atoms, alkoxyalkyl wherein the alkoxy moiety contains one to about four carbon atoms and the alkyl moiety contains one to about six carbon atoms, benzyl, (phenyl)ethyl and phenyl, the benzyl, (phenyl)ethyl or phenyl substituent being optionally substituted on the benzene ring by a moiety selected from the group consisting of methyl, methoxy, and halogen; and morpholinoalkyl wherein the alkyl moiety contains one to about four carbon atoms; R 67 and R 77 are independently selected from the group consisting of hydrogen and alkyl of one to about five carbon atoms, with the proviso that R 67 and R 77 taken together contain no more than six carbon
• the inventive pharmaceutical composition comprises as imidazoquinoline or its derivative imidazoquinolin(amines) from following formula (IX): IX
• Z is selected from the group consisting of: -(CH 2 ) P - wherein p is 1 to 4;
• R D is hydrogen or alkyl of one to four carbon atoms
• R E is selected from the group consisting of alkyl of one to four carbon atoms, hydroxy, -OR F wherein R F is alkyl of one to four carbon atoms, and - NR G R' C wherein R G and R' G are independently hydrogen or alkyl of one to four carbon atoms; and -(CH 2 ) a -(Y)-(CH 2 ) b - wherein a and b are integers and a+b is 0 to 3, and Y is O, S, or
• R is hydrogen or alkyl of one to four carbon atoms; and wherein q is 0 or 1 ;
• R 8 is selected from the group consisting of alkyl of one to four carbon atoms, alkoxy of one to four carbon atoms, and halogen,
• the inventive pharmaceutical composition comprises as imidazoquinolin(amine) the specific compound imiquimod, preferably having the specific formula 1 -isobutyl-1 /-£imidazo[4,5-c)quinolin-4-amine, even more preferably comprising the following specific structure:
• the inventive pharmaceutical composition comprises as imidazoquinolin(amine) the specific compound resiquimod, preferably comprising the following specific structure:
• the pharmaceutical composition according to the invention may also comprise a mixture of more than one imidazoquinolin(amine) as defined above, typically 2 to 4 imidazoquinolin(amines), which may e.g. be selected from any of the above disclosed compounds according formulae (I) to (Xl).
• at least one of the therapeutically active components in such a pharmaceutically active composition comprising more than one imidazoquinolin(amine) corresponds to formula (Xl): imiquimod.
• the substituents Ru-Ri 7 above are generally designated "1 -substituents" herein.
• the 1 -substituents are preferably alkyl containing one to six carbon atoms and hydroxyalkyl containing one to six carbon atoms, e.g., the 1- substituent is 2-methylpropyl or 2-hydroxy-2-methylpropyl.
• the substituents R 2I -R 27 above are generally designated "2-substituents" herein.
• the 2-substituents are preferably hydrogen, alkyl of one to six carbon atoms, alkoxyalkyl wherein the alkoxy moiety contains one to four carbon atoms and the alkyl moiety contains one to four carbon atoms, and hydroxyalkyl of one to four carbon atoms, e.g., the 2-substituent is hydrogen, methyl, butyl, hydroxymethyl, ethoxymethyl or methoxyethyl.
• ,alkyl preferably includes straight or branched C,. 1O alkyl groups, e.g., methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, 1 -methylpropyl, 3- methylbutyl, hexyl, and the like.
• Jower alkyl preferably includes straight or branched C 1 6 alkyl groups, e.g., methyl, ethyl, propyl, 1 -methylethyl, butyl, 1 -methylpropyl, 2-methylpropyl, 1 ,1 dimethylethyl, pentyl, 1 -methylbutyl, 2-methylbutyl, 3-methylbutyl, 1 ,1 dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, and the like.
• alkylene preferably refers to a divalent straight or branched hydrocarbon chain (e.g. ethylene -CH 2 -CH 2 -).
• cycloalkyl preferably includes groups such as, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, and alkyl-substituted C 3 . 7 cycloalkyl group, preferably straight or branched C,_ 6 alkyl group such as methyl, ethyl, propyl, butyl or pentyl, and C 5 . 7 cycloalkyl group such as, cyclopentyl or cyclohexyl, and the like.
• Jower alkoxy preferably includes C,. 6 alkoxy groups, such as methoxy, ethoxy or propoxy, and the like.
• Jower alkanoyl preferably includes C,. 6 alkanoyl groups, such as formyl, acetyl, propanoyl, butanoyl, pentanoyl or hexanoyl, and the like.
• ,C 7-11 aroyl includes groups such as benzoyl or naphthoyl
• Jower alkoxycarbonyl preferably includes C 2 . 7 alkoxycarbonyl groups, such as methoxycarbonyl, ethoxycarbonyl or propoxycarbonyl, and the like.
• Jower alkylamino group preferably means an amino group substituted by C,_ 6 alkyl group, such as, methylamino, ethylamino, propylamino, butylamino, and the like.
• ,di(lower alkyl)amino group preferably means an amino group substituted by the same or different and C,_ 6 alkyl group (e.g. dimethylamino, diethylamino, ethylmethylamino).
• Jower alkylcarbamoyl group preferably means a carbamoyl group substituted by C 1 6 alkyl group (e.g. methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, butylcarbamoyl).
• ,Di(lower alkyl)carbamoyl group preferably means a carbamoyl group substituted by the same or different and C,. 6 alkyl group (e.g. dimethylcarbamoyl, diethylcarbamoyl, ethylmethylcarbamoyl).
• halogen atom as defined herein preferably means a halogen atom such as fluorine atom, chlorine atom, bromine atom or iodine atom.
• ,aryl as defined herein preferably refers to a C 6 ., 0 monocyclic or fused cyclic aryl group, such as phenyl, indenyl, or naphthyl, and the like.
• heterocyclic as defined herein preferably refers to monocyclic saturated heterocyclic groups, or unsaturated monocyclic or fused heterocyclic group containing at least one heteroatom, e.g., 0-3 nitrogen atoms, 0-1 oxygen atom (-O-), and 0-1 sulfur atoms (-S-).
• non-limiting examples of saturated monocyclic heterocyclic group includes 5 or 6 membered saturated heterocyclic group, such as tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperidyl, piperazinyl or pyrazolidinyl.
• unsaturated monocyclic heterocyclic group includes 5 or 6 membered unsaturated heterocyclic group, such as furyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl or pyrimidinyl.
• Non-limiting examples ofunsaturated fused heterocyclic groups includes unsaturated bicyclic heterocyclic group, such as indolyl, isoindolyl, quinolyl, benzothizolyl, chromanyl, benzofuranyl, and the like.
• alkyl, aryl, and heterocyclic groups as defined herein can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include lower alkyl; C,. 6 alkoxy, such as methoxy, ethoxy or propoxy; carboxyl; C 2 . 7 alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl or propoxycarbonyl) and halogen; cycloalkyl and include C 3 6 cycloalkyl; hydroxyl; C 1 .
• 6 alkylthio such as, methylthio, ethylthio, propylthio or butylthio; substituted C,. 6 alkylthio, such as methoxyethylthio, methylthioethylthio, hydroxyethylthio or chloroethylthio; aryl; substituted C 6-10 monocyclic or fused-cyclic aryl, such as 4-hydroxyphenyl, 4- methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl or 3,4-dichlorophenyl; 5-6 membered unsaturated heterocyclic, such as furyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl or pyrimidinyl; and bicyclic unsaturated heterocyclic, such as indolyl, isoindolyl, quinolyl, benzothiazolyl, chro
• heterocyclic ring as defined herein can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include C,. 6 alkyl; hydroxy C,. 6 alkylene; C 1-6 alkoxy C 1-6 alkylene; hydroxyl; C 1-6 alkoxy; and cyano.
• imidazoquinolin(amines) as defined above in the context of the present invention also may have a chiral center and may be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism.
• the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of imidazoquinolin(amines) or their derivatives as defined above, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographie separation using a chiral stationary phase) and how to determine nicotine agonist activity using the standard tests described herein, or using other similar tests which are well known in the art.
• the pharmaceutical composition according to the present invention typically comprises an imidazoquinolin(amine) or a derivative thereof as defined herein, preferably imiquimod or a derivative thereof, in an amount of about 0.005 % (w/v) to about 5 % (w/v), preferably in an amount of about 0.01 % (w/v) to about 5 % (w/v), more preferably in an amount of about 0.1 % (w/v) to about 4 % (w/v), even more preferably in an amount of about 0.1 % (w/v) to about 3 % (w/v), even further preferred in an amount of about 0.2 % (w/v) to about 2 % (w/v), and most preferably in an amount of about 0.2 % (w/v) to about 1 % (w/v) or even in an amount of about 0.5 % (w/v) to about 1 % (w/v), wherein the amounts as defined in % (w/v) may either be determined on the basis of the amount of the
• the above amounts may be defined in % (w/w), wherein the amount as defined in % (w/w) may either be determined on the basis of the weight of imidazoquinolin(amine) or a derivative thereof as defined above, preferably imiquimod or a derivative thereof, with respect to the total weight of the inventive pharmaceutical composition.
• the pharmaceutical composition according to the present invention typically comprises an imidazoquinolin(amine) or a derivative thereof as defined herein, preferably imiquimod or a derivative thereof, in an amount of about 0.005 % (w/v) to about 1 , 2, 3, 4, or 5 % (w/v), in an amount of about 0.01 % (w/v) to about 1 , 2, 3, 4, or 5 % (w/v), in an amount of about 0.1 % (w/v) to about 1 , 2, 3, 4, or 5 % (w/v), in an amount of about 0.2 % (w/v) to about 1 , 2, 3, 4, or 5 % (w/v), in an amount of about 0.3 % (w/v) to about 1 , 2, 3, 4, or 5 % (w/v), in an amount of about 0.4 % (w/v) to about 1, 2, 3, 4, or 5 % (w/v), in an amount of about 0.5 % (w/v) to about 1, 2, 3, 4, or 5 % (w/v
• the pharmaceutical composition according to the present invention typically comprises an imidazoquinolin(amine) or a derivative thereof as defined herein, preferably imiquimod or a derivative thereof, in an amount of about 0.005 % (w/v) to about 0,5 % (w/v), in an amount of about 0.01 % (w/v) to about 0,5 % (w/v), in an amount of about 0.1 % (w/v) to about 0,6 % (w/v), in an amount of about 0.2 % (w/v) to about 0,7 % (w/v), in an amount of about 0.3 % (w/v) to about 0,8 % (w/v), in an amount of about 0.4 % (w/v) to about 0,9 % (w/v), in an amount of about 0.5 % (w/v) to about 1 ,0 % (w/v), in an amount of about 0.6 % (w/v) to about 1 ,1 % (w/v), in an
• imidazoquinolin(amine) or a derivative thereof as defined herein may be dissolved in such a
• the (stock) aqueous solution comprsing either lactic acid or acetic acid or a mixture of both as defined above typically in a higher concentration as indicated above for the final inventive pharmaceutical composition.
• the (stock) solution may comprise an amount of about 0.005 % (w/v) or 0.01 % (w/v) to about 30 % (w/v), preferably an amount of about 1 % (w/v) to about 25 % (w/v), more preferably an amount of about 5 % (w/v) to about 25 % (w/v), even more preferably an amount of about 10 % (w/v) to about 25 %
• the inventive pharmaceutical composition may also comprise additives or further components.
• additives or further components enhance the solubility and/or membrane penetration of the imidazoquinolin(amine) or a derivative thereof as defined herein in the inventive pharmaceutical composition.
• such additives or further components allow to provide a more suitable formulation for a specific disease to be treated, confer a better tolerance to the inventive pharmaceutical composition, etc.
• the inventive pharmaceutical composition may comprise cyclodextrines, which are also designated cycloamyloses.
• cyclodextrines may be used to enhance solubility and, advantageously, membrane penetration of the imidazoquinolin(amine) or a derivative thereof as defined herein in the inventive pharmaceutical composition, even though the imidazoquinolin(amine) or a derivative thereof may not be sufficiently dissolved by just cyclodextrines.
• solubility of the imidazoquinolin(amine) or a derivative thereof as defined herein is not only enhanced in the final inventive pharmaceutical composition using cyclodextrines but also in an intermediate (stock) solution formed by imidazoquinolin(amine) or a derivative thereof as defined herein and an organic acid as defined above, e.g. lactic acid, acetic acid or a mixture thereof.
• cyclodextrin in combination with lactic acid and/or acetic acid leads to a small, but significant increment of the solubility of imidazoquinolin(amine) or a derivative thereof, particularly of imiquimod, of at least 10%, more preferably of at least 15% or at least about 18 % compared to the solubility of that therapeutically active agent in combination with lactic acid alone.
• cyclodextrines may be used in any stage of preparation of the pharmaceutical composition to enhance the solubility of the imidazoquinolin(amine) or a derivative thereof as defined herein.
• cyclodextrines are preferably understood as members of a family of cyclic oligosaccharides, composed of 5 or more ⁇ -D- glucopyranoside units linked between positions 1 and 4, as known for amylose, a fragment of starch.
• cyclodextrines particularly comprise ⁇ - cyclodextrins, which form six membered sugar ring molecules, ⁇ -cyclodextrins, which form, seven sugar ring molecules, ⁇ -cyclodextrins, which form eight sugar ring molecules, ⁇ -cyclodextrins and ⁇ -cyclodextrins.
• the inventive pharmaceutical composition comprises ⁇ -cyclodextrins, ⁇ -cyclodextrins, and/or ⁇ -cyclodextrins, even more preferably, ⁇ -cyclodextrins, such as hydroxypropyl- ⁇ -cyclodextrin (HP- ⁇ -CD).
• ⁇ -cyclodextrins such as hydroxypropyl- ⁇ -cyclodextrin (HP- ⁇ -CD).
• the inventive pharmaceutical composition as defined above may comprise cyclodextrines as defined above in an amount of about 0.1 % (w/v) to about 30 % (w/v), typically in an amount of about 1 % (w/v) to about 20 % (w/v), preferably in an amount of about 2 % (w/v) to about 20 % (w/v), more preferably in an amount of about 5 % (w/v) to about 20 % (w/v), even more preferably in an amount of about 5 % (w/v) to about 15 % (w/v), and most preferably in an amount of about 10 % (w/v) to about 15 % (w/v), or in an amount of about 0.1 % (w/v) to about 4 % (w/v), 0,1 to 2%, more prefwerably in an amount of 0.5 to 2% or, alternatively, in an amount of about 2 % (w/v) to about 6 % (w/v), in an
• the above amounts may be defined in % (w/w), wherein the amount as defined in % (w/w) may either be determined on the basis of the weight of the cyclodextrine with respect to the total weight of the inventive pharmaceutical composition or on the basis of an intermediate stock solution.
• the pharmaceutical composition according to the present invention further comprises at least one thermo-sensitive agent.
• thermo-sensitive agent typically refers to a compound, preferably a polymer, which is able to change its state of aggregation or its viscosity at a defined point of transition (cooperative transition) from a liquid or semi-liquid state into a solid or semi solid state, preferably to a solid state.
• thermal-sensitive agent typically refers to a compound, preferably a(n) (organic) polymer, which is able to change its state of aggregation from a liquid or semi-liquid state into a solid or semi solid state (e.g.
• thermo-sensitive agent as defined above allows an in situ gel formation of the thermo- sensitive agent and any compound or composition formulated therewith, e.g.
• thermo-sensitive agent typically occurs directly upon or directly subsequent to administration of the inventive pharmaceutical composition to the site of affliction of the patient to be treated, i.e. not prior to administration of the inventive pharmaceutical composition.
• an in situ gel formation is in particular advantageous for specific applications, which are intended to release imidazoquinolin(amine) or a derivative thereof of the inventive pharmaceutical compoition as defined herein over an extended period of time.
• Such applications are usually directed to place the formulation in a body cavity, e.g. of a tissue or organ, such as the bladder, and may therefore be particularly suitable for, e.g., vesical administration in the therapy of bladder diseases.
• thermo-sensitive agents are the ease of its administration due to selection of a transition point at a temperature range as defined above. More specifically, the selection of a transition point at a temperature range as defined above allows not only for preparation or storage of the inventive pharmaceutical composition in a liquid or semi-liquid aggregate state. It also allows for administration of the (preferably liquid) inventive pharmaceutical composition by e.g. injection, since the inventive pharmaceutical composition directly solidifies or undergoes gelation subsequent to administration due to the increased temperature of the surrounding tissue or organ, which is preferably higher than the temperature of the transition point. Accordingly, gel formation is induced within the tissue or organ.
• Administration thereby may be carried out using non-invasive methods (without surgery), such as an injection needle having a cannula of a suitable diameter, an injection tube, endoscopic methods, etc.
• non-invasive methods such as an injection needle having a cannula of a suitable diameter, an injection tube, endoscopic methods, etc.
• gel formation results in increased bioadhesive properties of the inventive pharmaceutical composition of the present invention leading to a prolonged exposure of the imidazoquinolin(amine) or a derivative thereof of the inventive pharmaceutical composition to TLR7-expressing cells and less systemic drug penetration.
• the exposure of imidazoquinolin(amine) or a derivative thereof to TLR7-expressing cells induces an immunological response exerting the desired therapeutic effect.
• thermo- sensitive agents advantageously avoids or at least significantly reduces systemic side effects of imidazoquinolin(amine) or a derivative thereof due to local administrationat the site of affliction, increased in vivo viscosity of the inventive pharmaceutical composition, reduced diffusion of the biologically active agentto surrounding tissues, and, in addition, in some cases also due to increased bioadhesive properties.
• the pharmaceutical compositions of the present invention comprising thermo- sensitive agents advantageously allow for a so called “extended release” (or sometimes termed “long term release") of the imidazoquinolin(amine) or a derivative thereof contained therein.
• extended release or sometimes termed “long term release”
• the gel formation of the inventive pharmaceutical composition results in a sustained drug release of imidazoquinolin(amine) or a derivative thereof in a zero-order kinetic which enhances the duration of the therapeutic effect.
• Such prolonged therapeutic effect of the imidazoquinolin(amine) or a derivative thereof contained in the inventive pharmaceutical composition also avoids repeated administration of the inventive pharmaceutical composition, particularly in short time intervals, which typically cannot be avoided when using pharmaceutical compositions without exhibiting sustained drug release.
• the depot effect of a pharmaceutical composition of the invention typically lasts at least 24h, more preferably48h, more preferably at least 7 days, typically releasing the active agent in app. constant amounts over time (e.g. comparable release amounts within 24 h on e.g day 2 and day 8 after administration).
• the inventive pharmaceutical composition as defined above contains a thermo-sensitive agent as defined herein in an amount of about 0.1 % (w/v) to about 40 % (w/v), preferably between 0.1 and 5% (more preferably 0.1 . and 2%) or, alternatively, typically in an amount of about 2 % (w/v) to about 30 % (w/v), preferably in an amount of about 5 % (w/v) to about 30 % (w/v), more preferably in an amount of about 10 % (w/v) to about 30 % (w/v), and most preferably in an amount of about 10 % (w/v) to about 25 % (w/v), e.g.
• % (w/v) in an amount of about 0.1 % (w/v) to about 10 % (w/v), in an amount of about 5 % (w/v) to about 15 % (w/v), in an amount of about 10 % (w/v) to about 20 % (w/v), in an amount of about 15 % (w/v) to about 25 % (w/v), in an amount of about 20 % (w/v) to about 30 % (w/v), in an amount of about 25 % (w/v) to about 35 % (w/v), or in an amount of about 30 % (w/v) to about 40 % (w/v), wherein the amounts as defined in % (w/v) may either be understood to be given on the basis of the weight of the thermo-sensitive agent as defined herein with respect to the total volume of the inventive pharmaceutical composition or an intermediate stock solution, when e.g.
• the above amounts may be defined in % (w/w), wherein the amount as defined in % (w/w) may either be understood to be based on the weight of the thermo-sensitive agent as defined herein with respect to the total weight of the inventive pharmaceutical composition.
• the inventive pharmaceutical composition as defined above contains as a thermo-sensitive agent a chitosan or a derivate thereof.
• a chitosan is preferably understood as a linear polysaccharide composed of randomly distributed ⁇ -(1 -4)-l inked D-glucosamine (deacetylated unit) and of N-acetyl-D-glucosamine (acetylated unit).
• Chitosan is typically produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans such as crabs, shrimps, etc.
• the degree of deacetylation is typically determined by NMR spectroscopy, wherein the %DA in commercial chitosans is typically in the range 60-100 %.
• Chitosans in the context of the present invention furthermore include derivatives thereof such as trimethylchitosan, wherein the amino group of chitosan has been trimethylated or oligomeric derivatives (3-6 kDa) of chitosans.
• the inventive pharmaceutical composition as defined above contains as a thermo-sensitive agent a poloxamer.
• Poloxamers solubilised at relatively high concentrations (> 17.5%) in water, are able to produce systems that are liquid at low temperature (below room temperature) and that form jelly-like, semi-solid or solid structures or gels at elevated temperatures. Poloxamers are commercially available under the trade name Pluronics (BASF AG, Ludwigshafen, Germany). Poloxamers are present in many marketed pharmaceutical products as solubilizers, surfactants, viscosizing and jellifying agents.
• the inventive pharmaceutical composition preferably does not comprise any further components exhibiting surfactant properties apart from such thermo-sensitive components, e.g. poloxamers, which additionally exhibit surfactant properties.
• Poloxamers in the context of the present invention are typically to be understood as a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer, also abbreviated "PEO-PPO-PEO".
• Such poloxamers are therefore nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).
• the molecular weight of such poloxamers is generally not specifically defined and may be varied suitably for each specific purpose. Because the lengths of the polymer blocks can be customized, a multitude of poloxamers may be provided having slightly different properties.
• P407 Poloxamer with a polyoxypropylene molecular mass of 4,000 g/mol and a 70% polyoxyethylene content.
• the first digit (two digits in a three-digit number) in the numerical designation, multiplied by 300, indicates the approximate molecular weight of the hydrophobe; and the last digit x 10 gives the percentage polyoxyethylene content (e.g., L61 Pluronic with a polyoxypropylene molecular mass of 1 ,800 g/mol and a 10% polyoxyethylene content).
• poloxamer 181 (P181 ) Pluronic L61.
• Poloxamers suitable for the inventive pharmaceutical composition as a thermo-sensitive agent preferably comprise any poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer or mixture of such copolymers suitable for the inventive purpose, i.e. any PEO-PPO-PEO polymer or mixture of such copolymers exhibiting thermo-sensitive properties as defined above.
• Such PEO-PPO-PEO polymers include also commercially available PEO-PPO-PEO polymers and mixtures thereof, e.g.
• poloxamers suitable for the inventive pharmaceutical composition as a thermo-sensitive agent include poloxamers or mixtures thereof, the poloxamers selected from Poloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338, Poloxamer 403, and Poloxamer 407.
• poloxamers suitable for the inventive pharmaceutical composition as a thermo-sensitive agent include poloxamers or mixtures thereof, the poloxamers selected from Poloxamer 188, Poloxamer 403, and Poloxamer 407.
• Poloxamer 407 is most preferably selected and represents the first choice-polymer for the production of thermo-responsive gels within the context of the present invention. Poloxamer 407 as a thermo-sensitive agent may be used either alone or in admixture with other poloxamers as described above, preferably with
• the inventive pharmaceutical composition comprises as a thermo-sensitive agent Poloxamer 407 in an amount as defined above in general for thermo-sensitive agents, more preferably in an amount of about 0.1, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 % (w/v) to about 30 % (w/v), even more preferably in an amount of about 5 % (w/v) to about 25 % (w/v), and most preferably in an amount of about 10 % (w/v) to about 25 % (w/v), e.g.
• the above amounts may be defined in % (w/w), wherein the amount as defined in % (w/w) may either be understood to be based on the weight of the thermo-sensitive agent as defined herein with respect to the total weight of the inventive pharmaceutical composition.
• the inventive pharmaceutical composition may comprise as a thermo-sensitive agent any of the poloxamers as defined above, preferably any of the poloxamers selected from Poloxamer 124, Poloxamer 188, Poloxamer
• Such poloxamers are preferably present in the inventive pharmaceutical composition as a thermo-sensitive agent in amounts as described above for thermo-sensitive agents in general or more specifically as described above for Poloxamer 407.
• the inventive pharmaceutical composition may also comprise as a thermo-sensitive agent a mixture of any of the thermo- sensitive agents as defined above.
• a mixture of thermo-sensitive agents preferably comprises an overall amount of the thermo-sensitive agent of such a mixture similar as described above in general for thermo-sensitive agents or more specifically as described above for Poloxamer 407.
• such a mixture of thermo-sensitive agents preferably comprises a ratio of the different poloxamers, which leads to a mixture of thermo-sensitive agents that "jellifies" at a desired temperature, preferably at a temperatur range as defined above.
• the ratio and/or the amount of different poloxamers in the mixture may influence the "Lower Critical Solution Temperature” (LCST) and thus the gel transition temperature.
• LCST Low Critical Solution Temperature
• the amount and/or ratio of the different poloxamers in the mixture is preferably selected with respect to the drug solubility.
• any of two of the thermo-sensitive agents as defined above may be contained in the inventive pharmaceutical composition as a mixture in a range of about 1 :10 to about 10:1, e.g. in a ratio of about 1 :10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, or 10:10 (i.e. 1 :1 ), or in a ratio of about 10: 9, 10: 8, 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, or 10:1, or in a range formed by any of two of the ratio values as defined above.
• the inventive pharmaceutical composition may comprise as a thermo-sensitive agent a mixture of Poloxamer 407 and any of the above described poloxamers, even more preferably selected from Poloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338, and Poloxamer 403.
• a mixture of thermo- sensitive agents preferably comprises an overall amount of the thermo-sensitive agents of the mixture similar as described above in general for thermo-sensitive agents or more specifically as described above for Poloxamer 407.
• the inventive pharmaceutical composition may comprise as a thermo-sensitive agent a mixture of Poloxamer 407 and Poloxamer 188.
• a mixture of Poloxamer 407 and Poloxamer 188 is contained in the inventive pharmaceutical composition in an overall amount as described above in general for thermo-sensitive agents or more specifically as described above for Poloxamer 407.
• a mixture of Poloxamer 407 and Poloxamer 188 also occurs in the inventive pharmaceutical composition in a ratio as described above in general for mixtures of polyoxamers used as a thermo-sensitive agent in the inventive pharmaceutical composition.
• such a ratio is selected from a ratio of Poloxamer 407 : Poloxamer 188 of about 1 :20, 1 :19, 2:18:, 3:1 7, 4:16, 5:15, 6:14, 7:13, 8:12, 9:1 1 , 10:10 (1 :1 ), 1 1 :9, 12:8, 13:7, 14:6, 15:5, 16:4, 17:3, 18:2, 19:1 or 20:1 , or a ratio formed by any of two of the values as defined above.
• such a ratio is selected from a ratio of Poloxamer 407 : Poloxamer 188 of about 7:3, 7.5:2.5, 8:2, 8.5:1 .5, 9:1 , or 9.5:0.5, or a ratio formed by any of two of these values.
• the absolute content of Poloxamer 188 and of Poloxamer 407 in the inventive pharmaceutical composition may be determined on basis of the overall amount and on the specific ratio of both poloxamers in the inventive pharmaceutical composition.
• the inventive pharmaceutical composition may comprise as a thermo-sensitive agent Poloxamer 407 in an (overall) amount of about 1 7.5 % (w/v)/(w/w) to about 22.5 % (w/v)/(w/w), more preferably in an overall amount of about 17.5 % (w/v)/(w/w), of about 18.0 % (w/v)/(w/w), of about 18.5 % (w/v)/(w/w), of about 19.0 % (w/v)/(w/w), of about 19.5 % (w/v)/(w/w), of about 20.0 % (w/v)/(w/w), of about 20.5 % (w/v)/(w/w), of about 21.0 % (w/v)/(w/w), of about 21 .5 % (w/v)/(w/w), of about 22.0 % (w/v)/(w/w), or of about 22.5 % (
• the inventive pharmaceutical composition may comprise as a thermo-sensitive agent a mixture of Poloxamer 407 and Poloxamer 188 in an overall amount of about 22.5 % (w/v)/(w/w) to about 27.5 % (w/v)/(w/w), more preferably in an overall amount of about 25 % (w/v)/(w/w), and preferably in a ratio of Poloxamer 407 : Poloxamer 188 of about 15:5, 16:4, 1 7:3, 18:2, 19:1 or 20:1 , or a ratio formed by any of two of these values, more preferably in a ratio of about 9.5:0.5, of about 9:1 , of about 8.5:1.5, or of about 8:2, or a ratio formed by any of two of these values.
• Poloxamer 407 when the inventive pharmaceutical composition comprises a mixture of Poloxamer 407 and Poloxamer 188, Poloxamer 407 may be present in the inventive pharmaceutical composition in an amount of about 15.5 % (w/v)/(w/w) to about 26.5 % (w/v)/(w/w), preferably in an amount of about 17.5 % (w/v)/(w/w) to about 22.5 % (w/v)/(w/w), whereas Poloxamer 188 may be present in the pharmaceutical composition in an amount of about 1.0 % (w/v)/(w/w) to about 6.0 % (w/v)/(w/w), preferably in an amount of about 2.5 % (w/v)/(w/w) to about 4.5 % (w/v)/(w/w). (The term (w/v)/(w/w) means either (w/v) or (w/w)).
• the inventive pharmaceutical composition may furthermore comprise additives or further components, such as e.g. cyclodextrin as defined above.
• additives or further components such as e.g. cyclodextrin as defined above.
• the content of additives, particularly cyclodextrin may have an influence on above defined so called “lower critical solution temperature” (LCST) or "gel transition temperature.
• LCST lower critical solution temperature
• increasing percentages of cyclodextrin result in an increase of LCST of Poloxamer 407 or a mixture of Poloxamer 407 and Poloxamer 188 as defined above. This may suitable applied to other poloxamers as defined above and to other thermo-sensitive agents as defined herein.
• the LCST of the pharmaceutical compositions of the present invention are differently influenced by acetic acid and lactic acid. Both acids generally increase the LCST of the compositions. However, while e.g. imiquimod does not influence at all the gel transition temperature of inventive pharmaceutical compositions comprising only acetic acid, the LCST of compositions comprising lactic acid is enhanced. Thus, the final percentage of thermo-sensitive agents as defined herein may depend on the used acid and whether cyclodextrin is present or not.
• the pharmaceutical composition according to the invention may further comprise a pharmaceutically acceptable carrier and/or vehicle.
• a pharmaceutically acceptable carrier typically includes a liquid basis of the inventive inventive pharmaceutical composition, e.g. pyrogen- free water; the free water solution may be combined in any appropriate ratio with a water- miscible, pharmaceutically acceptable organic solvent, e.g. an alcohol (e.g. ethanol or isipropanol); the following may also be used: isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrate, etc. buffered solutions, an aqueous buffered solution, containing e.g.
• a sodium salt preferably at least 50 mM of a sodium salt, a calcium salt, preferably at least 0.01 mM of a calcium salt, and/or a potassium salt, preferably at least 3 mM of a potassium salt.
• the sodium, calcium and/or potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.
• examples of sodium salts include e.g. NaCI, NaI, NaBr, Na 2 CO 3 , NaHCO 3 , Na 2 SO 4
• examples of the optional potassium salts include e.g.
• KCl, Kl, KBr, K 2 CO 3 , KHCO 3 , K 2 SO 4 , and examples of calcium salts include e.g. CaCI 2 , CaI 2 , CaBr 2 , CaCO 3 , CaSO 4 , Ca(OH) 2 .
• organic anions of the aforementioned cations may be contained in the composition of the invention.
• the composition of the invention suitable for injection purposes as defined above may contain salts selected from sodium chloride (NaCI), calcium chloride (CaCl 2 ) and optionally potassium chloride (KCI), wherein further anions may be present additional to the chlorides.
• CaCI 2 can also be replaced by another salt like KCI.
• composition of the invention may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the composition of the invention may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects.
• Reference media are e.g. liquids occurring in "in vivd' methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person.
• compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well for the inventive pharmaceutical composition, which are suitable for administration to a patient to be treated.
• the term "compatible” as used here means that these constituents of the inventive pharmaceutical composition are capable of being mixed with the imidazoquinolin(amines) or derivatives thereof as defined according to the present invention in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive pharmaceutical composition under typical use conditions.
• Pharmaceutically acceptable carriers, fillers and diluents must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated.
• Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
• sugars such as, for example, lactose, glucose and sucrose
• starches such as, for example, corn star
• emulsifiers such as, for example, Tween ® ; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet- forming agents; stabilizers; antioxidants; preservatives.
• the inventive pharmaceutical composition may comprise an adjuvant.
• an adjuvant may be understood as any compound, which is suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response.
• the inventive pharmaceutical composition when administered, typically elicits an innate immune response due to the adjuvant, optionally contained therein.
• Such an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal.
• the adjuvant may be selected from the group consisting of, without being limited thereto, any of the following including chitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMERTM (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1 .25%), phosphate-buffered saline, pH 7.4); AVRIDINETM (propanediamine); BAY R1005TM ((N-(2-deoxy-2-L-leucylamino-b-D- glucopyranosyl)-N-octadecyl-dodecanoyl
• TM liposomes
• LOXORIBINETM (7-allyl-8-oxoguanosine); LT oral adjuvant ⁇ E.coli labile enterotoxin- protoxin); microspheres and microparticles of any composition
• MF59TM (squalene-water emulsion);
• MONTANIDE ISA 51 TM purified incomplete Freund's adjuvant
• MONTANIDE ISA 720TM metabolicisable oil adjuvant
• MPLTM (3-Q-desacyl-4'-monophosphoryl lipid A);
• MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 ,2- dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt);
• MURAMETIDETM Nac-Mur-L-Ala-D-Gln-OCHj
• MURAPALMITINETM and D- MURAPALMITINETM Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl
• NISVs neuroionic surfactant vesicles
• PLEURANTM ⁇ -glucan
• PLA polymer of lactic acid and glycolic acid; microspheres/nanospheres
• PLURONIC L 121 TM PMMA (polymethyl methacrylate); PODDSTM (proteinoid microspheres); polyethylene carbamate derivatives
• poly-rA poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, AL); STIMULONTM (QS-21 ); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2- ethoxymethyl-1 H-imidazo[4,5-
• the inventive pharmaceutical composition may additionally contain one or more auxiliary substances in order to further increase its immunomoulatory effect.
• various mechanisms can come into consideration in this respect. For example, compounds that permit the maturation of dendritic cells (DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class of suitable auxiliary substances.
• DCs dendritic cells
• TNF-alpha or CD40 ligand form a first class of suitable auxiliary substances.
• auxiliary substance any agent that influences the immune system in the manner of a "danger signal" (LPS, GP96, etc.) or cytokines, such as GM-CSF, which allow for an immune response produced by the immune-stimulating adjuvant according to the invention to be enhanced and/or influenced in a targeted manner.
• a "danger signal” LPS, GP96, etc.
• cytokines such as GM-CSF
• auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, that further promote the innate immune response, such as IL-I , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31 , IL-32, IL-33, INF-alpha, IFN-beta, INF- gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.
• cytokines such as monokines, lymphokines,
• the inventive pharmaceutical composition can also additionally or alternatively contain any further compound, which is known to be immune-stimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRl O, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13.
• any further compound which is known to be immune-stimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13.
• CpG nucleic acids in particular CpG-RNA or CpG- DNA.
• a CpG-RNA or CpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA).
• the CpG nucleic acid is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA).
• the CpG nucleic acid preferably contains at least one or more (mitogenic) cytosine/guanine dinucleotide sequence(s) (CpG motif(s)).
• CpG motif(s) cytosine/guanine dinucleotide sequence(s)
• at least one CpG motif contained in these sequences that is to say the C (cytosine) and the G (guanine) of the CpG motif, is unmethylated. All further cytosines or guanines optionally contained in these sequences can be either methylated or unmethylated.
• the C (cytosine) and the G (guanine) of the CpG motif can also be present in methylated form.
• the inventive pharmaceutical composition typically comprises a "safe and effective amount" of the above components of the inventive pharmaceutical composition, particularly of the imidazoquinolin(amines) and derivatives thereof as defined according to the present invention.
• a "safe and effective amount” means an amount of these component, particularly of the imidazoquinolin(amines) and derivatives thereof, that is sufficient to significantly induce a positive modification of a disease or disorder as defined herein.
• a "safe and effective amount” is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.
• a "safe and effective amount" of the components of the inventive pharmaceutical composition will furthermore vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, their activity, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the accompanying doctor.
• the inventive pharmaceutical composition may be used for human and also for veterinary medical purposes, preferably for human medical purposes.
• the inventive pharmaceutical composition will contain or release sufficient active imidazochinolin(amine) or a derivative thereof to provide a dose of about 10, 20, 50, or 100 nanograms per kilogram (ng/kg) to about 50 milligrams per kilogram (mg/kg), preferably about 10 micrograms per kilogram ( ⁇ g/kg) to about 5 mg/kg, of the compound or a salt thereof to the subject.
• the method includes administering sufficient imidazochinolin(amine) or a derivative thereof to provide a dose of from about 0.0001 , 0.001 , 0.01 , or 0.1 mg/m 2 to about 2.0 mg/ m 2 to the subject, for example, a dose of from about 0.004, 0.04, or 0.4 mg/m 2 to about 1 .2 mg/m 2 .
• the inventive pharmaceutical composition may be administered locally.
• Routes for local administration in general include, for example, topical administration routes but also intravesical, intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections. More preferably, the inventive pharmaceutical composition may be administered by an intravesical route.
• the suitable amount of the inventive pharmaceutical composition to be administered can be determined by routine experiments with animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models.
• Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof.
• compositions of the invention being restricted to specific components.
• the inventive pharmaceutical composition as defined above comprises at least one imidazoquinolin(amine) or a derivative thereof as defined herein and at least one organic acid as defined above selected from lactic acid and/or acetic acid - preferably in such a concentration that its final pH is from pH 3 to 5, preferably 3.5 to 4.5.
• the components of the inventive composition form the inventive adduct structures.
• the inventive pharmaceutical composition as defined above comprises preferably at least one imidazoquinolin(amine) or a derivative thereof as defined herein and at least one organic acid as defined above selected from lactic acid and/or acetic acid, however containing no more than 4, 3, 2, 1 or most preferably no further organic and/or inorganic acid as defined herein or, alternatively, less than 2 inorganic acids and no further organic acid or, still aternatively, just on additional organic acid and no inorganic acids.
• the inventive pharmaceutical composition as defined above comprises at least one imidazoquinolin(amine) or a derivative thereof as defined herein, and at least one organic acid as defined above selected from lactic acid and/or acetic acid, and less than 4, or 3 thermo-sensitive agents or more preferably just one thermo-sensitive agent.
• the inventive composition may also comprise no thermosensitive agent.
• the combination of a restricted number of thermo-sensitive agents in the inventive pharmaceutical composition and a restricted number of acids as defined above are also provided.
• the inventive pharmaceutical composition as defined above may comprise e.g.
• the inventive pharmaceutical composition may comprise no surfactants apart from thermo-sensitive agents (if any), which may have additionally surfactant properties. Accordingly, the inventive composition may comprise also no surfactant and no thermo-sensitive agent.
• the inventive pharmaceutical composition may contain less than 4, 3, or 2 cyclodextrines or, alternatively, no cyclodextrine at all. These embodiments may be combined with the preferred above embodiments. Accordingly, the inventive composition may e.g. contain no thermosensitive agent, no cyclodextrine and no surfactant. In addition, the inventive composition may contain no further solubility enhancer, be it a surfactant, a cyclodextrin or be it another solubility enhancing compound, apart from lactic and/or acetic acid.
• the inventive composition comprises at least one imidaziquinoli(amine) compound, acetic and/or lactic acid and 1 , 2 or no thermosensive agent(s) and no further solubility enhancing compound and no further therapeutically active ingredient and no cellulose or cellulose derivative.
• the inventive composition may exclusively additionally contain one or more of the following standard components belonging to the class of stabilizers and preservatives.
• the object of the present invention is solved by the use of imidazoquinolin(amines) and derivatives thereof as defined herein (for the manufacture of a pharmaceutical composition, e.g. as defined herein,) for the prophylaxis, treatment and/or amelioration of any of the diseases and disorders as defined herein, e.g.
• precancerous conditions such as actinic keratosis, genital warts (condylomata), VIN (vulvar intraepithelial neoplasia), VAIN (vaginal intraepithelial neoplasia), etc., Molluscum contagiosum, skin cancers, such as basal cell carcinoma, Bowen's disease, squamous cell carcinoma, superficial malignant melanomas, etc., bladder diseases, such as, for example, bladder cancer and cystitis, etc, cancer, including peritoneal cancer, ovarian cancer, etc.
• precancerous conditions such as actinic keratosis, genital warts (condylomata), VIN (vulvar intraepithelial neoplasia), VAIN (vaginal intraepithelial neoplasia), etc., Molluscum contagiosum, skin cancers, such as basal cell carcinoma, Bowen's disease, squamous
• the object of the present invention is solved by the use of imidazoquinolin(amines) and derivatives thereof as defined herein, (for the manufacture of a pharmaceutical composition, e.g. as defined herein,) for the
• imidazoquinolin(amines) and derivatives thereof as defined herein are preferably provided in a formulation as described above for the inventive pharmaceutical composition.
• the present invention furthermore comprises methods of treatment of diseases and disorders as defined herein, including skin disorders including precancerous conditions, such as actinic keratosis, genital warts (condylomata), VIN (vulvar intraepithelial neoplasia), VAIN (vaginal intraepithelial neoplasia), etc., Molluscum contagiosum, skin cancers, such as basal cell carcinoma, Bowen's disease, squamous cell carcinoma, superficial malignant melanomas, etc., bladder diseases, such as, for example, bladder cancer and cystitis, etc, cancer, including peritoneal cancer, ovarian cancer, etc., using the inventive pharmaceutical composition, preferably as defined above.
• skin cancers such as basal cell carcinoma, Bowen's disease, squamous cell carcinoma, superficial malignant melanomas, etc.
• bladder diseases such as, for example, bladder cancer and cystitis, etc, cancer, including peritoneal cancer, ovarian cancer
• such methods of treatment as defined above preferably comprise administration of the inventive pharmaceutical composition using any administration mode as defined above, preferably including, topical administration routes but also intravesical, intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections.
• the present invention comprises methods of (intravesical) treatment of bladder diseases, such as, for example, bladder cancer and cystitis, etc., using the inventive pharmaceutical composition, preferably as defined above.
• inventive methods of (intravesical) treatment of bladder diseases typically comprises the intravesical administration of the inventive pharmaceutical composition.
• inventive pharmaceutical composition may be administered using non-invasive methods, such as an injection needle having a cannula of a suitable diameter, an injection tube, endoscopic methods, etc.
• Figure 1 shows the viscosity of systems containing different percentages of Poloxamer
• Figure 2 shows the viscosity of systems containing different percentages of HP- ⁇ -CD
• Figure 3 shows the viscosity of systems containing different proportions of Poloxamer
• Poloxamer 188 (0% to 30%) as a function of temperature.
• Figure 4 shows the viscosity of systems prepared with 20% Poloxamer 407 or 25% poloxamer mixture in an acetic acid solution (AA), in presence or in the absence of imiquimod (TMX, 0.4%) as a function of temperature.
• Figure 5 shows the viscosity of systems prepared with 20% Poloxamer 407 in water or acetic acid solution (AA), or in the presence of imiquimod (TMX) in acetic acid solution as a function of temperature.
• Figure 6 shows the viscosity of systems prepared with 25% poloxamer mixture (Poloxamer 407 and Poloxamer 188) in water or acetic acid solution, in the presence of imiquimod in acetic acid solution as a function of temperature.
• Figure 7 shows the viscosity of systems prepared with 20% Poloxamer 407 (PG) or
• Figure 8 shows the viscosity of systems prepared with 20% Poloxamer 407 in water or in lactic acid solution, or in the presence of imiquimod (TMX, 0.9) in lacrtic acid solution as a function of temperature.
• Figure 9 shows the viscosity of systems prepared with the 25% poloxamer mixture
• Figure 10 shows the erosion profile (dissolution) of PG-LA1 (Poloxamer 407 in lactic acid solution) without imiquimod (top) and with imiquimod (TMX PG-LA1 ) (bottom) over time.
• Figure 11 shows the imiquimod (TMX) diffusion profiles (g) passing the cellulose membrane as a result of gel formulations prepared with 0.1 M lactic acid and
• Poloxamer 407 or Poloxamer 407/Poloxamer 188 mixture (PPG): in the same figure, the diffusion of imiquimod in 0.1 M lactic acid solution without gel formulation is given for comparative purposes.
• Figure 12 shows the amount of imiquimod (TMX-101 ) recoverd in BE (bladder epithelium) after 4 hours of contact with the formulation (see Example 5), namely for 0.9% Imiquimod - 15% HP- ⁇ -CD dissolved in 0.1 M lactic acid solution, or in 19% Poloxamer 407 gel containing 15% HP- ⁇ -CD (0.1 M lactic acid) or in liposome dispersion (1 % soybean lecithin in 0.1 M lactic acid solution). It is shown that the inventive formulations show a considerably better effect in long term release than the liposomal formulation which is given herein for comparative purposes.
• Figure 13 shows imiquimod (TMX-101 ) solubility in presence of different lactic acid concentrations. Solubility increases as a function of the lactic acid concentration.
• Figure 14 In the top part bladders from pigs are shown which were excised, washed and transferred into NaCI solution at 37°C. Formulations according to the invention were applied to the bladder and the bladder sectioned after 10 minutes. The gel state of the formulation applied were observed. In the bottom part, the bladder after addition of an inventive formulation comprising imiquimod in 20% Polaxamer 4077 in lactic acid solution (0.1 M)), which was previously stained by Coomassie Blue, is shown and the gel is still observed. No adhesion of the gel is obserbed.
• inventive formulation comprising imiquimod in 20% Polaxamer 4077 in lactic acid solution (0.1 M)
• the drug is slightly soluble in hydrochloric acid 0.1 N, 0.2 N and 2 N.
• the system prepared with HCI 2 N is a transparent solution at 74 0 C.
• the theoretical concentration of the drug dissolved in it was 0.042 M corresponding to 1.0 %.
• the solubility of imiquimod at different pH values was determined. Two different procedures were used. The drug was dispersed in the buffer (without acetic acid as co- solvent). 72 mg imiquimod were dispersed with 10 ml citrate buffer, 155 mg imiquimod in 10 ml phosphate buffer and 192 mg ini O ml acetate buffer. After vortexing for 5 minutes, each sample was visually inspected. Imiquimod was completely dissolved in glacial acetic acid and 1 ml of this solution (2.41 % imiquimod) was diluted with 9 ml of water, 0.9 %w/w NaCI solution, phosphate buffer pH 7.0 (0.1 M), citrate buffer pH 6.0 (0.1 M) or acetate buffer pH 5.0 (0.1 M). A visual inspection of the systems was carried out.
• a weighed amount of imiquimod (0.0316 g) was dissolved in 250 ml of solvent (acetonitrile (ACN) ⁇ vater pH 3.5). The solution concentration was 0.0005 M. Different amounts of this solution were appropriately diluted with the same solvent to prepare standard solutions with concentrations in the range 2x10 6 - 3x10 s M (in particular 2x10 6 M, 3x10 6 M, 10x10 6 M, 20x10 6 M, 30x10 6 M). These solutions were analyzed by HPLC for imiquimod.
• Solution B solution containing 50 rtiM ammonium acetate (3.85 g in 1 1 of water)
• the solubility was experimentally determined weighing 1200 mg of drug, adding 10 ml of glacial acetic acid and gently shaking the dispersion for 24 hours. This sample, after filtration (0.22 ⁇ m Millipore membrane filter) was analyzed by HPLC for the imiquimod content and its pH was measured.
• Imiquimod (738 mg) was initially dissolved in glacial acetic acid (10 ml). A volume of this solution was appropriately diluted with phosphate buffer pH 7.0 (0.1 M), citrate buffer pH 6.0 (0.1 M) or acetate buffer pH 5.0 (0.1 M) to prepare systems containing 2.0 % of drug. The samples, after filtration by 0.22 ⁇ m Millipore membrane filter, were spectrophotometrically and chromatographically analyzed for the imiquimod content. The pH was determined.
• imiquimod Some inorganic and organic acids were considered as potential solubilizers of the drug.
• 100 mg imiquimod were dissolved in 10 ml of phosphoric acid 0.1 M, succinic acid 0.1 M, citric acid 0.1 M, acetic acid 0.01 M, 0.05 M, 0.1 M and lactic acid 0.01 M, 0.05 M, 0.088 M, and 0.1 M.
• These systems after filtration, were spectrophotometrically analyzed for imiquimod and their pH was measured.
• Imiquimod 200 mg was weighed and mixed with different amounts of surfactants (Tween
• the imiquimod present in solution in compositions prepared with different acids and surfactants is spectrophotometrically determined and is shown in Table 5 together with the pH value.
• Table 5 Composition, pH value and amount of imiquimod in the formulations
• Imiquimod was weighed (100 mg) and added of glycerol or propylene glycol to a volume of 10 ml. After shaking (at 120 0 C for 24 hours) and cooling, 10 ml of water were added: the dispersions were filtered and the imiquimod amount in the liquid phases was determined.
• the system prepared with glycerol as solvent showed a pH value of 5.42 and an imiquimod concentration of 0.02 %. When propylene glycol was used the pH was 3.03 and the concentration of imiquimod in solution was 0.02%.
• Imiquimod is appreciably soluble in pure acetic and lactic acid (>7.5 % w/v). These two acids can be used as co-solvent to prepare formulations containing relatively high amount of the active substance.
• solubilizer for imiquimod
• acetic and lactic acids confirm their ability to interact with the drug and solubilize it.
• the amount of drug in solution is directly related to the acid concentration (this is also confirmed by Example 6.1 .).
• solubilizer lactic acid is more efficient than acetic acid.
• the surfactants associated to lactic acid do not ameliorate the solubility of imiquimod.
• the obtained results demonstrate that for these systems, the drug solubility depends primarily from the lactic acid and indicate that the drug is not entrapped in the micelles the surfactants form.
• Poloxamer 407 was used to prepare semisolid systems that contained as liquid phase:
• thermo-sensitive agent 25% polymer (overal amount/concentration of thermo-sensitive agent)
• HPMC hydroxypropylmethylcellulose
• LCST Lower Critical Solution Temperature
• thermo-sensible semisolid systems CDG. Palmieri et al., 15 th Int.Symp. on Microencapsulation, Parma, Italy 18-21 September 2005.
• Hydroxypropyl- ⁇ -cyclodextrin (HP- ⁇ -CD) at high percentages, ranging from 0 to 20% was used in systems constituted of Poloxamer 407 and water and the LCST of these formulations was determined.
• Poloxamer 188 Mixtures of poloxamers as jellifying systems were also assessed. Poloxamer 188, PEO-PPO- PEO copolymer with MW lower than Poloxamer 407, can be used to modulate the LCST of the gel. Poloxamer 407 was in part substituted by Poloxamer 188 to obtain hydrogels that contain an amount of polymers corresponding to 25%.
• the liquid phase (water) was substituted by lactic or acetic acid: their effects and those of the drug on the rheological behavior of thermo-sensitive systems were evaluated.
• the jelled systems we selected contained:
• composition of the investigated gels was the following:
• Poloxamer 188 2.50 g
• Lactic acid solution (0.135 M) 74.02 g
• Poloxamer 188 2.50 g Acetic acid solution (0.135 M) 74.02 g
• Poloxamer 407 20.00 g Acetic acid solution (0.125 M) 79.02 g
• the LCST values cannot be determined by DSC: while systems constituted of pure Poloxamer 407 showed an endothermic peak that can be linked to the gel transition, using mixtures of poloxamers, HP- ⁇ -CD/Poloxamer 407 or HPMC/Poloxamer 407 as gelling agents, the DSC transition signal was lost. So, the gel transition temperature was determined by viscosity measurement. The viscosity of the systems was evaluated in the range of temperature 1 -40 0 C, using a Rheometer BROOKFIELD DV-II+, equipped with the Small Sample Adapter and the spindle S-25.
• HPMC in the range of concentrations considered ( ⁇ 2%), is found not to modify the gel transition temperature of the formulation containing pure Poloxamer 407. 2.2.3. Viscosity of systems containing poloxamer and cyclodextrin
• Viscosity of systems containing a mixture of Poloxamer 407 and 188 A relevant effect of Poloxamer 188 on LCST has been observed: increasing proportion of this component raises the gel transition temperature (note the linear relationship between P188 concentration and system viscosity) (see Figure 3).
• Lactic acid solution (0.125 M) 79.02 g
• Lactic acid solution (0.125 M) 79.02 g
• the test is illustrated in "Development and in-vitro evaluation of sustained release Poloxamer 407 (P407) gel formulations of ceftiofur" by L. Zhang -J. Control. Release, 85 (2002) 73-81.
• Poloxamer 188 2.50 g
• Lactic acid solution (0.135 M) 74.02 g
• Lactic acid solution (0.125 M) 79.02 g
• the receiving phase was completely withdrawn and substituted by fresh acid solution.
• the receiving solution was submitted to UV analysis for imiquimod.
• the flux of imiquimod trough the artificial barrier is high (1 .95x10 6 g-cm 2 -s "1 ) and constant (almost for the first hour) suggesting that the transfer of the drug through the membrane does not affect the imiquimod kinetics through the poloxamer gels.
• imiquimod profiles of gel formulations are very different from that of the solution, but very similar between them. Imiquimod diffuses through the gel matrix with relatively high rate: after 6 hours, about 44.4% for imiquimod PG-LA 01 formulation and about 48.3% for
• Imiquimod PPG-LA01 was in the receiving phase of the Franz cell.
• the drug diffusion profiles for both the gel formulations are linear, suggesting pseudo-zero order kinetics: the imiquimod fluxes through the gel matrix are 2.0OxI O "7 g-cm 2 -s ⁇ ' for imiquimod PG-LA 01 and 2.19x10 7 g-crnV for imiquimod PPG-LA01 system ( Figure 1 1 ).
• the polymer gradually dissolves in the liquid that passes the membrane.
• the flux of the liquid in counter-current to the drug, could alter the diffusion of the active molecule from donor to receiving compartment.
• this effect is negligible: the flux of imiquimod through the membrane is ten times higher than its flux through the poloxamer systems.
• compositions The compositions submitted to the test were:
• the bladder of 6-9 months-aged female pigs was used: immediately after the excision, the urethra was cut away and the bladder epithelium (BE) was sectioned to obtain pieces of about 3 cm 2 area. Each portion of epithelium was mounted between the compartments of a Franz cell with the internal surface faced up. As receiving phase (bottom compartment), 0.1 M phosphate buffer solution (pH 7.4) was used: it was maintained under stirring (about 300 rpm) at 37 0 C during the experiment.
• One of the reported formulations represents the donor phase (top compartment): the formulation (2 g) was introduced in the donor compartment after the temperature of the apparatus attains 37 0 C temperature.
• BE removed from the diffusion cell was thoroughly washed with distilled water to remove excess formulation and carefully wiped with tissue paper. Then BE was frozen and sectioned using a cryostatic microtome. Five successive BE sections (each of 100 m thickness) were introduced in a tube, added of 5 ml lactic acid (92 %) and maintained under shaking overnight. The liquid phase was filtered (0.22 ⁇ m) and assayed by HPLC for imiquimod.
• Figure 12 depicts the obtained results reporting the amount of imiquimod recovered in BE after 4 hours of contact with the formulation (data are normalized for the absorption area). Data are the mean of 3 experiments (solution and liposomes) or 6 experiments (gel formulation).
• the gel formulation affects the absorption of imiquimod into the bladder epithelium: the amount of active released from the gel and recovered in BE is lower than that released from the solution. It is interesting to notice that the amount of imiquimod formulated in liposomes and present in the BE is greater than that from solution: this result suggests a promotion effect of lipid vesicles on the absorption of the drug.
• Poloxamer 407 has a negative effect on the drug solubility: as the percentage of polymer increases, the concentration of imiquimod in solution decreases. • When both Poloxamer 407 and cyclodextrin are present in the formulation, the opposite effects of these components are balanced and imiquimod solubility was not substantially modified respect to that of 0.1 M lactic acid solution.
• the viscosity was determined on the following compositions:

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