Patent Application
20160114055
The present invention relates to novel chemical conjugates as well as their pharmaceutical compositions containing them as pharmaceutical agents and to their uses thereof in therapy and diagnosis and, more particularly, but not exclusively, to novel conjugates of polymers having attached thereto a targeting moiety and one or more therapeutic agents for the treatment of amyloidosis.
The present invention is referred to novel polymeric conjugates in which at least it is linked a fibril disruptor agent and/or a blocking agent of aggregates in addition of an optional targeting moiety and/or a probe for diagnosis and therapy.
Specifically, the present invention relates to a polymer-drug conjugate, where the polymeric platform transports at least one bioactive agent, selected from the group: anthracyclines antibiotics as the tetracycline, rolitetracycline, minocycline and/or doxycycline and their derivatives, etc. . . . , able to disaggregate or break the amyloid fibrils and/or block the aggregates. This conjugate could contain in its structure one or several targeting moieties, which will provide an effective targeting of the polymer-drug conjugate to the selected area for drug activity, increasing the therapeutic efficacy in the treatment of amyloidosis.
Amyloidosis is a group of diseases characterized by fibrillar protein deposition in the extracellular space named amyloid, of which Familial Amyloid Polyneuropathy (FAP) and Alzheimer's Disease (AD) are examples.
Familial Amyloidotic Polyneuropathy (FAP) is a neurodegenerative disorder characterised by systemic extracellular deposition of transthyretin (TTR) amyloid fibrils in several organs, mainly in the peripheral nervous system. This disease is characterised by an ascending sensorimotor polyneuropathy and progressive dysautonomia, becoming usually fatal 10 to 15 years after its onset. TTR, a homotetramer mainly synthesised in the liver and choroid plexus, is responsible for the transport of thyroxine and retinol. Over 100 point mutations have been identified in TTR and associated with FAP; this disease presents its largest focus in Portugal and thus, represents an important topic of research. The mechanism by which TTR deposits and becomes pathological is not fully understood but it is currently accepted that the protein undergoes a series of events that lead to its dissociation into monomers which aggregate culminating with the formation of amyloid fibrils. Although with variable results, orthotopic liver transplantation (OLT) is recognized as the only treatment modality available for FAP. Currently, there is no effective treatment for this disorder and the search for drugs capable of interfering with the amyloidogenic cascade has been quite intense.
Underlying therapeutic strategies is the need to identify and to characterise TTR species formed along the process and the identification of biomarkers to assess disease progression and follow up of treatments. Saraiva et al. previously contributed for the characterisation of the dynamics of TTR fibril formation, including the characterisation of the different TTR species generated along the process of fibril formation [1] which allowed the screening of different drugs in vitro, acting at different stages of the amyloidogenic [2, 3], and in vivo [4]. The drug selected in the present invention, doxycycline (Doxy), is the most advanced fibril disrupter agent under study, being the most effective compound at disaggregating TTR mature fibrils. Doxy was able to disrupt amyloid fibrils in vitro, in the treated animals and also achieved other improvements regarding amyloid markers [2, 4]. Combination of Doxy with Tauroursodeoxycholic Acid (TUDCA) is already in phase II of clinical trials. TUDCA is a biliary acid with antiapoptotic and antioxidant activity. In recent studies, the combination of doxycycline with TUDCA administered to mice with amyloid deposition was more effective than either parent drugs per separate. Significantly lowering of TTR deposition and associated tissue markers were observed. The observed synergistic effect of doxy/TUDCA in the range of human tolerable quantities, in the transgenic TTR mice models prompts their application in FAP, particularly in the early stages of disease. During this study, it was suggested that a possible mechanism for TTR extracellular aggregation is the influence of secreted metabolites generated by oxidative stress and apoptosis on TTR [5]. The neuroprotector effect of this class of compounds, in addition to its own anti-microbacterial properties, was described in several other disease models including cerebral ischemia, spinal cord injury, Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), and AD. Within this scope, Doxycycline in combination with rifampin were tested in clinical trials as antibiotic therapy in Alzheimer's disease [6] although there were no statistically significant decline/deterioration results in comparison with placebo [7, 8]. However, several publications evidence the beneficial properties of tetracyclines for AD treatment, such as their anti-amyloidogenic activity [2, 9-12]. It is noteworthy to remark that work carried out by Cardoso et al., where doxycycline has demonstrate prevention of amyloid beta (Aβ) toxicity in vitro and in vivo [13].
In addition, it has been described that TTR binds A-beta protein, which plays the major role in the Alzheimer's Disease (AD). It can act like a “chaperone” by preventing formation of A-beta amyloid aggregates and thereby it may halt progression of AD [14]. This disorder, also an amyloidotic disease, affects 5% of the population over the age of 65 years and 20% over 80 years of age.
However, the advances in the design of novel drugs for amyloidosis treatment still reveal the need of more effective treatments by means of enhancing drug specificity and decreasing its systemic toxicity.
The present invention provides a response to this necessity with novel polymer-drug conjugates able to enhance the activity already found with doxycycline in the treatment of diseases involving amyloidosis, e.g. FAP, showing better specificity and reduced systemic toxicity.
Polymer Therapeutics, first polymeric nanomedicines [15, 16], are the basis for the development of the invention which is based on conjugation of the aforementioned compounds (directly or through a linker to a polymeric carrier) by covalent binding in order to obtain novel polymer-drug conjugates with therapeutic potential to treat these neuropathic disorders for the first time in the field. Conjugation procedures have demonstrated greater specificity and increased or retained activity of the parent molecules, importantly, polymer multivalence allows to achieve synergistic effect with combination therapy [17-19]). Conjugation can also provide improvements on drug stability and can reduce drug toxicity yielding a better therapeutic value.
Polymer Therapeutics are well-known as effective drug delivery systems with demonstrated clinical benefits since the 90's [20, 21]. In particular polymer-drug conjugates are considered new chemical entities (NCEs) capable to improve bioactive compound properties (changing its pharmacokinetics at whole body (EPR effect) and at cellular level (endocytosis) allowing even to overcome cellular mechanisms of resistance [15, 22]) and decreasing their inherent limitations (short half life, non-specific toxicity, low solubility, poor stability, potential immunogenicity, . . . ). More than 16 polymer-drug conjugates have already achieved clinical development [23, 24], mainly as anticancer agents and currently, a second generation of conjugates focused on improved structures, combination therapy or new molecular targets are under development to move this platform technology further [20, 21, 23, 25, 26]. The conjugate paclitaxel-polyglutamate OPAXIO™ (PGA-PTX conjugate) already in Phase II trials is the most advanced [27, 28].
In the international patent application WO2007060524 it is also described certain compounds 1,4-diazepane-2,5-dione which are able to be conjugated to poly-glutamic acid through covalent binding such amide bond. They have been proposed for the treatment of PD, MS and stroke. The conjugate design described in this invention considers also the presence of a peptidic linker between the drug and the polymeric chain.
Furthermore, due to the molecular complexity of diseases (such as neurodegenerative disorders), combination therapy is becoming increasingly important for a better long-term prognosis and to decrease side effects. Therefore, one of the main targets of the present invention is the use of advanced nanopharmaceutics based on combination therapy looking at agent synergism. Whilst nanosized systems are well established for the delivery of a single therapeutic agent, only in very recent years has their use been extended to the delivery of multi-agent therapy. In fact, the only example in the clinics comes from a Canadian company, Celator Technologies Inc. who has developed a methodical approach for combination therapy within their liposomal technology [29-33]. This technology led to the development of different liposomal formulations that are now being assessed in Phase II clinical trials, namely CPX-1 and CPX-351. These early studies revealed the therapeutic potential of this application but raised new challenges that need to be addressed for a successful optimisation of the system towards clinical applications.
Following these concepts, novel specific nanoconjugates for the treatment of amyloidosis related polyneuropathies are shown in this invention, using as polymeric platform the poly-glutamic acid (PGA) and its derivates.
One of the major drawbacks that hinders peptides/proteins drugs application in therapies is their variable solubility, low bioavailability and limited stability, therefore one purpose of the present invention is to move a step further this therapy enhancing the activity already found with Doxy in FAP and AD with polymer-drug conjugates, increasing their specificity and diminishing their systemic toxicity. These vehicles present advantages not just for peptides but also for small chemical compounds, allowing the control of their release at the desired site. Moreover, the property of polymer multivalency allows the conjugation of several active compounds within the same polymeric carrier, the combination of doxy and other drug in the same polymer carrier would synergistically enhance the therapeutic value of these prodrugs. Synergy could also be effective when conjugates of each drug are administered per separate [18]. Due to the molecular complexity of FAP and AD it is expected to achieve better therapeutic output if more than one molecular pathway of disease is targeted.
The present invention, in some embodiments thereof, relates to novel chemical conjugates and to uses thereof in therapy and diagnosis and, more particularly, but not exclusively, to novel conjugates of polymers having attached thereto one or more therapeutic agents and optionally a targeting moiety and/or a labelling probe, for treatment and diagnosis of amyloidosis related diseases.
In particular, the present invention relates to a polymer-drug conjugate where the polymeric backbone bears, at least, a fibrillar disrupting agent selected from the group including: anthracyclines antibiotics like the tetracycline, rolitetracycline, minocycline and/or doxycycline or their derivates, etc, . . . able to disaggregate or break the amyloid fibrils and/or block the aggregates. This conjugate may contain also in its structure targeting moieties which allow an effective targeting of the polymer-drug conjugate to the diseased area, providing higher efficacy in the treatment and/or diagnosis of amyloidosis related diseases, including polyneuropathic disorders and neurodegenerative diseases like FAP and AD.
Polymer-drug conjugates of the present invention have shown a marked specificity retaining or enhancing the inherent activity of the selected bioactive molecules, allowing, thanks to the multivalency of the polymers, the obtaining of synergistic effects through the combination therapy, higher stability and lower side toxicity of the parent drug achieving a better therapeutic index.
Therefore, the present invention provides a polymer-drug conjugate represented in the general formula I, also represented in
Wherein:
R1 represent an alkyl group, defined C-terminal linking point (alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals, activated carboxylic groups, etc), ethylenglycol (EG) of different molecular weights including poly(ethylene glycol) (PEG from 100 to 20000 g/mol).
R2 represents a hydrogen atom, an alkyl group, defined C-terminal linking point (alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals, activated carboxylic groups, etc), ethylenglycol (EG) of different molecular weights including poly(ethylene glycol) (PEG from 100 to 20000 g/mol), PEG-thiol, PEG-4TP.
R3 represents the linking spacer or bond between the polymer main chain and the bioactive agent (R4) as itself or derivated. R3 is an alkyl group, defined C-terminal linking point (alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals, activated carboxylic groups), ethylenglycol (EG) of different molecular weights including poly(ethylene glycol) (PEG from n=2-16), aminoacids such as lysine, arginine, imidazol, histidine, cysteine and secondary and tertiary amino groups and aminoacid sequences.
R4 is the selected drug for amyloidosid treatment, selected from the group which comprises the anthracycline antibiotics such as tetracycline, rolitetracycline, minocycline, doxycycline and their derivatives. The drug can be covalent linked to the polymer chain as itself or previously derivatised (including the introduction of amine, thiol, carbonyl, vinyl, alcohol or carboxyl groups among others).
R5 represents the linking spacer or bond between the polymer main chain and the bioactive agent R6 as itself or derivatised. R5 is an alkyl group, defined C-terminal linking point (alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals, activated carboxylic groups), ethylenglycol (EG) of different molecular weights including poly(ethylene glycol) (PEG from n=2 to n=16), aminoacids such as lysine, arginine, imidazol, histidine, cysteine and secondary and tertiary amino groups and aminoacid sequences.
R6 is the second selected drug for amyloidosis treatment, selected from the group which comprises the anthracycline antibiotics such as tetracycline, rolitetracycline, minocycline, doxycycline and their derivatives (the drug can be covalent linked to the polymer chain as itself or previously derivated); or a labelling moiety exploited for conjugate monitoring, for biodistribution experiments or as a diagnostic probe where the labelling agent comprises fluorescent probes for optical imaging such as Cy5.5, coordination complexes for MRI, or tracers for PET and SPECT, including the quelating agents DTPA, DOTA, NOTA, NODA and metallic ligands such as gallium, technetium, gadolinium, indium, etc.
R2, R3 and R5 can be used for conjugation of bioactive agents (including low molecular weight drugs, peptides, proteins, antibodies), near infrared dyes, coordination complexes for MRI, PET and SPECT tracers.
In addition, the present invention there is provided the pharmaceutical compositions which comprise the conjugates herein described with a pharmaceutically acceptable carrier.
According to an aspect of embodiments of the invention there is provided a method of treating amyloidosis in a subject in need of thereof, the method comprising administering to the subject a therapeutically effective amount of the polymer-drug conjugate as described herein.
According to another aspect of embodiments of the invention there is provided a method of diagnose amyloidosis in a subject in need of thereof, the method comprising administering to the subject a therapeutically effective amount of the polymer-drug conjugate as described herein.
The present invention, in some embodiments thereof, relates to novel chemical polymer-drug conjugates and to uses thereof in therapy and, more particularly, but not exclusively, to novel conjugates of polymers having attached thereto a fibril disrupter agent and/or a aggregate-blocking agent and/or a targeting moiety and/or labelling moiety and to uses thereof in treating amyloidosis.
The present invention relates to a polymer-drug conjugate where the polymeric backbone bears, at least, a fibrillar disrupting agent able to disaggregate or break the amyloid fibrils and/or block the aggregates. This conjugate may contain also in its structure targeting moieties which allow an effective targeting of the polymer-drug conjugate to the diseased area, providing higher efficacy in the treatment and/or diagnosis of amyloidosis.
This invention provides novel polymer-drug conjugates and pharmaceutical compositions which contain them to their use like pharmaceutical agents against diseases involving amyloidosis, including amyloidosis related to polyneuropathic disorders and neurodegenerative disorders like FAP and AD.
In one embodiment of this invention it is described the use of the polymer-drug conjugates where the polymeric backbone bears, at least, a bioactive agent in the amyloidosis treatment and at least a targeting moiety and a labelling probe, with diagnostic purposes and to perform biodistribution studies of the bioactive agent in animal models with amyloidosis related diseases.
The present invention relates to polymer-drug conjugates which comprise a polymeric backbone to which is covalent linked at least a bioactive agent, with the capability of disrupting amyloid fibrils and/or blocking aggregates activity.
The phrase “amyloid disrupter/disrupting agent”, as used herein, describes any therapeutic agent that directly disaggregates or breaks the amyloid fibrils and/or their deposits and promotes a concatenate effect improving disease conditions as observed by amyloid markers studies.
Exemplary of amyloid disrupting agents include, without limitation, anthracyclines antibiotics such as tetracycline, rolitetracycline, minocycline, and/or doxycycline and their derivates, being preferable the use of doxycycline, as it is represented in the
The phrase “aggregates-blocking agent”, as used herein, describes any therapeutic agent that directly interacts with the misfolded protein in its aggregate stage being able to avoid interaction with the receptor, thus avoiding cell death cascades.
In some embodiments of the present invention, the described conjugates comprise a polymeric backbone that represents the total plurality of units of the main chain, from which, some of these units are linked to the first therapeutic agent, optionally other ratio of these units is linked to the second bioactive agent, and optionally other ratio has been labelled with a probe for the monitoring of the conjugate; and optionally some of the units of the polymeric backbone remain free and other may be linked to a targeting moiety to allow the whole system to cross the blood-brain barrier.
The bioactive agent or the bioactive agents are linked to the polymeric matrix directly or through a spacer or linker, being this linker biodegradable and selected from the group consisting of a pH-sensitive and an enzymatically-cleavable linker. In the case of enzymatic linkages, these are cleaved as a consequence of the presence of enzymes over-expressed in inflamed areas.
The union comprises a direct covalent bonding of the active agent, via amide, ester, acetal, hydrazone or disulphide bond; and it may include any amino acid sequence including those substrates of metalloprotases (i.e. MMP-9 (PVGLIG)), cathepsin B (GPLG; R; PL) and other peptide sequences able to be recognised and cleaved in presence of enzymes located in drug release area. Preferably, the secuences Gly-Gly and Leu-Gly are used in the present invention.
According to some embodiments of the invention, the polymeric backbone is derived from a polyglutamic acid (PGA). According to some embodiments of the invention, the polymeric backbone is derived from polyglutames such as block-co-polymers, triblocks, where the other blocks include without limitation polyethylenglycol (PEG), water soluble polyamino acid, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly(D,L-lactide-co-glycolide) (PLA/PLGA), poly(hydroxyalkylmethacrylamide), polyglyceron, polyamidoamine (PAMAM) and polyethyleneimine (PEI), and different structures of the polyglutamic acid (PGA) represented in the
The present patent includes all the possible polymer-drug linkages derived from PGA modification of its pendant chains (e.g. alkyne, azide, tiol, halides, activated esters, activated alcohols, protected amines, maleimide groups, acetals, ethylene glycol (EG) of several molecular weights including polyethyleneglycol (PEG from 100 to 20000 g/mol) as well as drug modification for achieving all the correspondent type of bond with the polymer chain, directly or through spacers. All the possible combinations are included in this invention.
Optionally, the polymer-drug conjugate of the present invention could contain a targeting moiety able to cross specific biological barriers, for example the blood-brain barrier (BBB). This targeting residue can be selected from the group which comprises peptide, proteins o a monoclonal antibody. Examples of targeting residues are any ligand capable to target the transferrin receptor (e.g. the transferrin protein (holo- and apo-transferrin), monoclonal antibodies (e.g. the monoclonal (mAb) OX26), the cyclic peptide CRTIGPSVC, etc.) or to target other receptor present in the BBB such as de low density lipoprotein receptor-related protein 1 (LRP-1) like the peptide Angiopep2.
According to some embodiments of the present invention it is considered the incorporation of a labelling probe such as a near infrared dye for optical imaging, coordination complexes for MRI, and tracers for PET and SPECT studies. The use of Cy5.5 as probe is preferred in the present invention.
As used herein, the term “mol %” describes the number of moles of an attached moiety per 1 mol of the polymeric conjugate, multiplied by 100. Thus, i.e. a 1 mol % load of a fibril disrupting agent describes a polymeric conjugate composed of 100 backbone units, whereby 1 backbone unit has the bioactive agent attached thereto and the other 99 units are either free or have other agents attached thereto.
The optimal degree of loading of the therapeutically active agent (or agents) and the targeting moiety for a given conjugate and a given use is determined empirically based on the desired properties of the conjugate (e.g. water solubility, therapeutic efficacy, pharmacokinetic profile, toxicity and dosage requirements), and optionally on the amount of the conjugated moiety that can be attached to a polymeric backbone in a synthetic pathway of choice.
According to some embodiments of the invention, the drug loading attached to the water soluble polymer can vary. In general, the polymer-drug conjugates are characterized by a load of the therapeutically active agent or the targeting moiety greater than 0.5 mol %.
In certain aspects of the invention, the amount of fibril disrupter drug conjugated per water-soluble polymer can vary. At the lower end, such a composition may comprise from about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8.degree./a, about 9%, or about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21% about 22%, about 23%, about 24%, to about 25% (w/w) fibril disrupter drug relative to the mass of the conjugate. At the high end, such a composition may comprise from about 26%, about 27%, about 28%, about 29%, about 30%, about 31% about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, to about 40% or more (w/w) fibril disrupter drug relative to the mass of the conjugate.
Therefore, the present invention provides a polymer-drug conjugate represented in the general formula I, also represented in
Wherein:
R1 represent an alkyl group, defined C-terminal linking point (alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals, activated carboxylic groups, etc), ethylenglycol (EG) of different molecular weights including poly(ethylene glycol) (PEG from 100 to 20000 g/mol).
R2 represents a hydrogen atom, an alkyl group, defined C-terminal linking point (alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals, activated carboxylic groups, etc), ethylenglycol (EG) of different molecular weights including poly(ethylene glycol) (PEG from 100 to 20000 g/mol), PEG-thiol, PEG-4TP.
R3 represents the linking spacer or bond between the polymer main chain and the bioactive agent (R4) as itself or derivated. R3 is an alkyl group, defined C-terminal linking point (alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals, activated carboxylic groups), ethylenglycol (EG) of different molecular weights including poly(ethylene glycol) (PEG from n=2-16), aminoacids such as lysine, arginine, imidazol, histidine, cysteine and secondary and tertiary amino groups and aminoacid sequences.
R4 is the selected drug for amyloidosid treatment, selected from the group which comprises the anthracycline antibiotics such as tetracycline, rolitetracycline, minocycline, doxycycline and their derivatives. The drug can be covalent linked to the polymer chain as itself or previously derivatised (including the introduction of amine, thiol, carbonyl, vinyl, alcohol or carboxyl groups among others).
R5 represents the linking spacer or bond between the polymer main chain and the bioactive agent R6 as itself or derivatised. R5 is an alkyl group, defined C-terminal linking point (alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals, activated carboxylic groups), ethylenglycol (EG) of different molecular weights including poly(ethylene glycol) (PEG from n=2 to n=16), amino acids such as lysine, arginine, imidazol, histidine, cysteine and secondary and tertiary amino groups and amino acid sequences.
R6 is the second selected drug for amyloidosis treatment, selected from the group which comprises the anthracycline antibiotics such as tetracycline, rolitetracycline, minocycline, doxycycline and their derivatives (the drug can be covalent linked to the polymer chain as itself or previously derivated); or a labelling moiety exploited for conjugate monitoring, for biodistribution experiments or as a diagnostic probe where the labelling agent comprises fluorescent probes for optical imaging such as Cy5.5, coordination complexes for MRI, or tracers for PET and SPECT, including the quelating agents DTPA, DOTA, NOTA, NODA and metallic ligands such as gallium, technetium, gadolinium, indium, etc.
R2, R3 and R5 can be used for conjugation of bioactive agents (including low molecular weight drugs, peptides, proteins, antibodies), near infrared dyes, coordination complexes for MRI, PET and SPECT tracers.
The compounds of the present invention may contain one or more basic nitrogen atoms and, therefore they may form salts with acids that also form part of this invention. Examples of pharmaceutically acceptable salts include, among others, addition salts with inorganic acids such as hydrochloric, hydrobromic, hydroiodic, nitric, perchloric, sulphuric and phosphoric acid, as well as addition of organic acids as acetic, methanesulfonic, trifluoromethanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, benzoic, camphorsulfonic, mandelic, oxalic, succinic, fumaric, tartaric, and maleic acid. Likewise, compounds of the present invention may contain one or more acid protons and, therefore, they may form salts with bases, which also form part of this invention. Examples of these salts include salts with metal cations, such as for example an alkaline metal ion, an alkaline-earth metal ion or an aluminium ion; or it may be coordinated with an organic with an organic or inorganic base. An acceptable organic base includes among others diethylamine and triethylamine. An acceptable inorganic base includes aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.
Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and similar ones.
There is no limitation on the type of salt that can be used, provided that these are pharmaceutically acceptable when they are used for therapeutic purposes. Salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reaction of the free acid or base forms of these compounds with a stochiometric amount of the appropriate base or acid in water or in an organic solvent, such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile or in a mixture of them.
Some of the compounds of formula I of the present invention may exist in unsolvated as well as solvated forms such as, for example, hydrates. The present invention encompasses all such above-mentioned forms which are pharmaceutically active.
Some of the compounds of general formula I may exhibit polymorphism, encompassing the present invention all the possible polymorphic forms, and mixtures thereof.
Various polymorphs may be prepared by crystallization under different conditions or by heating or melting the compound followed by gradual or fast cooling. The presence of polymorphs may be determined by solid NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, powder X-ray diffraction or such other techniques.
Another aspect of the present invention relates to a process for the preparation of polymer conjugate compounds of formula I, their derivatives, their analogues, their tautomeric forms, their stereoisomers, their polymorphs or their pharmaceutical acceptable salts and solvates.
According to some embodiments of the invention there is provided a pharmaceutical composition, comprising, as an active ingredient, the conjugate as described herein and a pharmaceutically acceptable carrier.
According to some embodiments of the invention, the composition is being packaged in a packaging material and identified in print, in or on the packaging material for use in the treatment of a medical condition associated with amyloidosis.
According to an aspect of embodiments of the invention there is provided a method of treating amyloidosis in a subject in need of thereof, the method comprising administering to the subject a therapeutically effective amount of the conjugate as described herein.
According to an aspect of embodiments of the invention there is provided a method of monitoring the conjugate within a body of a patient, the method comprising: administering to the patient the conjugate having a labelling agent as described herein attached thereto; and employing an imaging technique for monitoring a distribution of the conjugate within the body.
According to an aspect of embodiments of the invention there is provided a use of the conjugate as described herein as a medicament.
According to an aspect of embodiments of the invention there is provided a use of the conjugate as described herein in the manufacture of a medicament for treating amyloidosis.
The compounds of the present invention can be administered in the form of any pharmaceutical formulation. The pharmaceutical formulation will depend upon the nature of the active compound and its route of administration. Any route of administration may be used, for example such as oral, buccal, pulmonary, topical, parenteral (including subcutaneous, intramuscular, and intravenous), transdermal, ocular (ophthalmic), inhalation, intranasal, otic, transmucosal, implant or rectal administration. However oral, intranasal or parenteral administration are preferred. For the preparation of any of the different formulations included in the present description, it would be used the techniques and methods known in the state-of-the-art, as well as it would be selected the most common excipients utilized in pharmacy for the preparation of the different formulations.
Solid compositions for oral administration include among others tablets, granulates and hard gelatin capsules, formulated both as immediate release or modified release formulations.
Alternatively, the compounds of the present invention may be incorporated into oral liquid preparations such as emulsions, solutions, dispersions, suspensions, syrups, elixirs or in the form of soft gelatin capsules. They may contain commonly-used inert diluents, such as purified water, ethanol, sorbitol, glycerol, polyethylene glycols (macrogols) and propylene glycol. Aid compositions can also contain coadjuvants such as wetting, suspending, sweetening, flavouring agents, preservatives, buffers, chelating agents and antioxidants.
Injectable preparations for parenteral administration comprise sterile solutions, suspensions or emulsions in oily or aqueous vehicles, and may contain coadjuvants, such as suspending, stabilizing, tonicity agents or dispersing agents.
The compound can also be formulated for its intranasal application. Formulations include particles, powders, solutions wherein the compound is dispersed or dissolved in suitable excipients. In one embodiment of the invention the pharmaceutical composition is in the form of nanospheres, microparticles and nanoparticles.
The effective dosage of active ingredient may vary depending on the particular compound administered, the route of administration, the nature and severity of the disease to be treated, as well as the age, the general condition and body weight of the patient, among other factors. A representative example of a suitable dosage range is from about 0.001 to about 100 mg/Kg body weight per day, which can be administered as single or divided doses. However, the dosage administered will be generally left to the discretion of the physician.
As used therein the term “treatment” includes treatment, prevention and management of such condition. The term “pharmaceutically acceptable” as used herein refers to those compounds, compositions, and/or dosage forms which are, within the scope of medical judgement, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
According to an aspect of embodiments of the invention there is provided a process of synthesizing the conjugate described herein, the process comprising:
According to some embodiments of the invention, step (b) is performed subsequent to, concomitant with or prior to (c).
According to some embodiments of the invention, at least one of the monomer units terminating by the first or the second reactive group further comprises a linker linking the reactive group to the monomeric unit.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practices or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
All reactions requiring anhydrous conditions were performed under argon or nitrogen atmosphere.
Chemicals and solvents are either A.R. grade or purified by standard techniques.
Size Exclusion chromatography (SEC). Sephadex G25 resin/G10 resin columns, eluent H2O. SEC analysis was performed using a HPLC system from Waters (Milford, USA), composed by an Autosampler 717, a photodiode array detector unit 2996 (model Z996), 3 pumps 515 and a multi lambda fluorescence detector 2475, with two TSK-gel columns in series (G3000 and G25000 PWXL). A flow rate of 1 miL/min and a mobile phase 0.1M PBS buffer was used. Also for SEC analysis of PEGylated conjugates, Zorbax® column GF-250 (4.6×250 mm, 4 μm) from Agilent technologies (USA). For reverse phase (RP) chromatography a LIChroCART®, Cat.1.50943 LiChrospher® 100, RP-18 (125×4 mm, 5 μm) column purchased from Waters Ltd. (Hertfordshire, UK) was used. As mobile phase, different acetonitrile gradients in aqueous 0.1% TFA were used. The UV spectra were recorded on Jasco V-630 UV/Vis spectrophotometer.
Nuclear Magnetic Resonance (NMR) analysis 1H-NRM, 13C-NMR and two-dimensional diffusion-ordered NMR spectroscopy (DOSY) were performed with Bruker Advance AC-300 (300 MHz) or AV500 (500 MHz). The chemical shifts are expressed in δ relative to TMS (δ=0 ppm) and the coupling constants in J in Hz. The spectra are recorded in the deuterated solvent indicated in each case, at 300K.
Liquid Chromatography-Mass Spectrometry (LCMS). Equipment use was an Acquity Ultra Performance LC (Waters) with a PDA detector and a Micromass ZQ Waters 400 LC Single quadrupole mass spectrometer. Chromatography column used was a RP-18 Kinetex (2.6 μm×100 mm).
Transmission electron microscopy (TEM). Samples were adsorbed to glow-discharged carbon-coated collodion film supported on 200-mesh copper grids. For negative staining, the grids were washed with deionized water and stained with 1% uranyl acetate solution. The grids were visualized with a Zeiss microscope operated at 60 kV.
Dynamic Light Scattering (DLS). Particle size was measured using a Malvern Zetasizer Nano ZS instrument, equipped with a 532 nm laser at a fixed scattering angle of 90°. Particle Radious (nm) in solution was determined at 37° C. for Doxycycline conjugates and in PBS 0.020M PBS at 25° C. for RAGE peptide conjugates.
Absorbance and fluorescence detection of in vitro or in vivo processed samples was performed with the equipment Victor2 Wallac 1420 Multilabel HTS Counter Perkin Elmer (Northwolk, Conn., EEUU) using the corresponding 96-well plates. Excitation wavelength was 595 nm and emission 680 nm.
Optical in vivo imaging. For fluorescence biodistribution studies, Xenogen IVIS® Spectrum platform was exploited with the Living Image®3.2 software (Caliper Life Sciences, Hopkinton, Mass.).
Small-angle neutron scattering (SANS) experiments were performed on the D11 instrument at the Institute Laue-Langevin in Grenoble (France). Scattering data are expressed in terms of the scattering vector, Q, which is given by Q=4π/λ·sin(θ/2) where λ is wavelength and θ the angle at which the neutrons are scattered. The incident neutron wavelengths were 6±1 Å and 12 Å, giving accessible Q-ranges of 0.0017 to 0.42 Å−1 using four different sample-detector distances. Sample solutions were prepared at a conjugate concentration of 0.5-2 ωt % on a 1 g scale in D2O (pH=5.5, 0.1M phosphate buffer) and placed in 2 mm path length quartz cells, mounted in a sample changer thermostatted at 37° C. (±0.2). These conditions allowed for the study of conjugates at pH, temperature and ionic strengths mimicking those experienced during drug release in vivo. Data were corrected for transmission intensity, electronic background and normalised against a flat scatterer according to the standard procedures for the instrument. The obtained scattering profiles I(Q) vs. Q were analysed according to I(Q) ∞φVp P(Q) S(Q)+Binc where φ is the volume fraction and Vp the particle volume. The FISH modelling suite was used for the analysis [35]. FISH incorporates parameterised form factors, P(Q) and structure factors, S(Q), to describe the dimensions of the scattering particle and inter-particle interactions.
Ethics Statement:
All animal procedures were performed in compliance with local legislation guidelines and protocols approved by the Institutional Animal care and Use Committee from Centro de Investigación Principe Felipe (CIPF, Valencia, Spain) and Instituto de Biologia Molecular e Cellular (IBMC, Porto, Portugal). Body weight was measured once a week.
Derivatisation Procedure of Doxycycline (Doxy-NH2)
The general derivatisation procedure of doxycycline which results in Doxy-NH2 obtaining (Compound
Doxycycline hydrochloride (0.5000 g, 512.94 g/mol) was slowly added to concentrated H2SO4 (1.75 mL). After gas evolution had stopped, the orange solution was slowly precipitated into 100 mL of cooled diethylether in an ice bath. The hydrosulfate salt was collected by filtration, washed with ether and dried under N2 flow. The orange powder (542.12 g/mol) was redissolved in H2SO4 (5 mL), cooled to 0° C. and NaNO3 (1.56 eq, 101 mg, 84.99 g/mol) was added over 10 min while the reaction was stirring. After 3 h at 0° C., reaction was directly precipitated into 200 mL of cool diethylether in an ice bath and the mixture was filtered under vacuum. The precipitate was washed with ether and air-dried to give an orange powder which was used without further purification. 1H-NMR (MeOD) analysis was carried out (
Yield=70% (derivate in C9 80%). 1H NMR (300 MHz, MeOD) derivatised in C9 δ 7.57 ppm (d, J=8.3 Hz, 1H), 7.06 ppm (d, J=8.4 Hz, 1H), derivate in C7 δ 7.15 ppm (s, 1H). MS analysis. [M+1]=460.1180
Synthesis of PGA-X-Doxy Conjugates
Doxycyline conjugation to PGA has been accomplished through different type of linker chemistry, directly to the polymer main chain as well as through cleavable spacers. Synthesis explained below show examples such as ester and amide bonds.
Molar percentage of conjugated drug was varied in all the synthesis described below, in order to obtain a family of conjugates and exploit the different properties (activity, conformation in solution, stability) achieved in each final conjugate. Results vary from 1 to 60 w %.
In certain aspects of the invention, the amount of fibril disrupter drug conjugated per water-soluble polymer can vary. At the lower end, such a composition may comprise from about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8.degree./a, about 9%, or about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21% about 22%, about 23%, about 24%, to about 25% (w/w) fibril disrupter drug relative to the mass of the conjugate. At the high end, such a composition may comprise from about 26%, about 27%, about 28%, about 29%, about 30%, about 31% about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, to about 40% or more (w/w) fibril disrupter drug relative to the mass of the conjugate.
Synthesis of PGA-COO-Doxy
The general synthesis of PGA-COO-Doxy conjugate is depicted in
Briefly, carboxyl groups of PGA were previously activated with N-hydroxysuccinimide. PGA-NHS (average MW unit depending on NHS activation, i.e. average MW unit of glutamic acid in case of 52% activation=179.6 g/mol) was added to a round bottom flask and dissolved in anhydrous DMF under N2 atmosphere. Separately, doxy (512.94 g/mol) was dissolved in the same conditions. Once dissolved, doxy was dropped in the solution of PGA-NHS. Next, a catalytic amount of DMAP was added and pH was adjusted to 8 with DIEA. After 12-16 h under agitation at room temperature, DMF was evaporated under high vacuum until product was dried. Non-reacted drug as well as reaction organic soluble subproducts were removed by washing the crude with a mixture of CHCl3:acetone (4:1) and placed in ice for 30 min. Thereafter, solvent was removed by centrifugation and the solid residue was dried under vacuum.
Synthesis of PGA-CONH-Doxy
The general synthesis of PGA-CONH-Doxy conjugate is depicted in
Method A (
Previous activated PGA (average MW unit depending on NHS activation, i.e. average MW unit of glutamic acid in case of 52% activation=179.6 g/mol) was added to a round bottom flask and dissolved in anhydrous DMF under N2 atmosphere. Separately, doxy-NH2 (557.13 g/mol) was dissolved in the same conditions. Once dissolved, doxy-NH2 was dropped in the solution of PGA-NHS. Next, a catalytic amount of DMAP was added and pH was adjusted to 8 with DIEA. After 5 h under agitation at room temperature, DMF was evaporated under high vacuum until product was dried. Non-reacted drug as well as reaction organic soluble subproducts were removed by washing the crude with a mixture of CHCl3:acetone (4:1) and placed in ice for 30 min. Thereafter, solvent was removed by centrifugation and the solid residue was dried under vacuum.
Method B (
PGA (average MW unit of glutamic acid=129.1/mol) was added to a round bottom flask and dissolved in anhydrous DMF under N2 atmosphere. Depending on the molar percentage of drug loading, reagents amount was varied. Next protocol is detailed for 15% mol DIC (0.035 mmol, 1.5 eq per carboxyl group, 0.836 g/cm3, 126.20 g/mol) was added to the reaction and after 5 min, HOBt (0.035 mmol, 1.5 eq per carboxyl group, 135.10 g/mol) was incorporated as a solid. 10 min later, doxy-NH2 (0.035 mmol, 557.13 g/mol) dissolved in anhydrous DMF was dropped to the reaction. pH was adjusted to 8 with DIEA. After 12-16 h under agitation at room temperature, DMF was evaporated under high vacuum until product was dried. Non-reacted drug as well as reaction organic soluble subproducts were removed by washing the crude with a mixture of CHCl3:acetone (4:1) and placed in ice for 30 min. Thereafter, solvent was removed by centrifugation and the solid residue was dried under vacuum.
Method C (
Previously, sodium salt form of PGA was placed in a round bottom flask and dissolved in ddH2O. Separately, and as example for a 30 mol % carboxyl group activation doxy-NH2 (0.6 eq, 557.53 g/mol) and DMTMM.CI (0.3 eq, 276.77 g/mol)) were dissolved in also ddH2O and then added to the flask. pH=8. Reaction was left under agitation 24 h at room temperature and protected from light. Then, product was dried by lyophilisation and purified.
Synthesis of PGA-CONH-AA-Doxy
The general synthesis of PGA-CONH-AA-Doxy conjugates (
Synthesis of Leu-Gly-NH-Doxy (
Z-Leu-Gly-OH (0.064 mmol, 322.36 g/mol) was added to a round bottom flask and dissolved in 0.5 mL anhydrous DMF under nitrogen atmosphere and continuous agitation. Afterwards, DMTMM.BF4 (0.07 mmol, 1.1 eq, 328.07 g/mol) was added dissolved in 0.5 mL of anhydrous DMF. After 10 minutes, doxy-NH2 (0.014 mmol, 2.2 eq, 557.13 g/mol) was added dissolved in 2 mL of anhydrous DMF. pH was checked to be 8. The reaction was allowed to proceed for 14 h stirring at room temperature and protected from light. After this, solvent was removed under vacuum and residue was washed several times with 2 mL of ddH2O through centrifugation (4° C., 4000 rpm, 10 min). Supernatant was collected and lyophilised. Solid product obtained (28.3 mg) was analysed by NMR-1H and afterwards it was dissolved in 5 mL of MeOH for proceeding to remove the protecting group of the amino acid sequence. Solution was placed in a round bottom flask fitted with a septum and a stirring bar. Then a catalytic amount of Pd(OH)2 Charcoal was added and the flask was purged with N2 to remove the air and later flask was purged with H2. Reaction was left stirring for 12 h under H2 pressure. Afterwards, reaction was filtered though a celite column and precipitated over cold diethylether. Ether was removed by centrifugation and the gel obtained was dried over vacuum.
Yield: 70%. 1H-NMR Doxy-NH-Gly-Leu-Z (300 MHz, MeOD) δ 7.64 (m, 1H, C8 Doxy-NH—), 7.40-7.08 (m, 5H+1H C7 Doxy-NH—), 5.19-4.93 (m, 2H), 4.14 (dd, J=9.5, 5.3 Hz, 3H), 4.02-3.63 (m, 3H), 1.80-1.41 (m, 3H), 0.88 (t, J=6.5 Hz, 6H), etc.
MS analysis: Doxy-NH-Gly-Leu (deprotected): [M+1]=673.26 g/mol.
Synthesis of Gly-Gly-Doxy (
Z-Gly-Gly-OH (0.079 mmol, 266.25/mol) was added to a round bottom flask and dissolved in 0.5 mL anhydrous DMF under nitrogen atmosphere and continuous agitation. Afterwards, DMTMM.BF4 (0.087 mmol, 1.1 eq, 328.07 g/mol was added dissolved in 0.5 mL of anhydrous DMF. After 10 minutes, doxy-NH2 (0.18 mmol, 2.2 eq, 557.13 g/mol) was added dissolved in 2 mL of anhydrous DMF. pH was checked to be 8. The reaction was allowed to proceed for 14 h stirring at room temperature and protected from light. After this, solvent was removed under vacuum and residue was washed several times with 2 mL of ddH2O through centrifugation (4° C., 4000 rpm, 10 min). Supernatant was collected and lyophilised. Solid product obtained (27.1 mg) was analysed by NMR-1H and afterwards it was dissolved in 5 mL of MeOH for proceeding to remove the protecting group of the amino acid sequence. Solution was placed in a round bottom flask fitted with a septum and a stirring bar. Then a catalytic amount of Pd(OH)2 Charcoal was added and the flask was purged with N2 to remove the air and later flask was purged with H2. Reaction was left stirring for 12 h under H2 pressure. Afterwards, reaction was filtered though a celite column and precipitated over cold diethylether. Ether was removed by centrifugation and the gel obtained was dried over vacuum.
Yield: 70%. 1H-NMR: Doxy-NH-Gly-Gly-Z (300 MHz, MeOD) δ 7.64 (m, 1H, C8 Doxy-NH—), 7.26 (5H+1H C7 Doxy-NH—), 4.96 (s, 2H), 3.69 (d, 2H), 3.57 (2H), etc.
MS analysis: Doxy-NH-Gly-Gly (deprotected): [M+1]=617.20 g/mol.
Synthesis of PGA-CONH-AA-DOXY (
PGA (52 mg, 0.4 mmol unit of glutamic acid) was placed in a round bottom flask and dissolved in anhydrous DMF (5 mL) under agitation and N2 atmosphere. Carboxyl pendant groups were activated with DMTMM.BF4 (i.e. for 30% activation, 19 mg, 0.06 mmol), which were added to main reaction previously dissolved in anhydrous DMF. After 10 min, AA-doxy-NH2 was added already dissolved in anhydrous DMF. pH was assessed to be 8. Reaction was left under agitation for 24 h protected from light at room temperature. Afterwards, solvent was removed by evaporation and residue was washed with MeOH at 4° C. After remove the supernatant by centrifugation, solid product was dried.
Yield: 50%.
Purification of PGA-X-Doxy Conjugates
PGA-X-Doxy encompasses all doxycycline conjugates, independently of the type of linkage/spacer between the drug and the polymeric carrier.
After obtaining the polymer conjugate salt form (NaHCO3 1M), crude was purified by SEC chromatography (G25/G10; ddH2O as eluent). Fractions were analysed by HPLC/GPC techniques (wavelength=273 nm). Retention time=12 min. Method: isocratic gradient with PBS 0.1M pH=7.4 as mobile phase, flow rate=1 mL/min, 50 min. Fractions containing the conjugate were mixed and after lyophilisation, compound was characterised. Example of product chromatogram is shown in
In this example, it is determined the drug loading, the polymer-drug conjugates stability in different media (plasma, hydrolytical conditions, etc) and their conformation in solution. PGA-X-Doxy encompasses all doxycycline conjugates, independently of the type of linkage/spacer between the drug and the polymeric carrier.
Determination of Total Drug Loading by UV Spectroscopy
For quantifying doxy content, drug weight percentage was determined. Previously, a calibration curve of the parent drug was done. A stock solution of PGA-X-Doxy conjugate in H2O was prepared (1 m/mL). To obtain appropriate and reproducible absorbance measurements, samples were diluted using H2O. Total drug loading of the conjugates was determined by measuring the optical density at 273 nm in H2O. PGA in the same concentration range as conjugate analysed (0-5 mg/mL) in H2O was used as blank.
Determination of Free Drug Content by LCMS
PGA-X-Doxy conjugates were dissolved in phosphate saline buffer (pH=7.4). Aliquots (100 uL, 6 mg/mL PGA-X-conj) were injected directly in the LC-MS without further extraction (approx. 20 uL, Kinetex column (2.6 um, C18, 100 A 100×4.6 mm, mobile phases ddH2O/ACN both 0.1% formic acid). Doxy and doxy-NH2 were able to be detected (Mw=444 g/mol, M+445 g/mol; Mw=459 g/mol, M+460 g/mol, respectively) at retention time around 10 minutes (
As a routine, no peaks presence determined a free drug concentration under the limit of detection (0.01 mg/mL) and rarely concentrations were approximately 0.098 wt %.
Drug Release Studies Under Hydrolytical Conditions
The ability of the conjugates of the present invention to release Doxy in the presence of different pHs was tested. pH 5.5 and 7.4 were evaluated. The results are presented in
Conjugates with amide bonding/spacer demonstrated stability under both pH conditions and time tested, while conjugates bearing ester bonds showed drug release in a time- and pH-dependent manner.
Stability of PGA-X-Doxy Conjugates in Plasma
Stability in plasma was assessed by incubating each of the conjugates (6 mg/mL), for 24 h at 37° C. in plasma freshly extracted from rodents. Free drug was used as a positive control and serum without compound as negative one. At several time points (0 min, 1 h, 4 h, 8 h and 24 h), aliquots of 100 uL were collected. To each sample, 100 uL of ACN was added to precipitate the serum proteins. After centrifugation (12000 rpm, 5 min, 4° C.), supernatants were collected and subjected to analysis by HPLC as described above. Pellets were washed with MeOH (100 uL) to extract the free drug out of the pellets. To redissolve the free drug in MeOH, the solid was vortexed and then sonicated. Subsequent to centrifugation (12000 rpm, 5 min, 4° C.), supernatant 2 was collected and analyzed by HPLC as reported below. The amount of Doxy release form the conjugates in the presence of serum was determined to be insignificant.
Stability of the PGA-X-Doxy Conjugates in In Vitro Conditions
Fibril disruption studies were carried out by incubation of the conjugates with TTR fibrils in PBS media at 37° C. To test if the drug was released from the polymer platform during the in these conditions, a PBS stability-test with the same parameters was set up. Conjugates (6 mg/mL) in PBS 0.1M were incubated at 37° C. at pH=7.4 for 16 days. Sterile conditions were used. 100 μL of the sample solutions were taken at different time points up to 16 days. MW loss was evaluated with GPC columns in a HPLC system using methodology and parameters described before. Furthermore, free drug extraction was performed and evaluated with LCMS system.
Stability of PGA-X-Doxy Conjugates in Plasma
Stability in plasma was assessed by incubating each of the conjugates (3-6 mg/mL), for 24 h at 37° C. in plasma freshly extracted from rodents. Free drug was used as a positive control and serum without compound as negative one. At several time points (0 min, 1 h, 4 h, 8 h and 24 h), aliquots of 100 uL were collected. To each sample, 100 uL of ACN was added to precipitate the serum proteins. After centrifugation (12000 rpm, 5 min, 4° C.), supernatants were collected and subjected to analysis by HPLC as described above. Pellets were washed with MeOH (100 uL) to extract the free drug out of the pellets. To redissolve the free drug in MeOH, the solid was vortex and then sonicated. Subsequent to centrifugation (12000 rpm, 5 min, 4° C.), supernatant 2 was collected and analysed by HPLC as reported below. The amount of Doxy release form the conjugates in the presence of serum was determined to be insignificant.
Conformation in Solution of PGA-CONH-Doxy Conjugates Through Small Angle Neutron Scattering (SANS) Studies.
Conformation in solution is a useful tool for explaining conjugates activity either in vitro or in vivo. Drug availability in the adopted conformation might be directly related with the observed activity. With the aim of elucidate in vitro activity differences observed for PGA-doxy conjugates, two compounds with different drug loading, 17 and 43%, were studied by SANS. The samples were evaluated at 0.5-2 wt % in a 1 g scale in D20 (pH=5.5, 0.1M PBS) and were place in quartz cells with an optical path of 2 mm, maintaining a the temperature of 37° C. (±0.2). The obtained results (
Preparation of Amyloid Fibrils
TTR Leu55Pro was dialysed against water at pH=7.4 during 24 h at 4° C. The solution was centrifuged and the pellet was washed, resuspended in sterile PBS and quantified by Lowry method. Sample concentration was adjusted to 1 mg/mL. Protein was later incubated at 37° C. for 10-13 days until fibril formation was ratified by Transmission Electron Microscopy (TEM).
Screening for TTR Fibril Disrupters
Previous findings revealed that Doxy acts as a TTR fibril disrupter in vitro and in vivo [4]. All PGA-X-Doxy conjugates synthesised were tested for their ability as disrupters compared with the parent drug (doxy). These includes (1) Doxy-PGA conjugates through ester bond, (2) Doxy-NH2 derivative PGA conjugates through amide bound and (3) through peptidic linkers, with different drug loadings.
Stock solutions of all compounds were prepared and filtered-sterile prior to use (3.6 mM in sterile H2O). As controls, PGA, Doxy and Doxy-NH2 were tested. Aliquots of TTR L55P fibrils were prepared and the conjugates and controls were added in a concentration of 6.7-fold molar excess of Doxy (180 μM). All aliquots were analysed by TEM and Dynamic Light Scattering (DLS) after 3 and 6 days to monitor fibril disruption.
Transmission Electron Microscopy (TEM)
For TEM analysis, sample aliquots were taken under sterile environment, vortexed and adsorbed to glow-discharged carbon-coated collodion film supported on 200-mesh copper grids. For negative staining, the grids were washed with deionized water and stained with 1% uranyl acetate solution. Example of the resulted images are shown in
Visualisation of samples by TEM revealed that, at a concentration of [6.7×10−5/100 μg fibrils] and in presence of the conjugates, fibrils were disrupted after 3 or 6 days due to the appearance of fibrils of smaller extension or a complete disaggregation (
Dynamic Light Scattering (DLS)
The same samples used in TEM analysis were also analysed by DLS as a complementary technique, in order to observe variations respect to fibrils control after treatment with PGA-X-Doxy conjugates. Results are shown in
As a proof of suitability of the nanoconjugates for i.v. injection in in vivo studies, the haemolytic activity of PGA-Doxy conjugates was tested.
Freshly prepared PGA-X-Doxy, dextran (Mw=74000 g/mol) and poly(ethyleneimine) (PEI; Mn˜60000, Mw˜750000, 50 wt % in H2O) solutions (range of concentrations 0-2 mg/mL), in PBS at pH 7.4 and 5.5 were plated (100 uL) into sterile 96-well microtitre plates. Blood was taken from adult mice by cardiac puncture immediately after death (by 4% CO2 asphyxiation) and placed in a heparinised tube on ice. Erythrocytes (RBC) were isolated by centrifugation at 3000 rpm for 10 min at 4° C. The plasma supernatant was discarded and the RBCs were washed three times using phosphate buffer saline solution. After the third wash, the RBC pellet was equally divided into two and resuspended in PBS solutions with the appropriate pH values (i.e. 7.4, 6.5 and 5.5). From each RBCs solution, it was added (100 uL) to the previously prepared microtitre plates containing the test compounds. As 100% reference of lysis, Tritron X-1% w/v was used as well as PBS as reference. Plates were incubated for 16 h at 37° C. Then, plates were centrifuged at 3000 rpm for 10 min at RT. The supernatant were then placed in a new 96-well microtitre plate and haemoglobin (Hb) release was measured spectrophotometrically (OD570). Hb release for each sample was expressed as percentage of the release produced by Triton X. PEI and dextran represented the positive and the negative control, respectively. All haemolysis experiments were carried out per triplicate.
Example of the conjugate PGA-CONH-Doxy 17 wt % is shown in
Synthesis of Fluorescently Labelled Conjugates
By means of conjugation of a near infrared dye (Cyane 5.5), conjugates were labelled for posterior biodistribution studies using optical imaging, monitoring in vivo as well as ex vivo fluorescence.
PGA-CONH-Doxy Conjugate Fluorescence Labelling
The general derivation procedure of PGA-DOXY fluorescence labelling (
Briefly, 15 mg of PGA-CONH-Doxy conjugate (19 ωt % (6.8 mol), average MW unit of glutamic acid=180.4/mol) was added to a round bottom flask and dissolved in 3 mL of borate buffer pH=9. Then, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC, 155.24 g/mol, 0.0032 mmol) were added and 10 min later, N-hydroxysulfosuccinimide (sulfo-NHS, 0.00013 mmol, 217.13 g/mol) was incorporated. Finally, Cy5.5 (6-SIDCC, 0.011 mmol, 1362 g/mol) diluted in 1 mL of buffer solution was poured into the reaction. Reaction mixture was left under agitation at RT for 24 h. After freeze-drying the mixture, residue was purified with a PD-10 column packed with Sephadex™ G-25 (fractions of 500 μL) and fluorescence was measured (595/680 nm) and fractions with the conjugate were joined. To ensure the absence of free Cy in the conjugate, final product was washed with ddH2O with Vivaspin® MWCO 3000 g/mol.
Yield: 60%. Labelling efficiency: 0.94 mol % Cy5.5.
Polymer-Drug Conjugates Biodistribution Studies in Mice by Optical Imaging Technique
Healthy Balb/c mice (9-12 month old) were used as mouse model for the in vivo biodistribution studies. For in vivo monitoring experiments, animals were first anesthetised and shaved to remove hair interference in fluorescence signal. The in vivo whole-body biodistribution as well as ex vivo monitoring of the labelled conjugate was performed through noninvasively fluorescence imaging (FLI) using the IVIS® Spectrum Imaging System. Animal care has handled in accordance to guidelines for the Care and Use of Laboratory Animals of the Centro de Investigación Principe Felipe, and the experimental procedures were approved by the Animal Experimentation Ethical Committee of the institution.
PGA-CONH-Doxy-Cy5.5 Conjugate Biodistribution
Biodistribution of cyane5.5 labelled PGA-CONH-Doxy conjugate (17 wt %) (
Preliminary Activity Studies in FAP Mice Model.
All animals were kept and used strictly in accordance with national rules and European Communities Council Directive (86/609/EEC), and all studies performed were approved by the Portuguese General Veterinarian Board (authorization number 024976 from DGV-Portugal). Transgenic mice for human V30M TTR in a TTR null background were kindly provided by Professor Suichiro Maeda from Yamanashi University. In previous studies, these animals were previously analysed and ˜60% of the animals over 1 year of age were found to have TTR deposition as amyloid, i.e., Congo red (CR)-positive material [36] having non-fibrillar TTR deposits at younger ages.
A group of animals (9-11 month of age, n=8) were treated intravenously with the conjugate PGA-CONH-Doxy detailed in Table 1. Animals were given 2 doses per week during 6 weeks. Non-treated mice were used as controls.
Animals were killed after anesthesia with ketamine/xylazine. Organs (including liver, kidney, esophagus, stomach, small and large intestines, heart, spleen, pancreas) were immediately excised and processed. It is important to note that in this model the gastrointestinal track present the majority of fibril accumulation. Tissues were fixed in 4% neutral buffered formalin and embedded in paraffin or immediately frozen, for light microscopy or for total protein extraction, respectively.
Immunohistochemistry (IHC) Studies of the Organs from the Treated Mice
TTR-non fibrilar deposition in the extracted tissues was evaluated by immunohistochemistry as well as possible toxicity of these deposits (apoptosis).
5 μm-thick sections were deparafinated in xylol and dehydrated in a descendent alcohol series. Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide/100% methanol and sections were blocked in 4% bovine serum and 1% bovine serum albumin in phosphate buffered solution (PBS). Primary antibodies used were rabbit polyclonal anti-TTR (Dako, 1:1000), rabbit polyclonal anti-Fas (St Cruz, 1:200), and the rabbit polyclonal anti-Bip (St Cruz, 1:50), which were diluted in blocking solution and incubated overnight at 4° C. Antigen visualization was performed with the biotin-extravidin-peroxidase kit (Sigma Aldrich), using 3-amino-9-ethyl carbazole (Sigma) or diaminobenzidine as substrates. In
In the FAP animal model used, the gastrointestinal (GI) tract is the most affected organ regarding fibrillar deposition. Immunohistochemistry results (
Briefly, Fas plays a key role in apoptotic cell death regulation (induced in this experiment by the non-fibrillar TTR). Fas is a tumour-necrosis receptor that binds the Fas ligand resulting in death-inducing signalling complex ending in the apoptosis cascade. BiP is a molecular chaperon synthesised in higher levels when protein folding is disturbed. Then, the process is directly related to cell stress. When drug stops disease advance, levels of Fas and Bip shoud decrease.
Immunostainning experiments of Fas and BiP did not produce any profitable result. Treated animals had the same pattern than control animals with disease.
Histological Analysis of the Organs from the Treated Mice
For evaluating conjugates toxicity, histological analysis of the tissue sections was performed. This technique enables a better assessment of severe toxicity. Paraffin-embedded sections were stained with hematoxylin/eosin solution (H&E). H&E-stained slice sections were thoroughly examined under low (25× and 50×) and medium (100× and 200×) magnification. Organs analysed were liver, small and large intestine and liver.
Following examination, staining slices from all groups and organs showed a normal morphology with non toxic evidences due to the treatment. Exception was found for liver of the control group with disease, where focal necrosis, moderate inflammatory infiltrate and vascular congestion was observed.
Summarising, the findings indicate that treated animals did not show toxicity attributed to the administered conjugate. Furthermore, all animals treated showed normal patterns of liver regarding tissue morphology than the disease control animals. Although TTR is not affected by TTR deposits in this organ, liver is the main producer of the protein. Conjugate administered did not provoke toxicity in the analysed organs. The illustrated groups in
Group A: group treated with the conjugate PGA-Doxy.
Group B: control group (animals with disease).
Group C: control group (animals without disease, healthy mice).
| Number | Date | Country | Kind |
|---|---|---|---|
| 13382184.3 | May 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/058856 | 4/30/2014 | WO | 00 |
