The presence of trans and gauche isomers, which differ in the rotation angle of the ester group is conceivable Figure 1 b. Another possibility is the formation of different protomers Figure 1 a , with the proton residing on either the amine nitrogen N-protonated or on the carbonyl oxygen O-protonated. In aqueous solutions, the N-protonated species is certainly favored because of the basicity of the amino group, however, the situation can be very different in the absence of solvent molecules.
Figure 3 Figure 3. IR photofragmentation of drift-time-separated species of benzocaine ions. Each of these signals is preceded by a small rectangular signal, which stems from electrical noise.
Data from two individual scans per species are depicted as gray dots. The solid lines represent an average of these two individual scans.
The identity of the fragments was confirmed by collision-induced dissociation experiments where identical fragmentation patterns were observed. The theoretical spectra were scaled by a factor of 0. Intramolecular vibrations strongly depend on the molecular structure, which makes vibrational spectroscopy, especially at frequencies in the mid-IR range, a valuable tool to deduce structural information.
An action spectrum is recorded by monitoring the intensity of the precursor ion signal and the corresponding fragment signals as a function of the laser wavelength. In addition, identical fragmentation behavior is observed when dissociation is induced via slow collisional heating.
Figure 4 Figure 4. Albuferon is an example of a protein drug that achieves an extended half-life by permanent covalent modification with another protein that in itself has a long half-life. The corresponding fusion protein of albumin and interferon alpha, Albuferon, exhibits a significantly extended half-life as compared to interferon alpha. Many small molecule medicinal agents, like alkaloids and anti-tumor agents, show low solubility in aqueous fluids. One way to solubilize these small molecule compounds is to conjugate the small molecule compounds to hydrophilic water-soluble polymers.
A variety of water-soluble polymers, such as human serum albumin, dextran, lectins, poly ethylene glycol PEG , poly styrene-co-maleic anhydride , poly N- hydroxypropylmethacrylamide , poly divinyl ether-co-maleic anhydride , hyaluronic acid have been described for this purpose R. Duncan, Nature Rev. Drug Disc, , 2, Covalent modification of biological molecules with poly ethylene glycol has been extensively studied since the late s.
So-called PEGylated proteins have shown improved therapeutic efficacy by increasing solubility, reducing immunogenicity, and increasing circulation half-live in vivo due to reduced renal clearance and proteolysis by enzymes see, for example, Caliceti P. Drug Deliv. However, many biological molecules such as IFN alfa 2, saquinavir or somatostatin are inactive or show decreased biological activity when a carrier is covalently conjugated to the drug T.
Peleg-Shulman et al, J. In order to avoid shortcomings imposed by either the non-covalent polymer mixtures or the permanent covalent attachment, it may be preferable to employ a prodrug approach for chemical conjugation of the drug to the polymer carrier. In such polymeric prodrugs, the biologically active moieties drugs, therapeutic, biological molecule, etc. Prodrugs are therapeutic agents that are almost inactive per se but are predictably transformed into active molecular entities see B.
Testa, J. The carrier prodrug approach may be applied in such a fashion that the drug is released in vivo from the polymer in order to regain its biological activity. The reduced biological activity of the prodrug as compared to the released drug is of advantage if a slow or controlled release of the drug is desired. In this case, a relatively large amount of prodrug may be administered without concomitant side effects and the risk of overdosing.
Release of the drug occurs over time, thereby reducing the necessity of repeated and frequent administration of the drug. Prodrug activation may occur by enzymatic or non-enzymatic cleavage of the temporary bond between the carrier and the drug molecule, or a sequential combination of both, i. In an enzyme-free in-vitro environment such as an aqueous buffer solution, a temporary bond such as an ester or amide may undergo hydrolysis, but the corresponding rate of hydrolysis may be much too slow and thus outside the therapeutically useful range.
In an in vivo environment, esterases or amidases are typically present and the esterases and amidases may cause significant catalytic acceleration of the kinetics of hydrolysis from twofold up to several orders of magnitude see, for example, R.
Greenwald et al. Prodrugs fall in two classes, bioprecursors and carrier- linked prodrugs. Bioprecursors do not contain a carrier group and are activated by the metabolic creation of a functional group. In carrier-linked prodrugs the active substance is linked to a carrier moiety by a temporary linkage. The carrier may be biologically inert for instance PEG or may have targeting properties for instance antibodies.
This invention is concerned with polymeric carrier-linked or macromolecular prodrugs, where the carrier itself is a macromolecule such as a carrier protein or polysaccharide or poly ethylene glycol.
Cleavage of a carrier prodrug generates a molecular entity drug of increased bioactivity and at least one side product, the carrier. After cleavage, the bioactive entity will reveal at least one previously conjugated and thereby protected functional group, and the presence of this group typically contributes to the drug's bioactivity. In order to implement a prodrug strategy, at least one selected functional group in the drug molecule is employed for attachment of the carrier polymer.
Preferred functional groups are hydroxyl or amino groups. Consequently, both the attachment chemistry and hydrolysis conditions depend on the type of functional group employed. Numerous macro molecular prodrugs are described in the literature where the temporary linkage is a labile ester bond.
In theses cases, the functional group provided by the bioactive entity is either a hydroxyl group or a carboxylic acid e. Greenwald, A. Pendri, CD. Conover, H. Zhao, Y. Choe, A. Martinez, K. Shum, S. Guan, J. Especially for therapeutic biomacromolecules but also for certain small molecule drugs, it may be desirable to link the carrier to amino groups of the bioactive entity i. N-terminus or lysine amino groups of proteins.
This will be the case if masking the drug's bioactivity requires conjugation of a certain amino group of the bioactive entity, for instance an amino group located in an active center or a region or epitope involved in receptor binding. Also, during preparation of the prodrug, the amino groups may be more chemo selectively addressed and serve as a better handle for conjugating the carrier and the drug because of their greater nucleophilicity as compared to hydroxylic or phenolic groups.
This is particularly true for proteins and peptides which may contain a great variety of different reactive functionalities, where non-selective conjugation reactions lead to undesired product mixtures which require extensive characterization or purification and may decrease reaction yield and therapeutic efficiency of the product.
Amide bonds are usually much more stable against hydrolysis than ester bonds, and the rate of clevage of the amide bond would be too slow for therapeutic utility in a carrier- linked prodrug. Therefore it is advantageous to add structural chemical components such as neighbouring groups in order to exert control over the cleavability of the prodrug amide bond. Such additional cleavage-controlling chemical structures that are provided neither by the carrier entity nor by the drug are termed "linkers".
Prodrug linkers can have a strong effect on the rate of hydrolysis of a given temporary bond. Variation of the chemical nature of these linkers allows the engineering of the properties of the linker to a great extent. Several examples have been published of the prodrug activation of amine-containing biologically active moieties by specific enzymes for targeted release. A prerequisite for enzymatic dependence is that the structure of the linker displays a structural motif that is recognized as a substrate by a corresponding endogenous enzyme.
In these cases, the cleavage of the temporary bond occurs in a one-step process which is catalyzed by the enzyme. Cavallaro et al. Bioconjugate Chem. Enzymatic release of cytarabin is effected by the protease plasmin which concentration is relatively high in various kinds of tumor mass. Enzyme-catalyzed acceleration of prodrug cleavage is a desirable feature for organ or cellular targeting applications. Targeted release of the bioactive entity is effected, only if an enzyme, that selectively cleaves the linkage, is specifically present in the organ or cell- type chosen for treatment.
A major drawback of predominantly enzymatic cleavage is interpatient variability. Enzyme levels may differ significantly between individuals resulting in biological variation of prodrug activation by the enzymatic cleavage. The enyzme levels may also vary depending on the site of administration.
For instance it is known that in the case of subcutaneous injection, certain areas of the body yield more predictable therapeutic effects than others. To reduce this unpredictable effect, non-enzymatic cleavage or intramolecular catalysis is of particular interest see, for example, B.
Furthermore, it is difficult to establish an in vivo-in vitro correlation of the pharmacokinetic properties for enzyme-dependent carrier-linked prodrugs. In the absence of a reliable in vivo-in vitro correlation optimization of a release profile becomes a cumbersome task. Other carrier prodrugs employing temporary linkages to amino groups present in the drug molecule are based on a cascade mechanism.
Cascade cleavage is enabled by linker compounds that are composed of a structural combination of a masking group and an activating group. The masking group is attached to the activating group by means of a first temporary linkage such as an ester or a carbamate. The activating group is attached to an amino-group of the drug molecule through a second temporary linkage, for instance a carbamate. The stability or susceptibility to hydrolysis of the second temporary linkage e.
In the presence of the masking group, the second temporary linkage is highly stable and unlikely to release the drug with therapeutically useful kinetics. In the absence of the masking group, this linkage becomes highly labile, causing rapid cleavage and drug release.
The cleavage of the first temporary linkage is the rate-limiting step in the cascade mechanism. This first step may induce a molecular rearrangement of the activating group such as a 1,6-elimination. The rearrangement renders the second temporary linkage so much more labile that its cleavage is induced. Ideally, the cleavage rate of the first temporary linkage is identical to the desired release rate for the drug molecule in a given therapeutic scenario.
Furthermore, it is desirable that the cleavage of the second temporary linkage is substantially instantaneous after its lability has been induced by cleavage of the first temporary bond. Examples of polymeric prodrugs based on 1,6-elimination have been described by R. DeGroot et al. Shabat et al. Examples of polymeric amino-containing prodrugs based on trimethyl lock lactonization were described by R.
In this prodrug system, substituted o-hydroxyphenyl- dimethylpropionic acid is linked to PEG by an ester, carbonate, or carbamate group as a first temporary linkage and to amino groups of drug molecules by means of an amide bond as second temporary linkage. The rate-determining step in drug release is the enzymatic cleavage of the first linkage.
This step is followed by fast amide cleavage by lactonization, liberating an aromatic lactone side product. The disadvantage in the abovementioned prodrug systems described by Greenwald, DeGroot and Shabat is the release of highly reactive and potentially toxic aromatic small molecule side products like quinone methides or aromatic lactones after cleavage of the temporary linkage.
The potentially toxic entities are released in a 1 : 1 stoichiometry with the drug and can assume high in vivo concentrations. A different group of cascade produgs with aromatic activating groups based on 1,6- elimination structurally separates the masking group and the carrier.
This may be achieved by employing a permanent bond between the polymer carrier and the activating group. This stable bond does not participate in the cascade cleavage mechanism. If the carrier is not serving as a masking group and the activating group is coupled to the carrier by means of a stable bond, release of potentially toxic side products such as the activating group is avoided.
The stable attachment of the activating group and the polymer also suppresses the release of drug-linker intermediates with undefined pharmacology. Antczak et al. Bioorg Med Chem 9 describe a reagent which forms the basis for a macromolecular cascade prodrug system for amine-containing drug molecules. In this approach an antibody serves as the carrier, a stable bond connects the antibody to an activating group, carrying an enzymatically cleavable masking group.
Upon enzymatic removal of the ester-linked masking group, a second temporary bond cleaves and releases the drug compound. In this system the masking group is linked to the activating group by a carbamate bond.
The activating group is conjugated permanently to a polyacrylamide polymer via an amide bond. After enzymatic activation of the masking group by a catalytic antibody, the masking group is cleaved by cyclization and the drug is released.
The activating group is still connected to the polyacrylamide polymer after drug release. Lee et al. Nevertheless, in these linkers the 1,6-elimination step still generates a highly reactive aromatic intermediate. Even if the aromatic moiety remains permanently attached to the polymeric carrier, side reactions with potentially toxic products or immunogenic effects may be caused. For these reasons, there is a need to provide novel linker technologies for forming polymeric prodrugs of amine containing active agents using aliphatic prodrug linkers that are not enzyme-dependent and do not generate reactive aromatic intermediates during cleavage.
Garman et al. Garman, S. A disadvantage of the maleamic acid linkage is the lack of stability of the conjugate at lower pH values. This limits the applicability of the maleamic acid linkage to active agents which are stable at basic high pH values, as purification of the active agent polymer conjugate has to be performed under basic high pH conditions to prevent premature prodrug cleavage.
More recently, R. In this system two PEG carrier molecules are linked via temporary bonds to a bicine molecule coupled to an amino group of the drug molecule. The first two steps in prodrug activation is the enzymatic cleavage of the first temporary linkages connecting both PEG carrier molecules with the hydroxy groups of the bicine activating group. Different linkages between PEG and bicine are described resulting in different prodrug activation kinetics.
The second step in prodrug activation is the cleavage of the second temporary linkage connecting the bicine activating group to the amino group of the drug molecule. Dipeptides are frequently utilized for prodrug development for targeting or targeted transport as they are substrates for enzymes or biotransport systems.
Less studied is the non-enzymatic route for dipeptide prodrug formation, namely the ability to undergo intramolecular cyclization to form the corresponding diketopiperazine DKP and release the active drug.
In this case, the cyclization reaction consists of a nucleophilic attack of the N-terminal amine of the peptide on the ester carbon atom to form a tetrahedral intermediate. This is followed by a proton transfer from the amine to the leaving group oxyanion with simultaneous formation of a peptide bond to give the cyclic DKP product and free drug.
This method is applicable to hydroxyl- containing drugs in vitro but has been found to compete with enzymatic hydrolysis of the ester bond in vivo, as corresponding dipeptide esters released paracetamol at a much faster rate than in buffer Gomes et al, Molecules 12 The problem of susceptibility of dipeptide-based prodrugs to peptidases may be addressed by incorporating at least one non-natural amino acid in the dipeptide motif.
Corresponding prodrugs of cytarabine Wipf et al, Bioorg. Peptide Res. Still, endogenous enzymes capable of cleaving ester bonds are not limited to peptidases, and the enzyme-dependence of such prodrug cleavage still gives rise to unpredictable in vivo performance. Enzyme-dependence by design was engineered into DKP prodrugs as described in US 7,,, where dipeptide ester prodrugs were formylated at the amino terminus of the dipeptide, and enzymatic deformylation was used as a trigger to set off diketopiperazine formation and subsequent cleavage of the ester-dipeptide bond followed by drug release.
Similarly, vinblastine conjugates bearing an oligopeptide were described Brady et al, J. Here, an octapeptide was attached by an ester linkage to the 4-hydroxyl group of vinblastine and found to undergo ester bond cleavage by DKP formation after specific enzymatic removal of the N-terminal hexapeptide. Recently the scope of the DKP formation reaction was extended to amide prodrugs.
US 5,, details prodrug activation using diketopiperazine formation for dipeptidyl amide prodrugs of cytarabine. In this case, the temporary linkage was formed between the carbonyl of a dipeptide and the aromatic amino group of cytarabine.
In another study, the utility of diketopiperazine activation was demonstrated for even more stable aliphatic amide prodrugs G.
In this case, the cyclization reaction consists of a nucleophilic attack of the N-terminal amine of the peptide on the ester carbon atom to form a tetrahedral intermediate. The term 9 to 1 1 membered heterobicycle also includes spiro structures of two rings like l,4-dioxaazaspiro[4. More preferably, D-H is a protein selected from the group of proteins consisting of antibody fragments, single chain antigen binding proteins, catalytic antibodies and fusion proteins. The crosslinks provide the network structure and physical integrity. In the present application the following terms are used as described below. The carrier prodrug approach may be applied in such a fashion that the drug is released in vivo from the polymer in order to regain its biological activity.
In particular detail, it was surprisingly found that diketopiperazine formation can be used for carrier-linked amide prodrugs. The identity of the fragments was confirmed by collision-induced dissociation experiments where identical fragmentation patterns were observed. The term "water- insoluble" refers to a swellable three-dimensionally crosslinked molecular network framing the hydrogel. Also, during preparation of the prodrug, the amino groups may be more chemo selectively addressed and serve as a better handle for conjugating the carrier and the drug because of their greater nucleophilicity as compared to hydroxylic or phenolic groups.
The carrier prodrug approach may be applied in such a fashion that the drug is released in vivo from the polymer in order to regain its biological activity. Prodrugs which contain one or more basic groups, i. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Pendri, CD.
Figure 3 Figure 3. For instance it is known that in the case of subcutaneous injection, certain areas of the body yield more predictable therapeutic effects than others. Furthermore, it is difficult to establish an in vivo-in vitro correlation of the pharmacokinetic properties for enzyme-dependent carrier-linked prodrugs. In these cases, the cleavage of the temporary bond occurs in a one-step process which is catalyzed by the enzyme.
The formation of the stabilized six-membered ring structure is facilitated through a cis- amide conformation inducing pseudoproline.
Duncan, Nature Rev. The identity of the fragments was confirmed by collision-induced dissociation experiments where identical fragmentation patterns were observed. Suaifan et al, Tetrahedron 62 Typically, the drug is mixed with carrier material and processed in such fashion, that the drug becomes distributed inside the bulk carrier. The term polymer describes a molecule comprised of repeating structural units connected by chemical bonds in a linear, circular, branched, crosslinked or dendrimeric way or a combination thereof, which can be of synthetic or biological origin or a combination of both. An amount adequate to accomplish this is defined as "therapeutically effective amount".
The present invention also includes all salts of the prodrugs which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts. This limits the applicability of the maleamic acid linkage to active agents which are stable at basic high pH values, as purification of the active agent polymer conjugate has to be performed under basic high pH conditions to prevent premature prodrug cleavage.
Stabilisation is achieved by strengthening of the protein- stabilising forces, by destabilisation of the denatured state, or by direct binding of excipients to the protein. The second step in prodrug activation is the cleavage of the second temporary linkage connecting the bicine activating group to the amino group of the drug molecule. Thus, the prodrugs which contain acidic groups can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. Suitable substituents are alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl or halogen moieties such as those described above. However, in some cases, one excipient may have dual or triple functions.
Buffering capacity may be adjusted to match the conditions most sensitive to pH stability. Each of these signals is preceded by a small rectangular signal, which stems from electrical noise. The term "hydrolytically degradable" or "biodegradable" refers within the context of the present invention to linkages which are non-enzymatically hydrolytically degradable under physiological conditions aqueous buffer at pH 7. Prodrugs fall in two classes, bioprecursors and carrier- linked prodrugs.