Calorimetric Investigation of the Phase Behaviour of the Binary System 7-Mpeg 5000-Succinyloxymethyloxy- Carbonyl-Paclitaxel (PP7)/Water

http://www.e-polymers.org e-Polymers 2005, no. 007. ISSN 1618-7229

Calorimetric investigation of the phase behaviour of the binary system 7-mPEG 5000-succinyloxymethyloxy- carbonyl-Paclitaxel (PP7)/water

Dedicated to Dr. E. A. Hemmer, Professor emeritus of the Gerhard-Mercator University Duisburg, Germany, on the occasion of his 75th birthday

Jung-Sun Sohn 1, Soo-Kyung Choi 1, Byung-Wook Jo 1, Kirsten Schwark 2, Michael Hess 2 *

1 Department of Polymer Engineering and Science, and Department of New Materials in Biology, Chosun University, 375, Seosok-dong, Dong-gu, KwangJu, 501-759, South Korea; [email protected] 2 Department of Physical Chemistry, University Duisburg-Essen, Campus Duisburg, 47048 Duisburg, Germany; [email protected]

(Received: October 22, 2004; published: February 2, 2005)

This work has been presented at the 12th Annual POLYCHAR World Forum on Advanced Materials, January 6-9, 2004, in Guimaraes, Portugal

Abstract: Paclitaxel is an antineoplastic agent derived from the bark of the Pacific Yew Tree (Taxus brevifolia). Oral administration of the pure drug is problematic as it has a poor absorption due to its poor solubility in aqueous media. A specific water-soluble prodrug – PP7 – has been introduced by coupling the drug with a water-soluble polymer. For any kind of medical application and administration, knowledge of the binary isobaric phase-diagram is important since it gives information about solubility, phase transitions and the corresponding compositions. The

system PP7 + H2O was studied calorimetrically from -20°C and shows the typical behaviour of an eutectic system. The properties of the phase diagram are discussed in view of other solution properties of the system presented earlier.

1. Introduction and scope The application of pharmaceutically active substances is frequently faced with problems of solubility, stability and bioavailability of the active species. In the case of the anti-cancer drug Paclitaxel [1] (Fig. 1) – an antineoplastic substance that can be isolated from the bark of the Pacific Yew Tree (Taxus brevifolia) – the first problem is its hydrophobicity. Despite several polar sites the molecule is only poorly soluble in aqueous media. Also, the pump function for Paclitaxel of the multi-drug pump trans- porter P-glycoprotein of the gastrointestinal tract is poor. There are several ways to overcome this problem. One possible way is substitution [2,3] at the reactive sites on carbons 2' or 7 (Fig. 1). The first such derivatives of Paclitaxel with poly(oxyethylene) 1 (PEO), however, were too stable so that a high percentage of the polymer drug was excreted before the drug could be made bioavailable by esterases on-site of their intended interaction. This problem was solved by the development of the prodrug PP7, which connects the hydrophilic PEOS – a succinic acid monoester of PEO monomethyl ether – with a self-immolating linker [4-7] at carbon 7 (Fig. 1). Some results about properties of PP7 in aqueous solution have been published earlier [8-10]. The term ‘prodrug’ indicates that PP7 is not the finally active species. Enzy- matic cleavage of the bond with the polymer substituent is necessary so that the Paclitaxel molecule can interfere in the late phase of G2 mitosis by stabilising the microtubule through non-covalent interactions.

The thermodynamic properties of the system PP7/H2O, cumulated in the isobaric phase diagram, give important information about different aspects of the system, e.g., when the physical properties or the polymers themselves have to be characterised. Many of the experimental techniques can only be performed in homogeneous solution, and knowledge about the state of the polymer drug is essential for the method of its application and bioavailability.

Fig. 1. Structure of Paclitaxel with the substitution sites 2' and 7

The state of the prodrug is also important for its capability to interfere with malign tissue. Here it is of particular importance that the PEO chains – depending on their molar masses – show affinity to certain tumour types [11]. Finally, the phase diagram of the polymer-solvent system contains information about interaction parameters and thermodynamic equations of state and gives useful hints for the alteration of the molecular structure of the water-soluble drug in order to cause certain behaviour in aqueous solution like complex formation and aggregation, which might me desired to increase the local concentration of the drug in solution. Know- ledge of the phase behaviour of pharmaceutical systems is therefore of great importance far beyond just academic interest. The investigation of the phase diagram is one more step on the way to understand the mechanism of drug activity with respect to conformation, shape and size in solution, transport mechanisms, and interaction with other constituents of body fluids.

2 2. Experimental part

2.1 Materials Paclitaxel obtained from Brystol-Myers Squibb Co., NY, USA, was modified with α- methyl-terminated ω-OH-functional PEO (Fluka) (〈Mw〉/〈Mn〉 = 1.05 and molar mass averages of 5000 g/mol) and linked to carbon 7 (see Fig. 1) with succinic acid through a self-immolating linker. The modified drug (the prodrug) is termed PP7 (5000), the α-methyl- and ω-succinyl-terminated PEO is termed PEOS in the following text. The additive ‘-5000’ indicates the molar mass of the PEO since also other degrees of polymerisation can be used. However, the yield decreases with increasing length of the polymer chain. The synthesis of the prodrug has been described elsewhere [4-7,12].

2.2 Methods Calorimetric measurements were carried out with a Perkin-Elmer DSC-7 differential scanning calorimeter. A total mass of approximately 20 mg was placed in a sealed pressure pan and heated up to 80°C for complete solution. After keeping the solution at 80°C for about 5 min, the sample was cooled down to -20°C with a cooling rate of 20 K/min and the glass transition temperature of the pure PEO (〈Mw〉= 5000 g/mol) at -63°C was not reached. The samples were heated with a heating rate of 20 K/min. Cold crystallisation during heating was not observed.

Fig. 2. Construction of a phase diagram from DSC experiments

The phase diagram was constructed according to Fig. 2 by taking the end-point of the first-order transition, which is supposed to be close to the equilibrium transition temperature since the largest crystals of the polymer melt at that temperature. The classification of thermodynamic transitions follows Ehrenfest [13]. Within the temper-

3 ature range of observation no cold crystallisation was observed. The crystallisation of non-equilibrium structures usually occurs at temperatures closer to the glass transition (around -40°C for PEO) [14].

3. Results and discussion A phase diagram shows thermodynamic transitions depending on the composition of the system given as the volume fraction or the mass fraction at constant pressure. A typical example of a binary system is given in Fig. 3. In most cases, phase diagrams show equilibrium situations only. However, it has been argued [15] that non-equilibrium but long-living phases deserve to be included in phase-diagrams, thus providing more information.

Fig. 3. General scheme of a common phase diagram. Further explanations see text

Region I is the homogeneous isotropic liquid mixture (one phase) of components 1 and 2. Regions II and III represent solid one-phase regions consisting of mixed crystals rich in component 1 (II) or rich in component 2 (III), respectively. These two phases can be very narrow or even absent. Sometimes these regions are addressed as representing a ‘border solubility’. Regions II and III vanish when the liquid phase I is in equilibrium with a solid pure component 1 or 2, respectively. The two solid one- phase regions II and III are separated by the solid two-phase region VI. At temperatures between Te and Tm,1 or Tm,2, the solid one-phase region II is separated from the liquid one-phase region I by the two-phase region IV, and the liquid one-phase region I is separated from the solid one-phase region III by the 2-phase region V. As a consequence of Gibbs phase rule two one-phase regions have always to be separated by one two-phase region. Inside the two-phase regions the lever rule is valid, e.g., between the points a and a' and b and b'. The curves Tm,1 – e - Tm,2 – e

(liquidus curve), Tm,1 – c - Tm,2 – d (solidus curve), c – c', and d – d' ( solvus curve) are equilibrium curves. e is the eutectic point and has no degree of freedom. c – e – c' is a 4 monovariant line. The other lines are bivariant. The lines can continue within the phases as indicated by the dotted lines describing metastable states. The solid lines describe the coexistence curves of the thermodynamic equilibrium state. In a real solid-liquid equilibrium system, e.g., a melt with the composition x2,a is in thermodynamic equilibrium with mixed crystals of composition x2,a' at the temperature Ta. Thermodynamic melting equilibrium requires infinitely large crystals. In polymers and in dynamic measurements this situation is approximated at a low heating rate and at the end of the DSC melting curve. Systems containing a solvent and a polymer have always to be considered as quasi-binary ones because of the non-uniformity of polymers.

Fig. 4. Examples of DSC traces of mixtures of PP7 (5000) + H2O of different composition. The melting transition of the PEO chain is clearly identified at higher temperatures. The melting of water and the invariant temperature of the solidification temperature of the eutectic mixture follow with decreasing temperature. Boundary solubility cannot unequivocally be identified. Xp is the mass fraction of PP7

Fig. 5 shows the experimental phase diagram of PP7 (5000) and water in comparison with PEO 6000 that was described by Dobnik [14]. Apparently, PP7 shows the same general behaviour as a corresponding PEO although some differences are observed. Pure Paclitaxel (recrystallized from hexane) shows a melting transition around 215°C. At lower temperatures amorphous Paclitaxel (prepared by evaporation of the solvent from CH2Cl2 solution) shows a glass transition temperature of about 155°C. Neither in pure PP7 nor in water-containing PP7 any transition referring to Paclitaxel was found. The only transitions are related to the melting of the PEO chain. A physical mixture (1:1) of PEO and Paclitaxel, on the contrary, shows unchanged melting of PEO and the glass transition of Paclitaxel.

Fig. 5. Phase diagram of PP7 (5000) + H2O and PEO (6000) + H2O

The quasi-binary phase diagram of the system PP7 (5000) + H2O constructed from a series of compositions like in Fig. 2 shows an eutecticum at about 64%(w/w) PP7 at -18°C. Compared with PEO (6000) the eutectic point is shifted to higher polymer concentration (about 20% higher) with almost the same transition temperature. The behaviour of PP7 (5000) is very similar to a much shorter PEO chain (about 1000 g/mol). A higher molar mass of the PEO results in a shift of the eutectic composition to a more water-rich composition and an increase in the eutectic temperature. The observed behaviour can be explained if it is assumed that a significant part of the polymer chain interacts with parts of Paclitaxel and has a restricted mobility and accessibility for water molecules. Consequently, the ‘active’ polymer chain appears to be shorter. The decreased transition temperature, which is observed with PP7 compared with pure PEO, can be explained by a decrease of the polymer-solvent interaction parameter χ, see below. While no mixed crystals of ice and PP7 were observed there is some indication of a mixed PP7-H2O crystal. The melting of the mixed crystal is difficult to identify and has to be deduced from the first deviation of the DSC signal from the baseline. Compared with the results obtained by Dobnik [14] for a series of PEOs of different chain length, the presence of a mixed crystal of the polymer chain on Paclitaxel with water is likely. The glass-transitions, which were described by Dobnik [14], could not be observed because they occur below the lowest temperature that could be reached with the available equipment. A thermodynamic description of the liquidus curve is possible in principle and delivers the freezing-point depression of the water and the polymer-water mixed crystal. For this purpose, a non-ideal, non-athermic approach has to be used. While in the case of ‘simple’ polymer-chains calculations on the basis of the Flory-Huggins-Stavermann lattice theory are possible, the prodrug is too complex and only some general conclusions can be drawn from this equation:

1 1 R ⋅ M lnw  1   − =− 2u  2 − 1− w + χ ⋅ 2 0 0   1 w w2 Tm2 T m2 ∆m H2u ⋅ M1  rw  rw  {  123  temperature−,concentration− andmolar mass dependent  temperature dependent 6 0 Tm2 = Melting temperature of the polymer in equilibrium with the melt (solution); T m2 = melting temperature of the pure polymer (ideal crystal); R = gas constant, M2u = 0 molar mass of a monomer unit; ∆mH 2u = molar enthalpy of fusion of a monomer unit (temperature-dependent); M1 = molar mass of the solvent; w2 = mass fraction of the polymer; rw = degree of polymerization of the polymer; χw = Flory-Huggins-Staverman polymer-solvent interaction parameter on the mass fraction scale (temperature- and concentration-dependent). 0 In addition to the temperature dependence of ∆mH 2u and χw the use of the mass fraction for the concentration suffers from the fact that the mass of the molecules is not proportional to the number of occupied lattice sites. Because of the complex structure of the prodrug the assumption that a solvent molecule has a volume com- parable to a monomer unit is far from reality so that the use of the volume fraction or the base mole fraction appears not be justified. Finally, the enthalpy of fusion has to be known in order to calculate the liquidus curve on the polymer side and the theory only considers a pure polymer crystal. However, the crystalline phases of a polymeric crystal are usually metastable and not in thermodynamic equilibrium. The theory considers pure polymer crystals coexisting with the melt (solution), a fact that is not given in this system since a considerable boundary solubility can be assumed. This boundary solubility can be due to the formation of a mixed crystal but it can also be caused by water in the amorphous phases of the prodrug. The absence of any thermal Paclitaxel transition gives support to a particular model of the PP7 molecule. A first approach might suggest that the PEO chain coils inde- pendently and separate from the Paclitaxel part of the whole prodrug, Fig. 6a. Such a molecule separated into a hydrophilic PEO coil and the hydrophobic Paclitaxel appears to be structural closely related with detergent molecules. Consequently, the formation of superstructures in aqueous solution are to be expected such as micelles or layered or cylindrical structures. In the present case for example the polar polymer chain is very flexible and will most probably form a random coil. The formation of a micelle with the Paclitaxel part in the core and the PEO coils aggregating in an outer shell can be imagined so that larger particles with a high local Paclitaxel concentration would be formed. Any of these superstructures that can be imagined formed by an amphiphilic molecular structure as described above should be reflected in physical properties such as the (concentration-dependence) of the surface tension, the solution viscosity or the self-diffusion coefficient. In previous investigations [9,12,16] no indication of any type of such a self-aggregation of PP7 in water was found. An alternative model, Fig. 6b, shows a conformation where the hydrophilic PEO chain coils around the Paclitaxel in a single core-shell structure. This is a conformation frequently found in proteins. The polymer chain coils in a way so that a hydrophilic surface is formed with a hydrophobic core or hydrophobic pockets resulting in a water-soluble organic-phase nano-reactor (enzyme). The fact that no melting or glass transition of the Paclitaxel part was found in PP7 seems to support the second alternative. In this second model the individual Paclitaxel units are separated from each other and cannot form individual phases able to show any cooperative transitions. Further studies will concentrate on the possibilities to achieve water-solubility of Paclitaxel accompanied by the formation of superstructures that would provide a much higher local concentration of the active part of the prodrug, such as the micelles shown in Fig. 6c. As long as these structures do not go beyond a certain size they can still penetrate cell membranes. The high local drug concentration would

7 increase the bioavailability of the drug while decreasing its loss by excretion. Further studies about the requirements to force the prodrug into superstructures are being conducted.

Fig. 6. a) Model of the prodrug with a conformation similar to a detergent molecule. b) Core-shell model of the prodrug with the polymer chain coiled around the hydrophobic drug. The drug is shielded so that no crystallisation of the Paclitaxel parts of the molecules is possible. c) Formation of superstructures like micelles require special properties of length, mobility and shape of the hydrophilic part of the prodrug relative to the hydrophobic part. Molecular modelling can give support in engineering of the prodrug

The study of the self-diffusion coefficient and the viscosity of the mPEG-ylated Paclitaxel in aqueous solution has revealed the important information that these molecules, although they consist of a lipophilic and a hydrophilic part, show no surface activity and do not form supramolecular structures such as micelles or vesicles. Consequently, a core-shell structure with a conformation akin to certain proteins appears to be highly likely for PP7 in aqueous solution. This model is supported by studies of the quasi-binary phase-diagram PP7/water. However, aggregation of the modified drug is desired because it would significantly increase the bioavailability of Paclitaxel. The formation of larger aggregates could also lead to 8 enhanced targeting effects of the drug since the blood vessels in malign tissue frequently show a higher permeability for larger particles. Intelligent molecular engineering of the drug could contribute to a higher efficiency of the active principle in PP7 so that the local density of Paclitaxel is increased by formation of water- soluble aggregations. Until now, the properties of PP7 have only been studied in water. In the blood serum, however, proteins, lipoprotein, and other biopolymers are present. Future investigations will therefore focus on the study of possible interactions of PP7 with blood constituents and on the modification of the prodrug so that a conformation in solution is obtained that favours the formation of molecular aggregates of controlled size combined with the ability to accumulate in tumour tissue.

Acknowledgement: The author (M. H.) expresses his thanks to Deutsche For- schungsgemeinschaft, DFG (Bonn, Germany) and KOSEF (Seoul, South Korea) for supporting his sabbatical at Chosun University, Kwang-Ju, South Korea, hosted by Prof. Byung-Wook Jo. C. P. Lee (Cambridge, UK) is thanked for helpful discussions.

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