Kinetics of solid state stability of glycine derivatives as a model for peptides using differential scanning calorimetry

Transcription

1 Biophysical Chemistry 97 (00) 3 10 Kinetics of soli state stability of glycine erivatives as a moel for pepties using ifferential scanning calorimetry a, b b c c M.I. Ab-Elrahman *, M.O. Ahme, S.M. Ahme, T. aboul-fal, A. El-Shorbagi a Department of Physics, Faculty of Science, Assiut University, Assiut 71516, Egypt b Department of Inustrial Pharmacy, Faculty of Pharmacy, Assiut University, Assiut, Egypt c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt Receive 6 November 001; receive in revise form 1 December 001; accepte 1 December 001 Abstract Kinetics of soli state stability of seven erivatives of 3,5-isubstitute tetrahyro-h-1,3,5-thiaiazine--thione (THTT) of glycine as a moel for amino acis an peptie rugs were stuie using ifferential scanning calorimetry (DSC). Each DSC curve for each erivative showe an enothermic peak followe by an exothermic one, which coul be attribute to the melting an ecomposition, respectively. The ecomposition activation energy of each erivative was calculate using the Augis an Bennet, Kissinger equations an Mahaevan approximation. Also, the melting activation energies as well as the thermoynamic parameter (enthalpy) for the investigate erivatives were evaluate. The relative stability of the erivatives in the soli state accoring to the calculate values of the ecomposition activation energy, frequency factors an half-life for each erivative coul be etermine. 00 Elsevier Science B.V. All rights reserve. Keywors: Organic crystals; Chemical synthesis; Calorimetry; Thermal analysis 1. Introuction Development of peptie rugs is presently a major area in rug research an in recent years, several biologically active pepties have been introuce. Proteins an pepties have become an important class of potent therapeutic rugs. The application of these pepties as clinically useful rugs is still seriously hampere ue to substantial elivery problems. Most pepties are rapily *Corresponing author. Tel.: q ; fax: q aress: mostafa.ia@euoramail.com (M.I. Ab-Elrahman). metabolize by proteolysis at most routes of aministration, they are non-lipophilic showing poor member penetration an they posess a short biological half-life w1 4x. As a part of the stuies starte in our laboratory, the moiety, tetrahyrothiaiazine--thione (THTT), was synthesize an use as a rug elivery system for peptie rugs. The prepare peptie erivatives were reaily bioreversible, lipophilic an more stable against peptiase enzymes in comparison with the parent peptie w5,6x. Differential scanning calorimetry (DSC) has a long an extensive application in the pharmaceutical inustry as a metho for the investigation of /0/$ - see front matter 00 Elsevier Science B.V. All rights reserve. PII: S Ž

2 4 M.I. Ab-Elrahman et al. / Biophysical Chemistry 97 (00) 3 10 purity, glass transition, stability, compatibility, etc. w7,8x. DSC offers a useful means of preicting the soli state stability of rugs w9x. The DSC experiment can be use to compute the Arrhenius preexponential factor, activation energy an orer of the reaction w10x. In this work, DSC was selecte to stuy the soli state stability of seven erivatives of tetrahyrothiaiazine--thione (THTT) of glycine. Also, the objective was concerne with the evaluation of the parameters of ecomposition kinetics applying the Augis an Bennet, Kissinger equations an Mahaevan approximation.. Experimental The seven erivatives of tetrahyrothiaiazine- -thione of glycine were prepare as mentione previously in our publications w5,6x. The DSC curves of the samples were obtaine using a Shimazu DSC-50 connecte with a TA-50I an TASYS programme computer. Samples of the erivatives ( 4 mg) in non-hermetically crimpe aluminum pans were heate at three ifferent heating rates (a), namely 5, 10, an 0 8Cymin uner nitrogen purge at 40 mlymin. The peak temperatures of melting (T ) an of ecomposition m (T ) for each sample were measure from DSC traces. The temperature energy calibrations of the instrument were performe using the well-known melting temperature an melting enthalpy of high purity inium supplie with the instrument. The calorimetric sensitivity is 10 mwycm an the temperature precision is "0.1 K. Best fits for the correlation between T m, T an a were calculate by the least-squares metho. The arithmetic mean as well as the stanar eviation were calculate for the activation energies. 3. Results an iscussion Scheme 1 shows the chemical structures of the seven (a g) synthesize erivatives of 3,5-isubstitute tetrahyro-h-1,3,5-thiaiazine--thione (THTT). Accoring to the substitution in position three, these erivatives can be classifie into three classes, namely N-3 alkyl substituent (methyl gm, ethyl ge, propyl gp, an butyl gb), N-3 cycloalkyl Scheme 1. In formulae: (a) RsCH 3; (b) RsCH3CH ; (c) Rs CH3CHCH ; () Rsn-C4H 9; (e) Rscyclo-C6H ; (f) Rs C6H5CH ; (g) RsC6H5CHCH. substituent (cyclohexyl gc) an N-3 aralkyl substituents (benzyl gbz an phenethyl g). This moiety, THTT, was synthesize an foun to be a promising prorug approach of the rug elivery system for peptie rugs w5,6x. The DSC traces of these erivatives were taken at a heating rate (a) of 5, 10 an 0 8Cymin. Fig. 1 shows the typical DSC curves of g, ge an gc erivatives representing the three classes at heating rate of 0 8Cymin. Each DSC trace has two characteristic phenomena, an enothermic peak followe by an exothermic one, in the stuie temperature range. The first one correspons to the peak temperature of the melting (T ) an the secon one to the peak m temperature of the ecomposition(t ) of each compoun. The exothermic peaks of g, ge an gc erivatives at heating rate (a) of 5, 10 an 0 8Cy min are shown on their DSC curves in Fig.. From these curves, it was clearly the shifting of the exothermic peak to a higher temperature by increasing the heating rate for each erivative. Generally, it is foun that the two characteristic temperatures of each erivative increase with increasing the heating rate as shown in Table 1. The ecomposition activation energy of the erivatives can be calculate using the following equation propose by Augis an Bennet wx: lnž a. syeyrtqln k 0, (1) where R is the gas constant an k is the frequency 0 factor. This equation is use specifically for nonisothermal crystallization wx. It can be applie here, for the first time, to obtain the ecomposition kinetics of the erivatives of THTT. In this case, E represents the activation energy for the ecomposition, E, an T epicts the ecomposition peak

3 M.I. Ab-Elrahman et al. / Biophysical Chemistry 97 (00) Fig. 1. Typical DSC traces of g, ge an gc erivatives of THTT at a heating rate of 0 8Cymin. temperature, T. The constant k0 measures the probability of molecular collisions effective for the formation of the activate complexes. The relation between ln(ayt ) an (1yT ) obtaine from the thermograms of a given erivative is linear an inicates the valiity of this relationship for all erivatives (Fig. 3). The ecomposition activation energies are calculate from the slopes of this function fitte to the ata. In aition, the frequency factor (k 0) for a given erivative is calculate from the original intercept of the straight line with vertical axis an given together with the E in Table. The results in this table show a corresponence between the values of E an k0 for a specific erivative. Accoring to the efinition of k 0, this corresponence seems to be reasonable. The interpretation of the DSC ata is provie by the formal theory of transformation kinetics as the ecomposition rate constant (K) is usually assume to be an Arrhenian temperature epenence Ksk0expŽ yeyrt., () where T is any temperature in the entire region of exothermic peak. The constant K escribes the ecomposition reaction rate at any temperature, T. Substituting the obtaine values of E an k from 0 Eq. (1) for each erivative, one can calculate K as a function of T for each heating rate. The calculate values of K(T) for three erivatives representing the three classes of the erivatives at a constant heating rate (as10 8Cymin) are plotte as lnk vs. 1yT an the straight lines are shown in Fig. 4. The valiity of Eq. () for each erivative confirms the results obtaine for E an k 0.From Fig. 4, it can be notice that the ge erivative thermally ecomposes before the erivative g which in turn ecomposes before gc. In other wors, gc is the most thermally stable erivative among the selecte ones followe by g then ge. This result was confirme by the obtaine values of E which relates to the stability for these erivatives as shown in Table. The orer of the reaction rate constant was foun to be a first orer reaction, an from which the half-life for each erivative coul be calculate by a simple relation: t1ys0.693yk w1x. Here t is the perio of time require for a rug to 1y

4 6 M.I. Ab-Elrahman et al. / Biophysical Chemistry 97 (00) 3 10 Table 1 The peak temperatures of enothermic an exothermic peak of THTT erivatives Derivative Enothermic peak (8C) at heating rate (8Cymin) Exothermic peak (8C) at heating rate (8Cymin) gm ge gp gb gc gbz g constant an the most stable erivative in the soli state. The mechanism of the hyrolysis of THTT in solution state to liberate the parent rug glycine, was previously reporte to occur via ring cleavage at N5 of the THTT moiety w5x. Fig.. Exothermic traces of g, ge an gc erivatives of THTT at heating rates of 5, 10 an 0 8Cymin. ecompose to one-half the original concentration. The half-lives of the THTT erivatives for glycine were calculate at 5 8C an presente in Table 3. In the case of N-3 alkyl substituents, the methyl group ecrease the half-life to 0. month, i.e. increase the ecomposition rate, whereas chain elongation (ethyl, propyl) was accompanie by increasing the half-life to 5 an 40 months, respectively. The soli state stability seeme to be affecte by substituents at N-3 of THTT moiety. However, the N-3 aralkyl substitution, benzyl group gave a higher half-life (57 months) than that of the phenethyl group (3 months). Cycloalkyl substituent, on the other han, reveale a varie pattern that gave the highest value of halflife (00 months), i.e. the least ecomposition rate Fig. 3. Augis an Bennet plots: ln(ayt ) vs. (1yT ) for the investigate seven erivatives of THTT.

5 M.I. Ab-Elrahman et al. / Biophysical Chemistry 97 (00) Table The ecomposition activation energies (E ) for the investigate erivatives calculate by ifferent methos an the corresponence frequency factors (k 0 ) Derivative Kissinger Mahaevan Augis an Bennet ln(ay T ) vs. 1yT 1yT vs. ln(a) ln(ayt) vs. 1yT E (kjymol) E (kjymol) -1 E (kjymol) k (min ) gbz 69.68" " " =10 "50 gm 86.61" " " =10 "4 ge " " "0.3.7=10 " gp " " " =10 "1 g " " " =10 "7 gb " " "0.16.5=10 " gc 4.76" " " =10 "6 0 The activation energy for ecomposition of the erivatives of THTT can also be calculate by the other two methos w13,14x. Accoring to Kissinger s formula w13x, the temperature of the exothermic peak epens on the heating rate by the following relation lnž ayt. syeyrtqconstant. (3) This expression has been originally suggeste for the stuying of the crystallization kinetics in materials having amorphous crystalline transformations. In which E is the activation energy for the crystallization an T is the crystallization peak temperature. However, it is possible to use this expression to stuy the ecomposition kinetics of materials which unergo thermal isintegration w15x. In this case, E an T replace E an T. Base on Eq. (3), plotting ln(ay T ) vs. (1yT ) gives a linear relation. The experimental points show a goo fit with the above relation, as shown in Fig. 5. The ecomposition activation energies for the erivatives were calculate from the slopes of the straight lines an liste in ascening orer in Table. The obtaine values of E values inicate that the more stable erivative is gc in the soli state, which has the largest value of E (4.76"1.70 kjymol). In aition, gc showe Table 3 The half-lives at 5 8C of the THTT erivatives Fig. 4. Temperature epenence of the ecomposition rate constant for gc, g an ge erivatives of THTT at a heating rate of 10 8Cymin. Derivative gm ge gp gb gc gbz g t 1y (months)

6 8 M.I. Ab-Elrahman et al. / Biophysical Chemistry 97 (00) 3 10 lnž a. sye yrt qconstant. (4) This equation can be rearrange in the form 1 R sy lnž a. qconstant. (5) T E Plots of (1yT ) vs. ln(a) for the seven erivatives gave straight lines, as shown in Fig. 6. The value of E for a given erivative was euce from the slope of the corresponence straight line. The obtaine E values of the erivatives of THTT are liste in Table. The results in this table show that the errors ue to the fitting of Eq. (3) to the experimental ata are much smaller than those ue to the fitting using Eq. (5). Thus, one can conclue that the Mahaevan et al. approximation is more suitable in the etermination of the E value an in the escribing of the ecomposition mechanism than the Kissinger equation. The melting activation energies Em values for the investigate erivatives of THTT can be evaluate using relations similar to those in Eqs. (3) an (5). However, in this case Em an Tm replace Fig. 5. Kissinger plots: ln(ay T ) vs. 1000yT for the investigate seven erivatives of THTT. the longest t1y an highest melting point. It was observe that the activation energy of ecomposition of alkyl substituents was increase by elongation of alkyl chain from methyl to propyl groups (Table ). These results are in agreement with the results of half-lives (Table 3) an their melting points (Table 1) which increase by the lengthening of the sie chain from methyl to propyl in the three positions of THTT, ue to progressive increasing of their intermolecular forces. On the other han, the erivative gbz having the smallest values of both E (69.68"1.70 kjymol) an t1y (0. month) can be consiere as the fast thermally ecompose erivative. The value of E can also be calculate by using the Mahaevan et al. approximation w14x. The T variation of ln(1y )with ln(a) is much slower than that of (1yT ) with ln(a). Thus, the Kissinger equation can be rewritten in the form of Mahaevan et al. as follows: Fig. 6. Maahevan plots: 1000yT vs. ln(a) for the investigate seven erivatives of THTT.

7 M.I. Ab-Elrahman et al. / Biophysical Chemistry 97 (00) Fig. 7. Kissinger plots: ln(ay T m ) vs. 1000yT m for the investigate seven erivatives of THTT. E an T, respectively. Fig. 7 shows a goo fit between the experimental points an the straight lines accoring to Kissinger s equation. Similar results were obtaine by applying Kissinger s equation for mercaptopurine ehyration w15x. The obtaine Em values using Kissinger s equation an Mahaevan approximation are presente in ascening orer in Table 4. The heat of fusion or enthalpy (DH), as a thermoynamic parameter, may be consiere as the heat require to increase the interatomic or intermolecular istance in crystals, thus allowing melting to occur w1x. A erivative that is boun together by weak forces generally has a low heat of fusion an a low melting point, whereas one boun together by strong forces has a high heat of fusion an a high melting point. The enthalpy of the investigate erivatives were obtaine from the computer program involve in the DSC apparatus an presente in Table 4. The value of DH was increase by increasing the length of sie chain from methyl (gm) to propyl group (gp), as foun to be Jyg, respectively. This coul be attribute to the relationship between the heat of fusion an intermolecular forces, which increase by increasing the sie chain in the N-3 position of THTT moiety. On the other han, cycloalkyl (gc) an aralkyl (gbz an g) substituents gave their enthalpy a varie value inepenently. 4. Conclusion From the results of DSC thermograms, it was foun that the cycloalkyl erivative of THTT of glycine was the most stable prorug as it has the highest activation energy an longest half-life among the teste erivatives. Moreover, in the case of the alkyl substituent, the stability was enhance upon the lengthening of the sie chain at position N-3. Generally, the soli state stability Table 4 The melting activation energies (E m) for the erivatives calculate by two ifferent methos an their enthalpy (DH) at heating rate as10 8ymin Derivative Kissinger Maahevan Enthalpy at ln(ay T m ) vs. 1yT m 1yTm vs. ln(a) as10 8Cymin E m (kjymol) E m (kjymol) DH (Jyg) gm 83." " gb " " gc " " gbz 160.5" " g " " gp " " ge " "

8 10 M.I. Ab-Elrahman et al. / Biophysical Chemistry 97 (00) 3 10 of such erivatives can be controlle by the choice of N-3 substituent. Finally, this work she some light on the soli state stability of potentially useful promoieties THTT as a rug elivery system for amino acis an peptie rugs. References w1x H. Bungar, in: S.S. Davis, L. Illum, E. Tomlinson (Es.), Delivery System for Peptie Drugs, Plenum Press, New York, 1986, pp wx V.H.L. Lee, R.D. Traver, M.E. Taub, in: V.H.L. Lee (E.), Peptie an Protein Drug Delivery, Marcel Dekker, New York, 1991, pp w3x A.K. Banga, Y.W. Chien, Systemic elivery of therapeutic pepties an proteins, Int. J. Pharm. 48 (1988) 15. w4x S.S. Davis, in: S.S. Davis, L. Illum, E. Tomlinson (Es.), Delivery Systems for Peptie Drugs, Plenum Press, New York, 1986, pp w5x T. aboul-fal, A. El-Shorbagi, New prorug approach for amino acis an amino-aci-like rugs, Eur. J. Me. Chem. 31 (1996) 165. w6x T. aboul-fal, A. El-Shorbagi, New carriers for representative pepties an peptie rugs, Arch. Pharm. Pharm. Me. 330 (1997) 37. w7x J.L. For, P. Timmins, Pharmaceutical Thermal Analysis, Techniques an Application, Ellis Horwoo, Chichester, w8x A.E. Beezer, S. Gaifor, A.K. Hills, R.J. Willson, J.C. Mitchell, Pharmaceutical microcalorimetry: application to long-term stability stuies, Int. J. Pharm. 179 (1999) 159. w9x A. Li Wan Po, Application of ifferential scanning calorimetry in pharmacy: preiction of soli state stability of rugs, Anal. Proc. 3 (1986) 391. w10x J.C.M. Torfs, L. Deij, A.J. Dorrepaal, J.C. Heijens, Determination of Arhenius kinetic constants by ifferential scanning calorimetry, Anal. Chem. 56 (1984) 883. wx J.A. Augis, J.E. Bennett, Kinetics of the transformation of metastable tin nickel eposits. I. Determination of the Avrami equation parameters by DSC or DTA, J. Elctrochem. Soc. 15 (1978) 330. w1x R.A. Martin, Physical Pharmacy, 4th e, Waverly International, New Delhi reprint, w13x H.E. Kissinger, Reaction kinetics in ifferential thermal analysis, Anal. Chem. 9 (1957) 170. w14x S. Mahaevan, A. Girihar, A.K. Singh, Calorimetric measurements on As Sb Se glasses, J. Non-Cryst. Solis 88 (1986). w15x S. Niazi, Thermoynamics of mercaptopurine ehyration, J. Pharm. Sci. 67 (1978) 488.