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Synthesis and biological studies of new lipid-soluble cisplatin analogues entrapped in liposomes
Synthesis and Biological Studies of New Lipid-Soluble Cisplatin Analogues Entrapped in Liposomes Salaam Al-Baker, Roman Perez-Soler, and Abdti R. Khokhar Department of Medical Oncology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas (USA)
ABSTRACT A series of highly lipophilic platinum(H) complexes of the type c~~-[(RNH~)~P~X,] have been synthesized, where R = ethyl, propyl, isopropyl, butyl, cyclopropyl, cyclopentyl, or neopentyl and X = either long-chain carboxylate, such as decanoate, (ClO), laurate (Cl2), myristate (Cl4), heptadecanoate (Cl7), stearate (Cl@, nonadecanoate (Cl9), or 2,2,3,3-tetramethylcyclopropylcarboxylate, or branched-chain carboxylate, such as neopentanoate, neohexanoate, neoheptanoate, neononanoate, or neodecanoate. These complexes have been characterized by elemental analysis, IR, and “C and ‘9sRt NMR spectroscopic techniques. The platinum complexes were entrapped in multilamellai vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) at a 7 : 3 molar ratio and tested for antitumor activity. The entrapment efficiency of liposomal platinum (L-F?) complexes ranged from 60 to 100%. The percentage of T/C obtained after a single i.p. injection of the optimal dose of L-Pt complexes tested against Ll210 leukemia ranged from 90 to 125%. These L-Pt preparations did not show significant antitumor activity in mice.
INTRODUCTION Cisplatin is one of the most effective antineoplastic drugs [l-3] against cancers of the head and neck, testes, ovaries, and bladder. The usefulness of cisplatin is, however, compromised by its propensity to cause several severe dose limiting toxicities, including nephrotoxicity, neurotoxicity, and ototoxicity [4-71. In recent years, there has been a growing interest in developing. new platinum complexes with greater antitumor activity, a broader spectrum of activity, and non-cross resistance to cisplatin. However, the development of some promising analogues has been
Address reprint requests to: Dr. Abdul R. Rhokhar, DeparWntofiMedical Oncology (Box 52), The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Journal of Inorganic Biochemistry, 47,99- 108 (1992) 0 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010
99 0162-0134/92/$5.00
100 S. Al-Baker et al.
hampered by their low solubility , poor stability, and difficult formulation, all of which decrease the potential of such analogues for clinical use [8]. An alternative approach to modifying the therapeutic index of cisplatin analogues may be the use of a drug carrier. Liposomes are particularly attractive as such because they are easy to prepare and are biodegradable [9, lo]. In general, studies with antineoplastic agents entrapped in liposomes have shown that liposomal entrapment of cytostatic drugs may result in preserved antitumor activity and reduced toxicity [ 111. However, due to its low lipophilicity, cisplatin is not a suitable drug for liposome formulation. All reported liposomal cisplatin preparations have a very low entrapment efficiency and high rate of drug leakage [ 12- 141. The objective of this study was to synthesize a series of highly lipid-soluble platinum(I1) complexes designed for liposome entrapment [ 15- 171. Such complexes should have a high entrapment efficiency and good antitumor activity [16, 18-201. In this paper we describe the chemical development of liposomal platinum and the biological activity of a series of highly lipid-soluble platinum(H) complexes containing a monodentate amine as the inert ligand and either long-chain or branched-chain aliphatic carboxylate as the leaving ligand. EXPERIMENTAL Materials and Methods All alkylamine and straight-chain carboxylic acids were purchased from Aldrich Chemical Co. (Milwaukee, WI). Neopentanoic acid, neohexanoic acid, neoheptanoic acid, neononanoic acid, and neodecanoic acid were obtained from Exxon Chemical Co. (Houston, TX). K,PtCl, was purchased from Aesar (Seabrook, NH). All chemicals obtained from commercial sources were used as received. Microanalysis of platinum complexes was performed by Robertson Laboratory Inc. (Madison, NJ). Thin-layer chromatography (TLC) was performed on precoated silica gel plates in a solvent system consisting of methanol-ethyl acetate (1 : 9). The plates were visualized as yellow spots after exposure to iodine vapor. Infrared spectra (4000-250 cm-‘) were recorded as a KBr pellet on a Beckman Microlab 250MX infrared spectrophotometer. All NMR spectra (i3C{‘H} and ‘95Pt(‘H}) were recorded on an IBM NR/200 AF NMR spectrometer. The ‘95Pt{‘H} spectra (43.055 MHz) were collected by using a 10 s (90) pulse and a 0.012 s acquisition time with a spectral width of 166 KHz (4 k data point). 19% chemical shifts were collected in CHCl, solution (about 30 n&I) for 10 hr at room temperature and were referenced to a D,O solution of Na,PtCl, (0.0 ppm), which was contained in a 5-mm NMR tube (0.5 g/O.5 ml D,O), which in turn was inserted into the lo-mm NMR tube. 13C{‘H} NMR spectra were measured in CDCl, solution, with the carbon-13 chemical shifts being referenced to the CDCl, peak at 77 ppm. The chromatographically pure (as determined by TLC) DMPC and DMPG used in this study were obtained from Avanti Polar Lipids (Birmingham, AL). L1210/0 leukemia cells were obtained from the DCT Tumor Repository (National Cancer Institute, Frederick, MD). BDFl mice weighing 18-20 g were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Synthesis of Platinum Complexes Synthesis of cis-bis(alkylaminc)diiodoplatlnum(II) complexes (cis[(RNH,),PtI,]). All platinum complexes of the type ci.~-[(RlW~)~Pt&] were synthesized according to an earlier reported procedure [21]. The preparation of
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101
cis-bis(isopropylamine)diiodoplatinum(II) is given here as an example. An aqueous solution of potassium iodide (53.0 g, 0.32 mol in 75 ml H,O) was added to a filtered aqueous solution of K,FtCl, (16.6 g, 0.04 mol in 250 ml H,O) and the reaction mixture was stirred for 10 min at room temperature; to this, isopropylamine (5.19 g, 0.088 mol) was added. An orange precipitate was obtained immediately and the reaction mixture was stirred for 1 hr at room temperature. The product was separated by filtration, then washed with dimethylformamide (DMF), with water, and finally with ethanol. The final product was dried under vacuum (yield: 95%). Synthesis of ds-bis(alkylamine)bis(carboxylato)platiaum(II) complexes (cis[(RNH,),PtX,]). cis-bis(isopropylamine)diiodoplatinum(II) (1.1336 g, 3.92 mmol) was dissolved in 250 ml chloroform at 45°C. To this, silver neopentanoate (0.818 g, 2 mmol), prepared in situ by reacting sodium neopentanoate with silver nitrate, was added. A clear yellow solution was formed within 5 min, and silver iodide started to precipitate as a yellow solid. The reaction mixture was stirred at room temperature for 20 hr (protected from light). Silver iodide was separated by filtration, and the filtrate was evaporated to dryness under reduced pressure at room temperature. A light yellow solid was obtained which was recrystallized from acetone. The final product (complex 3) was dried in vacua (yield: 70%). All other platinum complexes listed in Table 1 (complexes 1,2,4-18) were prepared in a similar manner. Liposomal-Platinum
Preparations
L-Ft preparations were prepared as reported for other platinum complexes [ 181. Briefly, chloroform solutions of DMFC and DMPG at a 7 : 3 molar ratio were mixed with the platinum complex at a drug:lipid weight ratio of 1 : 15. The chloroform was evaporated in a rotary evaporator, leaving a dry film containing the lipids and the platinum complex. Multilamellar liposomes containing the platinum complex were formed by adding 1 ml of 0.9% NaCl aqueous solution for each milligram of platinum complex to the dry lipid film and mildly shaking the solution by hand for a few minutes. To measure the entrapment efficiency, the liposome suspension was centrifuged at 30,008 g for 45 min, and the amount of the platinum complex in the supematant was determined. Quantitation of the platinum complexes was measured by ultraviolet (UV) spectrophotometry at a wavelength of 216 nm. The entrapment efficiency was calculated by the following formula: entrapment efficiency = [(total Pt complex - Pt complex in supernatant)/total Pt complex added] x 100. L-Pt complex vesicles were then sized in a Coulter counter (Coulter Electronics, Hialeah, FL). All preparations were regularly checked for the presence of free drug crystals in the pellet by optic microscopy. Biological Studies In vivo antitumor activity was screened against L1210/0 leukemia. First, L1210/0 leukemia cells were grown in vivo in the peritoneal cavity of BDFl mice and transplanted weekly. Then, groups of six B6D2Fl mice weighing 20-25 g each were inoculated with lo6 L1210/0 cells (i.p.) on day 0. Treatment was started on day 1 using the same route used for tumor inoculation. Doses of 12.5, 25, and 50 mg/kg were used for all L-Pt preparations since 12.5-50 mg/kg is the active dose range for other lipophilic cisplatin derivatives used in our previous studies [ 181. Toxic deaths were defined as those deaths occurring during the first 10 days in animals who showed no ascites or liver involvement by tumor at autopsy.
108
S. Al-Baker et al.
25. S. Neidle, I. M. Ismail, and P. J. Sadler, J. Inorg. Biochem. 13, 205 (1980). 26. T. G. Appleton, R. D. Berry, C. A. Davis, J. R. Hall, and H. A. Kimlin, Inorg. Chem. 23, 3514 (1984). 27. T. G. Appleton, J. R. Hall, and S. F. Ralph, Znorg. Chem. 24, 4685 (1985). 28. S. Al-Baker, R. Perez-Soler, and A. R. Khokhar, to be submitted for publication. 29. R. Perez-Soler, G. Lopez-Berestein, J. Lautersztain, S. Al-Baker, K. Francis, D. Macias-Kiger, M. N. Raber, and A. R. Khokhar, Cuncer Res. 50, 4254 (1990). 30. R. Perez-Soler, A. R. Khokhar, J. Lautersztain, S. Al-Baker, K. Francis, D. MaciasKiger, and G. Lopez-Berestein, J. Liposome Research l(4), 437 (1990). Received November IS, 1991; accepted January 9, 1992
ABSTRACT A series of highly lipophilic platinum(H) complexes of the type c~~-[(RNH~)~P~X,] have been synthesized, where R = ethyl, propyl, isopropyl, butyl, cyclopropyl, cyclopentyl, or neopentyl and X = either long-chain carboxylate, such as decanoate, (ClO), laurate (Cl2), myristate (Cl4), heptadecanoate (Cl7), stearate (Cl@, nonadecanoate (Cl9), or 2,2,3,3-tetramethylcyclopropylcarboxylate, or branched-chain carboxylate, such as neopentanoate, neohexanoate, neoheptanoate, neononanoate, or neodecanoate. These complexes have been characterized by elemental analysis, IR, and “C and ‘9sRt NMR spectroscopic techniques. The platinum complexes were entrapped in multilamellai vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) at a 7 : 3 molar ratio and tested for antitumor activity. The entrapment efficiency of liposomal platinum (L-F?) complexes ranged from 60 to 100%. The percentage of T/C obtained after a single i.p. injection of the optimal dose of L-Pt complexes tested against Ll210 leukemia ranged from 90 to 125%. These L-Pt preparations did not show significant antitumor activity in mice.
INTRODUCTION Cisplatin is one of the most effective antineoplastic drugs [l-3] against cancers of the head and neck, testes, ovaries, and bladder. The usefulness of cisplatin is, however, compromised by its propensity to cause several severe dose limiting toxicities, including nephrotoxicity, neurotoxicity, and ototoxicity [4-71. In recent years, there has been a growing interest in developing. new platinum complexes with greater antitumor activity, a broader spectrum of activity, and non-cross resistance to cisplatin. However, the development of some promising analogues has been
Address reprint requests to: Dr. Abdul R. Rhokhar, DeparWntofiMedical Oncology (Box 52), The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Journal of Inorganic Biochemistry, 47,99- 108 (1992) 0 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010
99 0162-0134/92/$5.00
100 S. Al-Baker et al.
hampered by their low solubility , poor stability, and difficult formulation, all of which decrease the potential of such analogues for clinical use [8]. An alternative approach to modifying the therapeutic index of cisplatin analogues may be the use of a drug carrier. Liposomes are particularly attractive as such because they are easy to prepare and are biodegradable [9, lo]. In general, studies with antineoplastic agents entrapped in liposomes have shown that liposomal entrapment of cytostatic drugs may result in preserved antitumor activity and reduced toxicity [ 111. However, due to its low lipophilicity, cisplatin is not a suitable drug for liposome formulation. All reported liposomal cisplatin preparations have a very low entrapment efficiency and high rate of drug leakage [ 12- 141. The objective of this study was to synthesize a series of highly lipid-soluble platinum(I1) complexes designed for liposome entrapment [ 15- 171. Such complexes should have a high entrapment efficiency and good antitumor activity [16, 18-201. In this paper we describe the chemical development of liposomal platinum and the biological activity of a series of highly lipid-soluble platinum(H) complexes containing a monodentate amine as the inert ligand and either long-chain or branched-chain aliphatic carboxylate as the leaving ligand. EXPERIMENTAL Materials and Methods All alkylamine and straight-chain carboxylic acids were purchased from Aldrich Chemical Co. (Milwaukee, WI). Neopentanoic acid, neohexanoic acid, neoheptanoic acid, neononanoic acid, and neodecanoic acid were obtained from Exxon Chemical Co. (Houston, TX). K,PtCl, was purchased from Aesar (Seabrook, NH). All chemicals obtained from commercial sources were used as received. Microanalysis of platinum complexes was performed by Robertson Laboratory Inc. (Madison, NJ). Thin-layer chromatography (TLC) was performed on precoated silica gel plates in a solvent system consisting of methanol-ethyl acetate (1 : 9). The plates were visualized as yellow spots after exposure to iodine vapor. Infrared spectra (4000-250 cm-‘) were recorded as a KBr pellet on a Beckman Microlab 250MX infrared spectrophotometer. All NMR spectra (i3C{‘H} and ‘95Pt(‘H}) were recorded on an IBM NR/200 AF NMR spectrometer. The ‘95Pt{‘H} spectra (43.055 MHz) were collected by using a 10 s (90) pulse and a 0.012 s acquisition time with a spectral width of 166 KHz (4 k data point). 19% chemical shifts were collected in CHCl, solution (about 30 n&I) for 10 hr at room temperature and were referenced to a D,O solution of Na,PtCl, (0.0 ppm), which was contained in a 5-mm NMR tube (0.5 g/O.5 ml D,O), which in turn was inserted into the lo-mm NMR tube. 13C{‘H} NMR spectra were measured in CDCl, solution, with the carbon-13 chemical shifts being referenced to the CDCl, peak at 77 ppm. The chromatographically pure (as determined by TLC) DMPC and DMPG used in this study were obtained from Avanti Polar Lipids (Birmingham, AL). L1210/0 leukemia cells were obtained from the DCT Tumor Repository (National Cancer Institute, Frederick, MD). BDFl mice weighing 18-20 g were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Synthesis of Platinum Complexes Synthesis of cis-bis(alkylaminc)diiodoplatlnum(II) complexes (cis[(RNH,),PtI,]). All platinum complexes of the type ci.~-[(RlW~)~Pt&] were synthesized according to an earlier reported procedure [21]. The preparation of
SYNTHESIS AND BIOLOGICAL STUDIES
101
cis-bis(isopropylamine)diiodoplatinum(II) is given here as an example. An aqueous solution of potassium iodide (53.0 g, 0.32 mol in 75 ml H,O) was added to a filtered aqueous solution of K,FtCl, (16.6 g, 0.04 mol in 250 ml H,O) and the reaction mixture was stirred for 10 min at room temperature; to this, isopropylamine (5.19 g, 0.088 mol) was added. An orange precipitate was obtained immediately and the reaction mixture was stirred for 1 hr at room temperature. The product was separated by filtration, then washed with dimethylformamide (DMF), with water, and finally with ethanol. The final product was dried under vacuum (yield: 95%). Synthesis of ds-bis(alkylamine)bis(carboxylato)platiaum(II) complexes (cis[(RNH,),PtX,]). cis-bis(isopropylamine)diiodoplatinum(II) (1.1336 g, 3.92 mmol) was dissolved in 250 ml chloroform at 45°C. To this, silver neopentanoate (0.818 g, 2 mmol), prepared in situ by reacting sodium neopentanoate with silver nitrate, was added. A clear yellow solution was formed within 5 min, and silver iodide started to precipitate as a yellow solid. The reaction mixture was stirred at room temperature for 20 hr (protected from light). Silver iodide was separated by filtration, and the filtrate was evaporated to dryness under reduced pressure at room temperature. A light yellow solid was obtained which was recrystallized from acetone. The final product (complex 3) was dried in vacua (yield: 70%). All other platinum complexes listed in Table 1 (complexes 1,2,4-18) were prepared in a similar manner. Liposomal-Platinum
Preparations
L-Ft preparations were prepared as reported for other platinum complexes [ 181. Briefly, chloroform solutions of DMFC and DMPG at a 7 : 3 molar ratio were mixed with the platinum complex at a drug:lipid weight ratio of 1 : 15. The chloroform was evaporated in a rotary evaporator, leaving a dry film containing the lipids and the platinum complex. Multilamellar liposomes containing the platinum complex were formed by adding 1 ml of 0.9% NaCl aqueous solution for each milligram of platinum complex to the dry lipid film and mildly shaking the solution by hand for a few minutes. To measure the entrapment efficiency, the liposome suspension was centrifuged at 30,008 g for 45 min, and the amount of the platinum complex in the supematant was determined. Quantitation of the platinum complexes was measured by ultraviolet (UV) spectrophotometry at a wavelength of 216 nm. The entrapment efficiency was calculated by the following formula: entrapment efficiency = [(total Pt complex - Pt complex in supernatant)/total Pt complex added] x 100. L-Pt complex vesicles were then sized in a Coulter counter (Coulter Electronics, Hialeah, FL). All preparations were regularly checked for the presence of free drug crystals in the pellet by optic microscopy. Biological Studies In vivo antitumor activity was screened against L1210/0 leukemia. First, L1210/0 leukemia cells were grown in vivo in the peritoneal cavity of BDFl mice and transplanted weekly. Then, groups of six B6D2Fl mice weighing 20-25 g each were inoculated with lo6 L1210/0 cells (i.p.) on day 0. Treatment was started on day 1 using the same route used for tumor inoculation. Doses of 12.5, 25, and 50 mg/kg were used for all L-Pt preparations since 12.5-50 mg/kg is the active dose range for other lipophilic cisplatin derivatives used in our previous studies [ 181. Toxic deaths were defined as those deaths occurring during the first 10 days in animals who showed no ascites or liver involvement by tumor at autopsy.
108
S. Al-Baker et al.
25. S. Neidle, I. M. Ismail, and P. J. Sadler, J. Inorg. Biochem. 13, 205 (1980). 26. T. G. Appleton, R. D. Berry, C. A. Davis, J. R. Hall, and H. A. Kimlin, Inorg. Chem. 23, 3514 (1984). 27. T. G. Appleton, J. R. Hall, and S. F. Ralph, Znorg. Chem. 24, 4685 (1985). 28. S. Al-Baker, R. Perez-Soler, and A. R. Khokhar, to be submitted for publication. 29. R. Perez-Soler, G. Lopez-Berestein, J. Lautersztain, S. Al-Baker, K. Francis, D. Macias-Kiger, M. N. Raber, and A. R. Khokhar, Cuncer Res. 50, 4254 (1990). 30. R. Perez-Soler, A. R. Khokhar, J. Lautersztain, S. Al-Baker, K. Francis, D. MaciasKiger, and G. Lopez-Berestein, J. Liposome Research l(4), 437 (1990). Received November IS, 1991; accepted January 9, 1992