Double-prodrugs of L-cysteine Differential protection against by pharmphresh28


									                                                                                                     J BIOCHEM MOLECULAR TOXICOLOGY
                                                                                                                Volume 16, Number 5, 2002

Double-Prodrugs of L-Cysteine: Differential Protection
Against Acetaminophen-Induced Hepatotoxicity in Mice
Daune L. Crankshaw,1 Lorelle I. Berkeley,1 Jonathan F. Cohen,2 Frances N. Shirota,1
and Herbert T. Nagasawa1,2
    Medical Research Laboratories, DVA Medical Center, Minneapolis, MN 55417, USA; E-mail:
    Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA

Received 2 June 2002; accepted 4 August 2002

       ABSTRACT: A series of double-prodrugs of L-cysteine,                   sulfhydryl or on the amino group of L-cysteine, or on
       designed to release L-cysteine in vivo and stimu-                      the secondary amino group of MTCA, appears to be a
       late the biosynthesis of glutathione (GSH), were                       poor “pro-moiety,” since these carbethoxylated double-
       synthesized. To evaluate the hepatoprotective ef-                      prodrugs of L-cysteine did not protect mice from ACP-
       fectiveness of these double-prodrugs, male Swiss-                      induced hepatotoxicity. C 2002 Wiley Periodicals, Inc.
       Webster mice were administered acetaminophen (ACP)                     J Biochem Mol Toxicol 16:235–244, 2002; Published on-
       (2.45 mmol/kg (360 mg/kg), intraperitoneally (i.p.)).                  line in Wiley InterScience (
       Prodrug (2.50 mmol/kg, i.p. or 1.25 mmol/kg, i.p., de-                 DOI 10.1002/jbt.10044
       pending on the protocol) was administered 1 h be-
       fore ACP as a priming dose. A supplementary dose                       KEYWORDS: L-Cysteine; Hepatotoxicity;         Acetamino-
       of prodrug (2.5 mmol/kg, i.p. or 1.25 mmol/kg, i.p.                    phen; Glutathione; Prodrugs
       depending on the protocol) was administered 0.5 h after
       ACP. The plasma alanine amino transferase (ALT) val-
       ues, 24 h after ACP administration were transformed to
       logs and the 95% and 99% confidence intervals of the log
       values were plotted and compared for each group. Hep-
       atoprotection was assessed by the degree of attenuation                L-Cysteine is the rate-limiting sulfhydryl amino

       of plasma ALT levels. With these multiple dose sched-             acid required for the first step in the two-step biosyn-
       ules, the use of 2% carboxymethylcellulose as vehicle             thesis of glutathione (GSH) [1]. Prodrugs of cysteine
       for the prodrugs was found to be detrimental; therefore,          are, therefore, also GSH precursors, since the cysteine
       the prodrugs were dissolved in dilute aqueous base                liberated in vivo from their prodrug forms stimulates
       and the pH adjusted for administration. When a prim-              GSH biosynthesis and is incorporated into this tripep-
       ing dose was given 1 h before ACP followed by a sup-              tide. It is now well established that L-cysteine prodrugs
       plementary dose 0.5 h after ACP, only N,S-bis-acetyl-L-           [2–7], or prodrugs of GSH itself [8,9], are effective hep-
       cysteine, where both the sulfhydryl and amino groups
                                                                         atoprotective agents that can greatly attenuate the liver
       of L-cysteine were functionalized with the acetyl group,
                                                                         toxicity elicited in rodents by high doses of toxic xenobi-
       was found to be effective in protecting mice against
       the hepatotoxic effects of ACP. This suggests that these          otics such as acetaminophen (ACP). The hepatotoxicity
       acetyl groups were rapidly hydrolyzed in vivo to lib-             of ACP is generally believed to be a consequence of
       erate L-cysteine. In contrast, N-acetylation of 2(R,S)-           the formation of a highly reactive metabolic oxidation
       methylthiazolidine-4(R)-carboxylic acid (MTCA) and                product of ACP, viz., N-acetyl- p-benzoquinoneimine
       its 2-n-propyl analog (PTCA), or N-acetylation of                 (NAPQI), which binds ubiquitously to tissue macro-
       2-oxothiazolidine-4-carboxylic acid (OTCA), reduced               molecules, thereby triggering a cascade of inflamma-
       the hepatoprotective effects relative to the parent               tory events eventually leading to necrosis [10]. GSH
       MTCA, PTCA, and OTCA, indicating that the re-                     effectively sequesters this reactive metabolite of ACP
       lease of L-cysteine in vivo from these N-acetylated               and protects the liver from toxicity [11].
       thiazolidine prodrugs was metabolically unfavorable.
                                                                              L-Thiazolidine-4-carboxylic acid (TCA) and its 2-
       The carbethoxy group, whether functionalized on the
                                                                         oxo- (OTCA) and 2-alkyl-substituted (MTCA, PTCA)
                                                                         derivatives (Chart 1) serve as prodrugs of L-cysteine.
    Correspondence to: Herbert T. Nagasawa.
                                                                         Bioactivation of TCA requires the intervention of
    Contract Grant Sponsor: Department of Veterans Affairs.              the hepatic mitochondrial enzyme, proline oxidase
c 2002 Wiley Periodicals, Inc.                                           [5,12,13], to give 2 -thiazoline-4-carboxylic acid, which

236    CRANKSHAW ET AL.                                                                        Volume 16, Number 5, 2002

                                                             CHART 2.

spontaneously hydrolyzes nonenzymatically to N-              These N-acylated thiazolidine-4-carboxylic acids can,
formyl-L-cysteine (structures not shown). The latter is      therefore, be considered to be double-prodrugs of L-
further hydrolyzed by a cytosolic enzyme to formate          cysteine, inasmuch as two sequential steps (enzy-
and L-cysteine as TCA can replace L-cysteine as a nu-        matic/nonenzymatic or double enzymatic) are re-
tritional component in rats [14]. Similarly, the enzyme      quired for the liberation of this sulfhydryl amino acid in
5-oxoprolinase is required to open the thiazolidine ring     vivo.
of OTCA to give L-cysteine, this process requiring 1 mol          N-Acetyl-L-cysteine (N-AcC, Chart 3), a well-
of ATP [15].                                                 known cysteine prodrug (2), provides the free
     In contrast, MTCA and PTCA are hydrolytically           sulfhydryl amino acid to cells following its rapid en-
labile, and, under physiological conditions of temper-       zymatic deacetylation in the liver [16]. N-AcC is the
ature and pH, undergo a nonenzymatic, hydrolytic             only FDA-approved antidote for the treatment of hep-
ring opening dissociation to liberate L-cysteine and         atotoxic drug overdoses, such as with ACP, a widely
acetaldehyde or propionaldehyde [4,5] (see Eq. (1)).         used (and misused) analgesic agent. While effective
A “demand-pull” dissociation is operational in vivo          clinically, N-AcC has an unpleasant taste and is known
when either the aldehyde or cysteine is removed by           to have poor bioavailability (9% in humans, hence
metabolic action. In vitro, the presence of L-cysteine       large doses are required) [17] and is extensively co-
in the medium suppresses this dissociation [5], while        valently bound to plasma proteins (>50%) because of
volatilization of the aldehyde (RCHO) or removal of          the presence of the free, reactive sulfhydryl group [10].
the cysteine, e.g., by air oxidation, promotes dissocia-     Except for the thiazolidine-4-carboxylic acids, where
tion. Indeed, the possibility of hydrolytic dissociation     this SH group of L-cysteine is latentiated, i.e., chemi-
of MTCA and PTCA mandates that, for chemical or              cally protected until uncovered either by metabolic ac-
biological                                                   tion or by nonenzymatic hydrolysis [5–7], sulfhydryl-
                                                             protected L-cysteine derivatives have not been studied
                                                             as protective agents against ACP-induced hepatotoxi-
                                                             city [10]. Accordingly, N,S-di-acylated derivatives of L-
                                                             cysteine were synthesized (Chart 3) as a second series of
                                                             sulfhydryl-protected, double-prodrugs for comparison
                                                             with the thiazolidine double-prodrugs listed in Chart 2.
                                                             N-Carbethoxy-L-cysteine (N-CbeC), although itself not
                                                             a double-prodrug, was also prepared for comparison
studies, H2 O solutions of these compounds be prepared
fresh, just prior to use. It follows that, unlike TCA or
OTCA, aqueous diet preparations containing these pro-
drugs cannot be preformulated, placing severe restric-
tions on any animal feeding experiments that may be
contemplated with MTCA and/or PTCA.
    However, by acylating the secondary amino (N H)
function of the thiazolidine, e.g., with an acetyl (Ac) or
a carbethoxy (Cbe) group, MTCA is converted to sta-
ble derivatives (Chart 2) that cannot hydrolyze spon-
taneously and now require enzymatic deacylation of
the N-acetyl or the N-carbethoxy group to convert
them back to MTCA. Similarly, enzymatic deacetyla-
tion of the N-acetylated OTCA (N-AcOTCA, Chart 2)
would result in reversion of this compound to OTCA.          CHART 3.
Volume 16, Number 5, 2002                                                                 DOUBLE-PRODRUGS OF L-CYSTEINE            237

TABLE 1. Prodrug/ACP Administration Protocols Using                       literature methods. The syntheses of the other double-
Various Vehicles                                                          prodrugs of L-cysteine are described later.
                                            Time (min)

Protocol (Vehicle)         −60                  0             +30             Physicochemical/Analytical Methods
1 (CMC)              Prodrug (2.50)         ACP (2.45)   Prodrug (1.25)       1
2 (CMC)              Prodrug (2.50)         ACP (2.45)         –
                                                                               H NMR spectra were recorded at ambient tem-
3 (pH adjusted       Prodrug (2.50)         ACP (2.45)   Prodrug (1.25)   perature on Varian Unity 200 and 300 MHz NMR spec-
   saline)                                                                trometers equipped with four nuclear probes. Chem-
4 (pH adjusted       Prodrug (1.25)         ACP (2.45)   Prodrug (2.50)   ical shifts are reported as δ values (ppm). Melting
   saline)                                                                points were taken on a Fisher–Johns hot-stage melting
 All doses ( ) are in mmol/kg throughout.                                 point apparatus and are uncorrected. For TLC anal-
                                                                          yses, Analtech silica gel GF plates were used. The
with N-AcC, the recognized standard for all cysteine                      plates were visualized by spraying with ninhydrin
prodrugs.                                                                 or CeSO4 –H2 SO4 solution and heating. Column chro-
     These two series of L-cysteine double-prodrugs                       matography was carried out using columns packed
(Charts 2 and 3) were evaluated for their hepatopro-                      with Kieselgel 60 silica gel (230–400 mesh, EM Science).
tective properties in mice treated with 2.45 mmol/kg of                   When the reactants or desired products contained a free
ACP, a dose somewhat lower than that used previously                      sulfhydryl group, the reactions were conducted under
[18] in order to allow for 24 h survival of the animals.                  a N2 atmosphere.
Because two sequential steps are necessary for cysteine
release by these double-prodrugs, and this was anti-
                                                                              Synthetic Procedures
cipated to span a finite time in vivo, a protocol using
divided doses of the hepatoprotective agent adminis-                          N-Acetyl-2(R,S)-methylthiazolidine-4(R)-
tered pre- and post-ACP was implemented (Table 1).                            carboxylic Acid (N-AcMTCA)
Also, since most of the double-prodrugs to be tested had
                                                                               This compound was prepared by acetylation of
limited aqueous solubility, 2% carboxymethyl cellulose
                                                                          MTCA (1.47 g, 10.0 mmol) dissolved in 25 mL of 6%
(CMC) was used initially as a suspending agent. How-
                                                                          aqueous Na2 CO3 (cooled on an ice bath) by dropwise
ever, we found, to our chagrin, that the use of 2% CMC
                                                                          addition of 2.04 g (20 mmol) of acetic anhydride over
for multiple dose schedules was detrimental (vide infra);
                                                                          2 min with stirring. After 1 h, the product was isolated
hence, the prodrugs were dissolved in dilute aqueous
                                                                          by acidification of the reaction mixture, saturating it
base and adjusted to pH 6–8 for administration.
                                                                          with NaCl and extracting twice with 50 mL of EtOAc.
     This structure–activity study demonstrated for the
                                                                          The combined EtOAc extracts were washed with satu-
first time that there is substantial differential efficacy
                                                                          rated NaCl, dried (Na2 SO4 ), and the solvent evaporated
among the thiazolidine double-prodrugs, as well as
                                                                          in vacuo to give a solid product, mp 164–165◦ C. Recrys-
among the open-chain N,S-diacylated double-prodrugs
                                                                          tallization from EtOAc gave N-AcMTCA as colorless
of L-cysteine, in protecting mice against ACP-induced
                                                                          crystals, mp 166–167◦ C (subl > 150◦ C) (71% yield). IR
hepatotoxicity. Indeed, the stringent functional group
                                                                          (KBr; cm−1 ) ν = 1730 (CO), 1599 (amide I). The NMR
(pro-moiety) requirement exhibited for maximal effi-
                                                                          spectrum indicated that the product was approximately
cacy in vivo suggests that differential bioactivation
                                                                          a 55:45 mixture of C-2 epimers, which, however, did not
mechanisms must play major roles in the ultimate re-
                                                                          separate on TLC, using n-BuOH/HOAc/H2 O(100:22:5)
lease of L-cysteine from these double-prodrugs.
                                                                          (bright yellow on exposure to I2 ). 1 H NMR (DMSO) δ
                                                                          1.42 (d, J = 6.2 Hz) and 1.53 (d, J = 6.4 Hz) (3H, CH3 ),
     MATERIALS AND METHODS                                                1.98 (s) and 2.09 (s) (3H, CH3 CO), 3.18–3.84 (m, 2H,
                                                                          CH2 ), 4.64 (t, 1H, CH), 5.27 (q, J = 6.2 Hz) and 5.35 (q,
     Chemicals                                                            J = 6.4 Hz) (1H, CH), 13 C NMR (DMSO) δ 22.3, 22.9,
                                                                          23.5, 24.7, 31.9, 33.7, 59.1, 60.2, 62.8, 63.3, 167.8, 168.6,
     TCA, N-AcC (Aldrich, Milwaukee, WI), and OTCA                        172.3, 172.6. Anal calcd for C7 H11 NO3 S: C, 44.43; H, 5.86;
(Chemical Dynamics, South Plaines, NJ) were commer-                       N, 7.40. Found: C, 44.60; H, 5.83; N, 7.43.
cial products used as received, while MTCA, PTCA
[5], N-AcOTCA, mp 155–158◦ C, [ ]22 D − 139.8◦ (c 1.9,
acetone/H2 O, 1:1) reported mp 153–154◦ C, [ ]20 D −
140.5◦ (c 1.8, acetone/H2 O, 1:1 [19]), and N,S-bis-
acetyl-L-cysteine (N,S-bis-AcC), mp 121.5–123.5◦ C (re-                       An aqueous solution of N-AcC (1.01 g, 6.18 mmol)
ported mp 120–121◦ C) [20], were prepared according to                    in an ice bath was stirred under N2 . To this was added
238      CRANKSHAW ET AL.                                                                              Volume 16, Number 5, 2002

a solution of ethyl chloroformate (0.60 mL, 0.68 g, 6.3           N2 . To this was added a solution of tris carboxyethyl
mmol) in ice-cooled THF, followed by 13 mL of an                  phosphine HCl [22] (532 mg, 1.86 mmol) in 50 mL of
ice-cooled aqueous solution of Na2 CO3 (663 mg, 6.26              N2 -purged H2 O. After 45 min, analysis of an aliquot
mmol) over 5 min. After ca. 10 min, an additional por-            showed no starting material by TLC and a spot with
tion of ethyl chloroformate (0.08 mL) was added for a to-         positive test for SH. The pH of the solution was raised
tal of 0.68 mL (0.77 g, 7.1 mmol). The reaction was stirred       from 2.8 to 10.5 with ∼13 mL of 1 N aq NaOH. Acetic
for an additional 30 min and the solvents were removed            anhydride (0.80 mL, 0.86 g, 8.4 mmol) was added over
in vacuo. The product was dissolved in ca. 50 mL of H2 O          1 min, after which time the pH of the solution was
(pH ∼8.0), and the solution was washed with 3 × 20                4.6. This was raised to 8.2 with additional 12 mL of
mL portions of EtOAc and then acidified to pH 3.0                  1 N aq NaOH. TLC analysis after ca. 30 min showed
with 1 N HCl. The aqueous solution was extracted with             that no products with free thiol remained. The solution
3 × 20 mL portions of EtOAc. The combined EtOAc                   was acidified to pH 2.7 with 6 N aq HCl and extracted
extracts were dried and evaporated to dryness in vacuo            with 3 × 70 mL portions of EtOAc. The combined
to give a viscous oil. This was lyophilized to give 940 mg        ETOAc extracts were dried (Na2 SO4 ) and concentrated
(64.7% yield) of S-Cbe-N-AcC as a viscous, colorless              to give 667 mg of oil. This was purified by column chro-
oil which solidified on standing, mp 68.5–71.0◦ C. 1 H             matography, elution being done with EtOAc/hexane
NMR (CDCl3 ) δ 1.30 (t, J = 7.1 Hz, 3H, CH3 CH2 ), 2.07           (1:1) to give S-Ac-N-CbeC as a colorless oil (334 mg,
(s, 3H, CH3 CO), 3.29 (dd, J = 6.8, 14.5 Hz) and 3.47             53.4% overall yield) which solidified on standing, mp
(dd, J = 4.2, 14.5 Hz, 2H, CH2 S), 4.28 (q, J = 7.1 Hz,           86–88◦ C. 1 H NMR (CDCI3 ) δ 1.25 (t, J = 7.1 Hz, 3H,
2H, CH2 CH3 ), 4.81 (m, 1H, CH), 6.92 (d, J = 7.3 Hz,             CH3 CH2 ), 2.37 (s, 3H, CH3 CO), 3.33 (dd, J = 7.0, 14.3
NH). 13 C NMR (CDCl3 ) δ 14.3, 22.8, 32.2, 52.9, 64.5,            Hz) and 3.47 (dd, J = 4.5, 14.2 Hz, 2H, CH2 S), 4.14 (q,
171.0, 172.1, 172.3. Anal calcd for C8 H13 NO5 S: C, 40.84;       J = 7.1 Hz, 2H, CH2 CH3 ), 4.6 (m, 1H, CH), 5.56 (d,
H, 5.57; N, 5.95. Found: C, 40.79; H, 5.47; N, 6.04.              J = 7.7 Hz, NH), 9.48 (br s, 1H, OH). 13 C NMR (CDCI3 )
                                                                  δ 14.5, 30.6, 30.9, 53.7, 61.8, 156.5, 174.2, 195.8. Anal calcd
                                                                  for C8 H13 NO5 S: C, 40.84; H, 5.57; N, 5.95. Found: C,
      N,S-bis-Carbethoxy-L-cysteine (N,S-bis-CbeC)
                                                                  40.94; H, 5.53; N, 5.96.
     L-Cysteine (3.03 g, 25.0 mmol) was stirred under N2
with cooling (ice bath) in 50 mL of H2 O. Ethyl chloro-
formate (9.60 mL, 10.9 g, 100 mmol) was added over                    N-Carbethoxy-L-cysteine (N-CbeC)
2 min, followed by NaOH (4.40 g, 110 mmol) in 50 mL                    N,N-bis-Carbethoxy-L-cystine (505 mg, 1.31 mmol)
of H2 O over 15 min. The solution was stirred for 30 min          in 50 mL of H2 O was reduced as aforementioned with
(the pH was 8.5), then extracted with 3 × 50 mL por-              500 mg (1.74 mmol) of the phosphine reagent, and the
tions of EtOAc. The aqueous layer (now pH 7.5) was                reaction solution (pH 2.1) was extracted with 3 × 50 mL
acidified to pH 2 with 10 mL of 6 N aqueous HCl and                portions of N2 -purged EtOAc. The combined EtOAc ex-
extracted with 3 × 50 mL portions of EtOAc. The or-               tracts were dried and concentrated to give 495 mg of a
ganic extracts were dried and concentrated to give 7.94           viscous oil. This was purified by chromatography, elu-
g of viscous oil. The oil was purified by column chro-             tion being done with EtOAc/hexane (1:1) and EtOAc to
matography, elution being done with EtOAc/hexane                  give N-CbeC as a colorless oil (262 mg, 52% yield). The
(1:1) to give N,S-bis-CbeC as a viscous oil (4.87 g, 73.6%        column was maintained under N2 and the chromatog-
yield). This product was subjected to lyophilization to           raphy solvents were all flushed with N2 before use. 1 H
remove traces of residual EtOAc. 1 H NMR (CDCl3 ) δ               NMR (CDCI3 ) δ 1.26 (t, J = 7.1 Hz, 3H, CH3 CH2 ), 3.0
1.30 (m, 6H, CH3 CH2 ), 3.27 (dd, J = 6.8, 14.4 Hz) and           (m, 2H, CH2 S), 4.14 (q, J = 7.1 Hz, 2H, CH2 CH3 ), 4.7
3.46 (dd, J = 4.6, 14.4 Hz, 2H, CH2 S), 4.13 and 4.28 (q,         (m, 1H, CH), 5.59 (d, J = 7.8 Hz, NH), 9.53 (br s, 1H,
J = 7.1 Hz, 4H, CH2 CH3 ), 4.60 (m, 1H, CH), 5.66 (d,             OH). 13 C NMR (CDCI3 ) δ 14.5, 27.0, 54.9, 61.8, 156.3,
J = 7.8 Hz, NH), 6.59 (br s, 1H, OH). 13 C NMR (CDCl3 )           174.3. Anal calcd for C6 H11 NO4 S: C, 37.30; H, 5.74; N,
δ 14.2, 14.4, 32.7, 53.8, 61.7, 64.3, 156.4, 170.6, 174.2. Anal   7.25. Found: C, 37.45; H, 5.53; N, 7.14.
calcd for C9 H15 NO6 S: C, 40.75; H, 5.70; N, 5.28. Found:
C, 40.62; H, 5.51; N, 5.26.
                                                                      Male, Swiss-Webster N4D mice (Harlan Sprague-
                                                                  Dawley, Indianapolis, IN) weighing 25–34 g were
    A solution of N,N -bis-carbethoxy-L-cystine, pre-             housed and cared for in conventional cages accord-
pared according to a literature procedure [21] (513               ing to the guidelines of our Institutional Animal Care
mg, 1.33 mmol), in 50 mL of H2 O was stirred under                and Use Committee (IACUC), with a 12:12 light/dark
Volume 16, Number 5, 2002                                                     DOUBLE-PRODRUGS OF L-CYSTEINE         239

photoperiod (lights on at 0700) in a temperature con-          to be toxic, except for those mice fully protected by the
trolled (21–22◦ C) room. Water and food (Harlan Rodent         most effective prodrugs (vide infra). The animals were
Chow) were allowed ad libitum except as noted later. All       allowed access to food following the last drug adminis-
of the following protocols were IACUC approved.                tration, observed for 6 h post-ACP, and, when deemed
                                                               necessary, were sacrificed on imminent death, or blood
                                                               was drawn shortly following death, for measurement of
                                                               alanine aminotransferase (ALT) levels (vide infra). ALT
    Drug Administration Protocols
                                                               levels were not determined for mice that died between
     Following an acclimatization period of 5–12 days,         6–24 h; however, the numbers of such deaths within a
the mice were fasted overnight (16 h) before drug ad-          group were recorded.
ministration by intraperitoneal (i.p.) injection. The de-
tails of the various drug dose protocols are listed in
Table 1. In order to manage the large numbers of pro-              Plasma ALT Levels
drugs to be tested and to minimize any fluctuations in
                                                                    Twenty-four hours following the administra-
animal responses on a given day, the animals were di-
                                                               tion of ACP, the mice were anesthetized with Ke-
vided into groups of 3 or 4 for each protocol, and the
                                                               tamine/Xylazine (3:1), and blood was collected by car-
experiment repeated several days later to reach the n re-
                                                               diac puncture. Samples were placed in heparinized
quired for statistical treatment. When excessive toxicity
                                                               Wintrobe tubes, centrifuged in a Sorvall RC-3B refrig-
was evident—as manifested by unexpected premature
                                                               erated centrifuge for 10 min (H2000 rotor, 1500 rpm,
deaths—the experiments with those prodrugs were ter-
                                                               5◦ C), and the plasma layer collected and kept refrig-
minated as required by our IACUC (Protocols 1 and 2).
                                                               erated until ALT activities (U/L) were determined (in
     The prodrugs were prepared as a suspension in ei-
                                                               duplicate) by kinetic assay (30◦ C), using Sigma Infin-
ther 2% carboxymethyl cellulose (CMC) or as a solution
                                                               ity ALT Reagent and a Beckman Model DU-70 spec-
in a pH adjusted aqueous medium (pH 6–8) as follows.
                                                               trophotometer. Plasma samples that required storage
Crystalline samples of the prodrugs were weighed and
                                                               longer than overnight were kept in an ultra-low freezer
ground to a fine powder in a quartz mortar. (This ini-
                                                               at −80◦ C. The ALT activities of samples stored at this
tial grinding was not necessary for prodrugs that were
                                                               temperature have been reported to be stable for at least
oils or gums.) To this was added 0.25 mL of 2% CMC
                                                               a week [23].
and a slurry was produced by grinding to ensure a fine
suspension. An additional 0.25 mL of suspending agent
was added, followed by further grinding. The suspen-
                                                                   Data Analysis
sion or slurry was brought to the final volume with 2%
CMC.                                                                The ACP dose selected for this animal model
     All of the prodrugs tested in this series could be sol-   (2.45 mmol/kg), although reduced from previous stud-
ubilized in dilute aqueous base. Accordingly, depend-          ies (see Drug Administration Protocols), induced high
ing on the alkaline stability of the compound, 0.25 mL         plasma ALT levels within 24 h. To determine whether
of either 10 N or 1 N NaOH was added to the finely              the prodrugs protected against ACP-induced hepato-
ground, preweighed sample. A slurry was produced by            toxicity, the averaged plasma ALT values from mice
grinding with a pestle and another 0.1 mL of base was          from each prodrug group were log transformed [24]
added to increase the volume, followed by incremental          and the 95% and 99% confidence intervals of the log
additions of base until clear. The solution, usually 0.4–      values were calculated, then plotted for each group and
0.5 mL, was transferred into a microfuge tube (1.5 mL)         compared to the ACP group and the vehicle control
and the pH determined using a microprobe. Additional           group [18]. The differences between groups (at the cor-
base was added to adjust pH, and when necessary, di-           responding p value) were considered significant when
lute HCl to back titrate to pH 6–8. The solution was           their confidence intervals did not overlap.
then brought to volume with saline to give the final
stock solution for injections.
     ACP, dissolved in warm saline, was administered               RESULTS
at zero time at 0.015 mL/g body weight, which cor-
responded to a dose of 360 mg (2.45 mmol)/kg. We                   The hepatoprotective properties of the thiazolidine
found it necessary to reduce the dose of ACP from the          double-prodrugs of L-cysteine administered pre- and
400 mg (2.65 mmol)/kg dose used previously [18] be-            post-ACP in divided doses (Protocol 1, Table 1), as re-
cause these mice (although of the same strain purchased        flected by plasma ALT levels measured 24 h following
from the same source) appeared to be much more sen-            the administration of ACP, are shown in Figure 1. Their
sitive to ACP toxicity. Even this lower dose appeared          relative degrees of efficacy are readily compared by
240      CRANKSHAW ET AL.                                                                                     Volume 16, Number 5, 2002

FIGURE 1. Protection from ACP-induced hepatotoxicity by thiazo-
lidine prodrugs and double-prodrugs of L-cysteine, using CMC as
vehicle (Protocol 1, Table 1). Survival rates (24 h) are as indicated.   FIGURE 2. Lack of hepatoprotection by thiazolidine prodrugs and
See Materials and Methods section for details on how the data were       double-prodrugs of L-cysteine when preadministered in CMC (Pro-
analyzed.                                                                tocol 2, Table 1). Also see legend to Figure 1.

visual inspection of the overlap of the 95% (unhatched)
and/or 99% (hatched) confidence intervals of the log                      tive in protecting mice against hepatotoxicity elicited by
transformed data [18] (use of a straight edge is sug-                    ACP.
gested). The rationale for the use of this paradigm for                       The use of CMC as a dispersing agent now con-
screening of large numbers of potential hepatoprotec-                    traindicated, it was necessary to employ another dis-
tive agents in mice was to keep the number of animals                    persing agent or to somehow solubilize these cysteine
required at minimal levels, yet be within the accept-                    double-prodrugs in order to facilitate their administra-
able parameters for valid statistical treatment of the                   tion. Fortunately, a uniform characteristic of all these
data.                                                                    prodrugs was the presence of a free carboxyl group
     The lack of protection by N-AcTCA, N-AcOTCA,                        in the molecule with potential for salt formation. Ac-
and N-Cbe-MTCA can be seen readily. Notable, how-                        cordingly, stock injection solutions of the prodrug were
ever, was the apparent poor efficacy displayed by                         prepared by dissolving them in dilute NaOH and then
the positive controls, MTCA and PTCA, compounds                          adjusting the pH to near neutrality (see Materials and
known to be highly effective in protecting mice against                  Methods section). The results of protection experiments
ACP-induced hepatotoxicity [4,25] as well as ACP-[26]                    using this Protocol 3 (Table 1) are presented in Fig-
and naphthalene-induced cataractogenesis [27]. This                      ure 3. N-AcC was included here as the positive control
raised the question whether this pretreatment proto-                     for the open-chain cysteine prodrugs. The results indi-
col, especially the use of CMC as vehicle for repeated                   cated that except for N,S-bis-AcC none of the double-
injections, was problematical. This suspicion was ver-                   prodrugs of cysteine, either as thiazolidine forms or as
ified when the results of experiments using Proto-                        open-chain derivatives, was effective in protecting mice
col 2 (Table 1) were analyzed. In this protocol, only                    against ACP-induced hepatotoxicity.
the pretreatment schedule with the prodrug was re-                            While these results were disappointing, most trou-
tained and the second, post-treatment dose following                     bling was the observation that the three positive con-
ACP was eliminated. The data of Figure 2 clearly in-                     trols, viz., MTCA, PTCA, and N-AcC, all displayed
dicate that a single pretreatment schedule with prodrug                  less than optimal protective activity with this proto-
suspended in CMC and administered 1 h prior to                           col. It was now clear from the results of Figure 3 that
ACP, even at a dose which was highly effective as a sin-                 the pre- and post-ACP doses of prodrugs, viz., the full
gle dose given 30 min post-ACP [18], was totally ineffec-                dose and half dose, respectively, of Protocol 3 (Table 1),
Volume 16, Number 5, 2002                                                                  DOUBLE-PRODRUGS OF L-CYSTEINE               241

FIGURE 3. Protective effects of a series of prodrugs and double-prodrugs of L-cysteine against ACP-induced hepatotoxicity, using Protocol 3
(Table 1). Also see legend to Figure 1. ∗: Blood from 5 animals only. ∗∗: Blood was available from 7 animals.

needed to be reversed for maximal efficacy, and Proto-                    positive controls for the thiazolidine double-prodrugs,
col 4 was established as the ultimate paradigm for im-                   were now not different from the ALT levels of the ve-
plementation. The results of this study are graphically                  hicle control animals at the 99% and 95% confidence
summarized in Figure 4. Significantly, the ALT levels                     levels, respectively, while the ALT levels of the N-AcC-
of ACP-mice treated also with MTCA and PTCA, the                         treated animals, the positive control for the open-chain

FIGURE 4. Differential protection against ACP-induced hepatotoxicity by double-prodrugs of L-cysteine (Protocol 4, Table 1). Also see legend
to Figure 1. ∗: Blood was available from 6 animals.
242     CRANKSHAW ET AL.                                                                      Volume 16, Number 5, 2002

double-prodrugs of cysteine, were not different from         the urinary recoveries were not quantitative, these data
vehicle control mice at the 99% confidence level.             suggest that the acetyl groups on N-AcTCA and N-
     The hepatoprotection displayed by the N-acylated        AcMTCA are not readily hydrolyzed in vivo, reflect-
thiazolidine double-prodrugs of L-cysteine was               ing the observed lack of hepatoprotection by N-AcTCA
marginal at best, (Figure 4), and while several of the       (Figure 1) and the marginal activity of N-AcMTCA
mice in each group were fully protected based on             (Figure 4). On the other hand, the acetyl group in N-
plasma ALT levels at 24 h, premature deaths were also        AcOTCA, being part of an imide structure, should have
recorded. Thus, the N-acyl group, in particular, the         been enzymatically more labile, the expected product
N-acetyl group (vide infra for discussion of the N-Cbe       here being OTCA, a known hepatoprotective cysteine
group) on the thiazolidines appears to be metabolically      prodrug [3]. The large variation in efficacy displayed by
much more stable than had been anticipated.                  this compound (Figure 4) suggests that individual ani-
     The standout among all of the double-prodrugs           mals must have shown considerable differences in the
tested was N,S-bis-AcC, which fully protected mice           double enzymatic deacetylation and thiazolidine ring
from hepatotoxicity (Figure 4). In marked contrast, N,S-     opening steps leading to the release of L-cysteine.
bis-CbeC and N-CbeC itself, where the acetyl groups               Conceptually, double-prodrugs should confer high
were replaced with the carbethoxy group, were totally in-    selectivity to the drugs being latentiated, since two se-
effective in protecting mice against ACP-induced hep-        quential enzymatic steps are required for their ulti-
atotoxicity. While no deaths were encountered with           mate liberation in vivo, and specificity for each step
MTCA, PTCA, N,S-bis-AcC, or N-AcC, which were all            should vary, depending on the enzymatic makeup of
fully protective, many of the animals treated with N,S-      the species, strain, etc. The use of random-bred Swiss-
bis-CbeC and/or with N-CbeC died prematurely (be-            Webster mice instead of an inbred mouse strain to
fore 6 h) requiring that blood be drawn soon after death     evaluate the efficacy of this series of cysteine double-
for the measurement of plasma ALT levels (Figure 4).         prodrugs was a deliberate effort to assess the universal-
                                                             ity of action of these compounds. Double-prodrugs that
                                                             exhibit high hepatoprotective properties in this outbred
      DISCUSSION                                             mouse strain would likely be more reflective of and ap-
                                                             plicable to the human situation, the goal strived for in
     In previous studies, MTCA, suspended in 2% CMC          drug research. Conversely, double-prodrugs with poor
and administered as a single dose 30 min post-ACP,           efficacy due to differential specificity at each step, a
protected against hepatotoxicity [18]. However, in the       consequence of genetic polymorphism [31], should be
present study, the levels of protection achieved by          avoided for further development.
MTCA and PTCA also suspended in 2% CMC but ad-                    A case in point is the carbethoxy (Cbe) group. This
ministered pre- and post-ACP (Protocol 1, Table 1) were      group, whether attached to divalent sulfur (as in S-
highly erratic (Figure 1). This lack of effective protec-    Cbe-N-AcC) or on nitrogen (as in N-CbeC or N-Cbe-
tion observed even with MTCA and PTCA appears to             MTCA) can, theoretically be hydrolyzed by esterases or
be due to the detrimental effects of CMC itself. For         be oxidatively removed by the cytochrome P450 enzymes
example, it has been reported that viscous additives         via ethyl group dealkylation, both routes giving rise to
such as CMC used as carrier can adversely affect drug        CO2 , as well as ethanol and acetaldehyde, respectively,
absorption [28]. Moreover, pretreatment of mice with         as by-products. The fact that those compounds func-
1% CMC before challenge with ACP (300 mg/kg) was             tionalized with the Cbe group had erratic, widely dif-
shown to reduce liver GSH concentration by 81% and           ferential hepatoprotective efficacy (Figure 4) suggests
dramatically increase liver injury [29]. Our results using   that polymorphism must play a major role in the liber-
Protocols 1 and 2 (Figures 1 and 2) could, therefore, be     ation of cysteine from these Cbe-functionalized double-
consequences of this CMC effect coupled to the inher-        prodrugs. In marked contrast, N,S-bis-AcC, where both
ent hepatotoxicity of ACP. Thus, as documented here,         the sulfhydryl and amino groups of L-cysteine are func-
the use of CMC as a dispersing agent in the evaluation       tionalized with the acetyl group, was highly effective
of hepatoprotective agents is contraindicated, despite       in protecting mice from the hepatotoxic effect of ACP
previous successes with its use.                             (Figure 4). It follows that enzymatic removal of the
     Roquebert et al. [30] reported that when the pyri-      acetyl groups from N,S-bis-AcC was facile and was not
doxine salt of N-acetyl-TCA (N-AcTCA, structure not          subject to differential metabolism because of genetic
shown) radiolabeled with 35 S was administered to            polymorphism.
rats, N-AcTCA was recovered unchanged in the urine.               Whether the activity of these cysteine prodrugs is
We have observed that a large dose of N-AcMTCA               limited to their stimulatory action on the biosynthesis
administered i.p. to rats (1.25 g/kg) can also be recov-     of GSH and its sequestration of NAPQI produced in
ered unchanged in the urine (unpublished). Although          the oxidative metabolism of ACP remains unclear. The
Volume 16, Number 5, 2002                                                     DOUBLE-PRODRUGS OF L-CYSTEINE             243

nullification by GSH of the various reactive oxygen and        2. Lauterberg BH, Corcoran GB, Mitchel JR. Mechanism of
nitrogen species, which are the toxic mediators in the           action of N-acetylcysteine in the protection against the
early stages of the inflammatory cascade, may also play           hepatotoxicity of acetaminophen in rats in vivo. J Clin
                                                                 Invest 1983;71:980–991.
a role. It is known that maintaining GSH homeosta-            3. Williamson JM, Boettcher B, Meister A. Intracellular cys-
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tecting the enzyme, AdoMet synthetase (methionine                moting glutathione synthesis. Proc Natl Acad Sci USA
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AdoMet synthetase, being a sulfhydryl enzyme, re-             4. Nagasawa HT, Goon DJW, Zera RT, Yuzon DL. Pro-
                                                                 drugs of L-cysteine as liver protective agents. 2(R,S)-
quires GSH homeostasis to maintain the integrity of its          Methylthiazolidine-4(R)-carboxylic acid, a latent cys-
active-site sulfhydryl groups [33]. It is also a key             teine. J Med Chem 1982;25:489–491.
enzyme in the methylation cycle with major cellu-             5. Nagasawa HT, Goon DJW, Muldoon WP, Zera RT. 2-
lar functions [34]. We have recently shown that ACP              Substituted thiazolidine-4(R)-carboxylic acids as pro-
administration to mice not only depletes hepatic mi-             drugs of L-cysteine. Protection of mice against ac-
                                                                 etaminophen hepatoxicity. J Med Chem 1984;27:591–596.
tochondrial GSH levels, but also compromises the ac-          6. Roberts JC, Nagasawa HT, Zera RT, Fricke RF, Goon
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tration of L-cysteine prodrugs protected the activity            acetaminophen-induced hepatotoxicity. 2-(Polyhydro-
of this enzyme [35]. Carbon tetrachloride [32] and N-            xyalkyl)- and 2-(polyacetoxyalkyl)thiazolidine-4(R)-car-
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                                                                 and conversion to glutathione. Arch Biochem Biophys
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can attenuate the cellular effects of cytokine activation        acetyl-L-cysteine in vivo: Studies with N-acetyl-D-cysteine
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                                                                   ¨                            a
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    ACKNOWLEDGMENT                                           15. Williamson JM, Meister A. Stimulation of hepatic
                                                                 glutathione formation by administration of L-2-
     We thank Michael A. Kuskowski, Ph.D., of the Geri-          oxothiazolidine-4-carboxylate,       a   5-oxo-L-prolinase
atric Research, Education and Clinical Center for sta-           substrate. Proc Natl Acad Sci USA 1981;78:936–939.
                                                             16. Chasseaud LF. Reactions with electrophiles after enzyme
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Ph.D., for the preparation of N-AcMTCA, and Terry T.         17. Olsson B, Johansson M, Gabrielsson J, Bolme P. Pharma-
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                                                             18. Nagasawa HT, Shoeman DW, Cohen JF, Rathbun WB.
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