Synthesis, Degradation, and Antimicrobial Properties of Targeted Macromolecular Prodrugs of Norfloxacin

Article abstract of DOI:10.1128/AAC.47.11.3435-3441.2003

Long-term antibiotic treatment is required to cure tuberculosis. Targeted antibiotics should improve the efficacy of treatment by concentrating the drugs close to the bacteria. The aim of the present study was to synthesize targeted conjugates. For this purpose, we used mannose as a homing device to direct norfloxacin into macrophages. Dextran was used as the polymer bearing both mannose and norfloxacin. Using different peptide spacer arms to link norfloxacin to dextran, we demonstrated that norfloxacin acts as an antibiotic only when it is released in its native form. Also, targeting by using mannose as a homing device is required to achieve antimycobacterial activity in vivo. Thus, norfloxacin, which is inactive against mycobacteria in its native form in vivo, can be transformed into an active drug by targeting.

Full text of DOI:10.1128/AAC.47.11.3435-3441.2003

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2003, p. 3435–3441
0066-4804/03/$08.00ϩ0 DOI: 10.1128/AAC.47.11.3435–3441.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 11
Synthesis, Degradation, and Antimicrobial Properties of Targeted
Macromolecular Prodrugs of Norfloxacin
Eveline Roseeuw,1 Veerle Coessens,1 Anne-Marie Balazuc,2 Micheline Lagranderie,2
Pierre Chavarot,2 Augusto Pessina,3 Maria Grazia Neri,3 Etienne Schacht,1
Gilles Marchal,2 and Dominique Domurado4*
Biomaterial and Polymer Research Group, Department of Organic Chemistry, University of Ghent, 9000 Ghent,
Belgium1; Laboratoire de R´ef´erence des Mycobact´eries, Institut Pasteur, 75724 Paris Cedex 15,2 and Groupe
de Pharmacocin´etique des Prodrogues et Conjugu´es Macromol´eculaires (INSERM), CRBA-UMR 5473
CNRS, Facult´e de Pharmacie, 34093 Montpellier Cedex 5,4 France; and Istituto de
Microbiologia, Universita` di Milano, 20133 Milan, Italy3
Received 13 December 2002/Returned for modification 14 April 2003/Accepted 11 August 2003
Long-term antibiotic treatment is required to cure tuberculosis. Targeted antibiotics should improve the
efficacy of treatment by concentrating the drugs close to the bacteria. The aim of the present study was to
synthesize targeted conjugates. For this purpose, we used mannose as a homing device to direct norfloxacin
into macrophages. Dextran was used as the polymer bearing both mannose and norfloxacin. Using different
peptide spacer arms to link norfloxacin to dextran, we demonstrated that norfloxacin acts as an antibiotic only
when it is released in its native form. Also, targeting by using mannose as a homing device is required to
achieve antimycobacterial activity in vivo. Thus, norfloxacin, which is inactive against mycobacteria in its
native form in vivo, can be transformed into an active drug by targeting.
A variety of bacterial and protozoan pathogens reside within
host macrophages. These pathogens are largely protected
against many of the host’s defense mechanisms, i.e., antibodies
and/or complement. This intracellular mode of life also means
that the pathogens can potentially become quiescent or latent.
Examples of facultative or obligate intracellular pathogens in-
clude the agents responsible for chronic bacterial diseases such
as brucellosis, trachoma, and tuberculosis and those responsi-
ble for protozoan diseases such as toxoplasmosis and leishman-
iasis.
In this report, we focus on bacterial infections and, more
precisely, on infections caused by mycobacteria, which are fac-
ultative intracellular pathogens. They escape killing during
phagocytosis by blocking phagosome-lysosome fusion. Intra-
cellular mycobacteria are also largely protected against drugs.
Consequently, it is difficult to destroy the pathogen while leav-
ing the host cells intact. The main aim of this work was to
design a conjugate able to target and to deliver a drug into the
phagosomal vacuole, such that it is brought into close contact
with the engulfed bacteria.
through a macromolecular carrier could induce the endocyto-
sis of the drug by the target cell via a specific receptor and,
following this first step, subcellular distribution of the drug to
sites where the bacilli are localized. The use of mannosyl li-
gands to target macrophages has been proposed (9).
Water-soluble polyfunctional polymers can accommodate
several homing device residues, thus increasing the affinity for
the target (10). Similarly, the linking of several drug molecules
may increase the rate at which the drug is delivered to the
target. A conjugate that is large enough to impair renal filtra-
tion may optimize drug delivery (1).
We report on the development of a macromolecular vehicle
based on dextran linked to mannose, to target the macrophage,
and norfloxacin, to be delivered into the phagosomal vacuole
in its active form. We describe the synthesis of conjugates in
which norfloxacin was linked to the macromolecular carrier
through two different peptide arms. The relative in vitro anti-
biotic efficacies of the different norfloxacin macromolecular
prodrugs were determined, and we tested the in vivo antibiotic
activities of the macromolecular prodrugs against intracellular
Mycobacterium bovis (BCG strain) in mice.
The targeting of drugs into cells, more specifically, into
phagocytic cells, was proposed by De Duve et al. (5), who
developed the notion of lysosomotropic drugs or carriers. Dif-
ferent vehicle molecules have been prepared and tested, but
the results were disappointing. Some of these molecules were
active in vitro, but they were frequently unable to find their
targets in vivo or to deliver the drug into the infected cells in an
active form.
MATERIALS AND METHODS
Materials. Norfloxacin was obtained from Sigma-Aldrich (Bornem, Belgium).
Gly-Phe, Z-Gly-paranitrophenylester, methoxy (OMe) Ser (Ser-OMe), and Ala-
Leu were purchased from Bachem (Bubendorf, Switzerland). para-Nitrophenyl-
chloroformate was obtained from Merck (Darmstadt, Germany), and bovine
cathepsin B was obtained from Fluka (Buchs, Switzerland). Dextran (Pharmacia,
Uppsala, Sweden) was repurified to get fractions with weight-average molar
masses of 64,000, 90,000, or 95,000 g molϪ1 and with a weight-average molar
mass/number-average molar mass ratio of 1.2. All other chemicals were pur-
chased from Acros (Beerse, Belgium).
The temporary conjugation of a drug to a homing device
* Corresponding author. Mailing address: CRBA, Facult´e de Phar-
macie, 15 Ave. Charles Flahault, BP 14491, 34093 Montpellier Cedex
5, France. Phone: (33) 467-418-268. Fax: (33) 467-520-898. E-mail:
domurado@univ-montp1.fr.
Synthesis of Leu-norfloxacin. (i) Synthesis of N-(tert-butoxycarbonyl)-L-Leu
(Boc-Leu). Leu (0.58 g, 4.5 mmol) was dissolved in a mixture of dioxane (10 ml)
and 0.5 M NaOH (10 ml). After the mixture was cooled to 0°C, di-tert-butylpy-
rocarbonate (1.08 g, 4.95 mmol) was added. The reaction mixture was stirred for
3435

3436
ROSEEUW ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Schematic representation of Gly-Phe-Gly-Gly-(␣-norfloxa-
cin)-OMe synthesis.
Ser-OMe was obtained with a yield of 93%. Rf (dichloromethane-isopropanol;
9/1) ϭ 0.4.
1H NMR (MeOD) ␦ 3.95 to 3.70 (7H, m, CH2 Gly ϩ CH2 Ser ϩ CH3 methyl
ester), 4.55 (1H, m, CH Ser), 5.15 (2H, s, CH2 benzyl), 7.35 (5H, m, Phe) ppm.
(b) Z-Gly-Gly(␣-OAc)-OMe. Z-Gly-Ser-OMe (200 mg, 0.65 mmol), lead ace-
˚
tate [Pb(OAc)4; 340 mg, 0.97 mmol], and molecular sieves (pore size, 4 A; 550
mg), which were used to trap water, were refluxed in dry ethyl acetate (20 ml) for
3 h. After the mixture was cooled, the mixture was filtered on Celite, and the
solvent was evaporated. Pure Z-Gly-Gly(␣-OAc)-OMe was obtained with a yield
of 95%. Rf (dichloromethane-methanol; 9/1) ϭ 0.5.
1H NMR (500 MHz, CDCl3) ␦ 7.35 (5H, m, phenyl), 6.40 (1H, d, CH of
acetoxy [OAc]-substituted Gly), 5.15 (2H, s, CH2 benzyl), 4.00 (2H, m, CH2 Gly),
3.80 (3H, s, methylester), 2.10 (3H, s, CH3 of OAc) ppm.
(c) Z-Gly-Gly(␣-norfloxacin)-OMe. Z-Gly-Gly(␣-OAc)-OMe (200 mg, 0.59
mmol) was dissolved in dimethylformamide (10 ml). Norfloxacin (189 mg, 0.59
mmol) dissolved in dimethylformamide and triethylamine (82 ␮l, 0.59 mmol)
were added, and the mixture was stirred for 36 h. The solvent was evaporated,
and Z-Gly-Gly(␣-norfloxacin)-OMe was extracted with dichloromethane, then
50 mM HCl solution (three times), and finally, water (three times). Pure Z-Gly-
Gly(␣-norfloxacin)-OMe was obtained with a yield of 89%. Rf (dichloromethane-
methanol-acetic acid; 95/5/0.1) ϭ 0.14.
1 h at 0°C and then for 4 h at room temperature. The dioxane was evaporated.
The resulting aqueous solution was acidified to pH 3 with a KHSO4 solution (1
M) and extracted with ethyl acetate (two times with 100 ml each time). The ethyl
acetate solution was dried on Na2SO4 and evaporated until it was dry.
(ii) Synthesis of Boc-Leu-pentafluorophenyl ester (Boc-Leu-PFP). Boc-Leu
(3.82 mmol) and pentafluorophenol (0.82 g, 4.44 mmol) were dissolved in dry
tetrahydrofuran (20 ml). After the mixture was cooled to 0°C, dicyclohexylcar-
bodiimide (0.86 g, 4.16 mmol) was added. The reaction mixture was stirred for
1 h at 0°C and overnight at room temperature. The precipitate that formed
during the reaction was filtered, and the filtrate was evaporated under vacuum.
The residue was dissolved in ethyl acetate (30 ml), and the solution was filtered.
The filtrate was evaporated. Rf (dichloromethane-methanol; 9/1) ϭ 0.65. Infra-
red (film), 1,790 cmϪ1: PFP ester.
(iii) Synthesis of Boc-Leu-norfloxacin. Norfloxacin was silylated in dichlo-
romethane by adding two equivalents of N-methyl-N-(trimethylsilyl)trifluoroac-
etamide. Silylated norfloxacin and Boc-Leu-PFP were dissolved in dichlorometh-
ane in equivalent quantities. The solution was stirred overnight. The residue was
dissolved in dichloromethane-methanol (9/1), extracted twice with water, and
then purified by chromatography on a silica (normal phase) column.
1H nuclear magnetic resonance (NMR) (CDCl3) ␦ 8.70 (1H, s, C-2 norfloxa-
cin), 8.15 (1H, d, C-5 norfloxacin), 6.85 (1H, d, C-8 norfloxacin), 4.70 (1H, m,
CH, Leu), 4.35 (2H, q, CH2 norfloxacin), 4.1 to 3.5 (8H, m, 4-CH2, piperazine),
1.75 (1H, m, CH, Leu), 1.61 (3H, t, CH3, norfloxacin), 1.5 (2H, m, CH2, Leu),
1.40 (9H, m, Boc), 0.95 to 0.85 (2-CH3, Leu) ppm.
1H NMR (CDCl3) ␦ 8.65 (1H, s, H on C-2 norfloxacin), 7.93 (1H, d, H on C-5
norfloxacin), 7.40 (5H, m, phenyl), 7.05 (1H, d, NH-substituted Gly), 6.77 (1H,
d, H on C-8 norfloxacin), 5.60 (1H, t, NH Gly), 5.45 (1H, d, CH), 5.15 (2H, s,
CH2 benzyl), 4.31 (2H, q, CH2 norfloxacin), 4.00 (2H, d, CH2 Gly), 3.83 (3H, s,
CH3 methyl ester), 3.30, 2.85, and 2.74 (8H, m, CH2 groups of piperazine), 1.60
(3H, t, CH3 norfloxacin) ppm.
(ii) Synthesis of Gly-Gly-(␣-norfloxacin)-OMe. Z-Gly-Gly(␣-norfloxacin)-OMe
(320 mg, 0.54 mmol) was dissolved in dry methanol (MeOH; 10 ml) and HBr in
acetic acid (0.2 ml). To this solution, 10% Pd/C (320 mg) was added. After 24 h
of stirring under H2 pressure, the catalyst was filtered and the filtrate was
concentrated. Pure Gly-Gly-(␣-norfloxacin)-OMe was obtained with a yield of
64%. Rf (dichloromethane-methanol-acetic acid; 95/5/0.1) ϭ 0.0.
1H NMR (dimethyl sulfoxide [DMSO]-d6) ␦ 8.97 (1H, s, H on C-2 norfloxa-
cin), 7.93 (1H, d, H on C-5 norfloxacin), 7.15 (1H, d, H on C-8 norfloxacin), 5.23
(1H, s, CH), 4.60 (2H, q, CH2 norfloxacin), 3.71 (3H, s, CH3 methyl ester), 3.55
to 2.7 (10H, m, CH2 Gly and 4-CH2 piperazine), 1.60 (3H, t, CH3 norfloxacin)
ppm.
(iii) Synthesis of Boc-Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe. Boc-Gly-Phe-O-
PFP (137 mg, 0.28 mmol) and Gly-Gly(␣-norfloxacin)-OMe (130 mg, 0.28 mmol)
were dissolved in dimethylformamide (10 ml), and N-methylmorpholine (500 ␮l)
was added. The dimethylformamide was evaporated after 48 h. Extraction with
dichloromethane was followed by evaporation and purification by chromatogra-
phy with a gradient (from dichloromethane-methanol [9/1] to dichloromethane-
To obtain Leu-norfloxacin, the Boc group was removed by using trifluoroacetic
acid.
Synthesis of Gly-Phe-Gly-Gly-(␣-norfloxacin)OMe (Fig. 1). (i) Synthesis of
benzyloxycarbonyl-Gly-Gly(␣-norfloxacin)-OMe [Z-Gly-Gly(␣-norfloxacin)-OMe].
(a) Z-Gly-Ser-OMe. Benzyloxycarbonyl-protected Gly-para-nitrophenylester (Z-
Gly-para-nitrophenylester; 1 g, 3 mmol), Ser-OMe (0.46 g, 3 mmol), and N-
methylmorpholine (3 ml) were dissolved in dimethylformamide (15 ml). The
solvent was evaporated after 48 h, and the residue was purified by chromatog-
raphy (dichloromethane-isopropanol; gradient from 98/2 to 88/12). Pure Z-Gly-

VOL. 47, 2003
TARGETED MACROMOLECULAR PRODRUGS OF NORFLOXACIN
3437
FIG. 2. Schematic representation of dextran-Gly-Phe-Gly-Gly-(␣-
norfloxacin)-OMe synthesis.
methanol-acetic acid [9/1/0.1]). Pure Boc-Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe
was obtained with a yield of 70%. Rf (dichloromethane-methanol-acetic acid;
9/1/0.1) ϭ 0.29.
1H NMR (DMSO-d6) ␦ 8.90 (1H, s, H C-2 norfloxacin), 8.61 (1H, m, NH),
8.48 (1H, m, NH), 8.15 (1H, m, NH), 7.85 (1H, m, H C-5 norfloxacin), 7.20 (6H,
m, phenyl ϩ H on C-8 norfloxacin), 6.88 (1H, m, NH), 5.23 (1H, m, CH), 4.50
(3H, m, CH Phe and CH2 norfloxacin), 3.85 (2H, m, CH2 Gly), 3.72 (3H, s, CH3),
3.60 to 3.40 (2H, m, CH2 Gly), 3.28 (4H, m, 2-CH2 piperazine), 3.05 (1H, m, HB
Phe), 2.75 (5H, m, HA Phe ϩ 2-CH2 piperazine), 1.35 (12H, m, Boc and CH3
norfloxacin) ppm.
(iv) Coupling of Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe to activated dextran
(Fig. 2). Chloroformate-activated dextran (13), with 15 mol% linear carbonates,
as measured by spectrophotometry (402 nm, ε ϭ 18,400 l molϪ1 cmϪ1), was
dissolved in DMSO-pyridine (1/1). Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe was
added to the solution. The Boc-protecting group was first removed by dissolving
Boc-Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe (50 mg, 65 ␮mol) in trifluoroacetic
acid (2 ml). The mixture was stirred for 30 min. After evaporation of the solvent,
the residue was dried and added to the activated dextran. After 48 h, the
conjugate was precipitated in ethanol-ether (1/1) and then dissolved in NaOH
(0.1 M, 8 ml) and purified by preparative gel permeation chromatography (Seph-
adex G25; Pharmacia). The substitution degree of norfloxacin was measured by
UV spectroscopy at 278 nm and 1H NMR.
EDTA (1 mM), and bovine cathepsin B (250 ␮g). Aliquots were taken at regular
intervals and analyzed by high-pressure liquid chromatography.
Bacteriology. (i) Bacteria. The bacteria used were Escherichia coli K12 (ATCC
25290), Brucella melitensis (ATCC 739), Staphylococcus aureus (ATCC 6538),
and M. bovis (BCG strain 1173P2; Institut Pasteur).
(ii) MICs and MBCs. The antimicrobial activities, i.e., the MICs and minimal
bactericidal concentrations (MBCs), of native norfloxacin and Leu-norfloxacin
were determined. The comparison of native norfloxacin and Leu-norfloxacin was
based on the use of equimolar amounts of norfloxacin. Norfloxacin was dissolved
in HCl (1 M), and Leu-norfloxacin was dissolved in water. The MIC was deter-
mined by adding 105 CFU of bacteria to tubes containing 1 ml of serial twofold
dilutions of norfloxacin and Leu-norfloxacin in broth (Mueller-Hinton broth;
Difco). After 18 h at 37°C, the MIC was defined as the lowest concentration of
the tested molecule that prevented bacterial growth. When no bacterial growth
was observed, samples of medium (100 ␮l) were plated on a suitable agar
medium. After incubation at 37°C, colonies were detected. The MBC was de-
fined as the lowest concentration of the tested molecule at which no viable
bacteria were present in the sample.
(iii) In vitro antimycobacterial activities of norfloxacin and of its macromo-
lecular conjugates. One hundred microliters of Middlebrook 7H9 medium
(Difco) containing 104 CFU of BCG (105 per ml) was placed in each well of a
microtiter plate (Nunc) containing 50 ␮l of appropriate dilutions of the different
preparations to be tested. After 4 days at 37°C, 50 ␮l of 7H9 medium containing
10 ␮Ci of [3H]uracil (Amersham) per ml was added for 18 h. The labeled
bacteria were harvested onto glass fiber filters and subjected to liquid scintillation
counting. The results are expressed as the mean Ϯ standard deviation counts per
minute for triplicate culture wells.
The degree of substitution was determined by comparison of integration of the
signals by 1H NMR (D2O) ␦ 4.95 ppm (H-1 dextran) and ␦ 7.20 ppm (6H, phenyl
and H on C-8 norfloxacin).
(v) Synthesis of the conjugate dextran-Gly-Phe-Ala-Leu-norfloxacin. The dex-
tran-Gly-Phe-Ala-Leu-norfloxacin conjugate was synthesized as described previ-
ously (4).
(vi) Mannosylation. Mannosylation (Fig. 3) was carried out as described pre-
viously (6, 13).
Release of norfloxacin from the conjugates. (i) Chemical release. One milli-
gram of the polymeric norfloxacin conjugate was dissolved in 1 ml of either 0.1
M phosphate buffer (pH 7.4) or 0.28 M citrate-phosphate buffer (pH 5.5) at 37°C.
At predetermined time intervals, samples were taken and analyzed by high-
˚
pressure liquid chromatography (PLRP-S column, 100 A, 5-␮m Achrom; Zulte)
with a mixture of acetonitrile-methanol-tetrahydrofuran-acetic acid-water (228/
39/16/138/1,582) as the eluent. The flow rate was 0.75 ml/min. The products were
detected spectophotometrically at 278 nm.
In vivo assays. (i) Animals. Specific-pathogen-free C57BL/6 mice (age, 6
weeks) were obtained from Iffa-Credo (Saint-Germain sur l’Arbesle, France).
(ii) Proteolysis. The conjugate (1 mg) was dissolved in 2 ml of a citrate-
phosphate buffer (0.28 M pH 5.5) containing reduced glutathione (5 mM),
FIG. 3. Schematic representation of mannosylated dextran-Gly-Phe-Gly-Gly-(␣-norfloxacin)-OMe synthesis.

3438
ROSEEUW ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Schematic representation of the structures of the different conjugates studied. Norfloxacin was linked to the carrier either through an
amide bond (a and b) or through an ␣ bond (c and d), and the conjugates were mannosylated (b and d) or not (a and c).
(ii) Microorganisms and infection. M. bovis BCG 1173P2 (Institut Pasteur)
two different chemical bonds, an amide bond and an ␣ bond,
borne by two different tetrapeptide linkers.
was grown on Sauton medium for 14 days. The bacilli were then homogenized
with stainless steel balls in the same medium to a concentration of 50 mg mlϪ1
When norfloxacin was linked to Gly-Phe-Ala-Leu through
an amide bond, Leu-norfloxacin was released in the presence
of cathepsin B. Thus, Leu-norfloxacin was synthesized to study
its in vitro antimicrobial efficacy.
In Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe, norfloxacin was
linked to the carrier through an ␣ bond. The structures of the
four norfloxacin conjugates, consisting of two different linkers
with or without mannose, are shown in Fig. 4. The chemical
characteristics of the four conjugates are summarized in Table
1.
Norfloxacin release. We first studied the macromolecular
conjugates bearing two different bonds to link norfloxacin, but
no mannose, in vitro to determine their capacity to release
norfloxacin. This was done in the presence and absence of
cathepsin B, because the peptide arms linking norfloxacin to its
macromolecular carrier were susceptible to this lysosomal en-
zyme. Less than 0.2% of the norfloxacin was spontaneously
released from the Gly-Phe-Ala-Leu tetrapeptide arm, i.e., the
arm bearing an amide bond, in 24 h at both plasma pH (pH
7.4) and lysosome pH (pH 5.5) (Fig. 5). Moreover, cathepsin
B, which degrades the peptide arm, did not hydrolyze the
amide bond between norfloxacin and the last amino acid
(Leu), meaning that Leu-norfloxacin was released (4). The
(107 viable units per mg) (8). Vials containing the BCG suspension were stored
at Ϫ70°C until use. Mice were infected by intravenous injection of 0.5 ml con-
taining 106 viable units.
(iii) Treatment of mice. Starting from the 6th day after infection, the mice (five
per group) were injected intraperitoneally twice a day for 5 days with the dif-
ferent test molecules. Phosphate-buffered saline (PBS) and isoniazid (0.5 mg per
mouse per day) were used as negative and positive control treatments, respec-
tively. The macromolecular conjugates containing norfloxacin were diluted in
PBS, and each mouse was injected with the equivalent of 0.25 mg of norfloxacin
per day. Native norfloxacin was injected as a control (0.5 mg per day).
(iv) BCG growth in lungs. BCG growth was monitored by counting the number
of viable units in the lungs of the mice 2 days after the end of treatment. This
2-day washout period allowed the elimination of persisting native antibiotics. The
lung tissues were homogenized, and suitable dilutions were plated on Middle-
brook 7H11 medium. The number of CFU was counted after 21 days at 37°C.
Statistics. The means for each group of five mice were tested by one-way
analysis of variance by the Tukey-Kramer multiple-comparison test (Instat;
GraphPad Software, San Diego, Calif.).
RESULTS
Synthesis. The conjugates designed to target an antibiotic
into macrophages included dextran as the carrier, norfloxacin
as the antibiotic, and, sometimes, mannose as a homing device.
To try to produce a carrier that was able to release native
norfloxacin, the antibiotic was linked to the carrier by means of

VOL. 47, 2003
TARGETED MACROMOLECULAR PRODRUGS OF NORFLOXACIN
TABLE 1. Chemical characteristics of the four norfloxacin conjugates
3439
Mannose concn
Norfloxacin concn
Norfloxacin concn
Product
Mw of dextran
(mol%)
(% [wt/wt])
(mol%)
Dextran-GFGG-␣-norfloxacin
95,000
95,000
64,000
90,000
4.9
7.1
8.2
8.3
3.3
4.3
5.5
5.2
Dextran-GFAL-norfloxacin
Mannosylated dextran-GFGG-␣-norfloxacin
Mannosylated dextran-GFAL-norfloxacin
2.3
1.7
antimicrobial efficacy of Leu-norfloxacin was found to be neg-
ligible (see below).
As the ultimate goal of this work was to design a macromo-
lecular prodrug active against intracellular bacteria, particu-
larly members of the genus Mycobacterium, we tested the ac-
tivities of the conjugates prepared during this work against M.
bovis BCG in vitro. The antibacterial activities of native nor-
floxacin and the dextran-norfloxacin conjugates containing ei-
ther the amide bond or the ␣ bond were compared at equal
norfloxacin concentrations (Fig. 6). The antibacterial activity
of the conjugate bearing an ␣ bond was similar to that of native
norfloxacin. The conjugate in which an amide bond links
leucine to norfloxacin was at least 20 times less active than the
other conjugate.
In a second series of in vitro experiments with M. bovis (Fig.
7), we compared the bactericidal activities of native norfloxacin
and the conjugate of norfloxacin containing an ␣ bond to the
activity of the reference antibiotic, isoniazid. Isoniazid was
about 10 times more active than norfloxacin at an equal molar
concentration. Likewise, the dextran-norfloxacin conjugate
was slightly less active than norfloxacin alone. As expected, the
presence of mannose on the dextran carrier had no effect on
the antibiotic activity in vitro, as no macrophages were present
in this test (results not shown).
In contrast to this behavior, about 25% of the norfloxacin
was spontaneously released from Gly-Phe-Gly-Gly(␣-norfloxa-
cin)-OMe, which bears an ␣ bond, after 24 h at pH 7.4,
whereas about 40% of the norfloxacin was spontaneously re-
leased at pH 5.5 (Fig. 5). In the presence of cathepsin B,
norfloxacin was released (only the release at pH 5.5 was stud-
ied because the optimum pH of cathepsin B is between 4.5 and
6) from the peptide bearing an ␣ bond about four times more
rapidly, as shown by the initial release rates (Fig. 5). Regard-
less of whether the release mechanism was chemical or enzy-
matic, the Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe peptide al-
ways released native norfloxacin.
Microbiology. As the conjugate consisting of norfloxacin
linked to the Gly-Phe-Ala-Leu tetrapeptide arm through an
amide bond released Leu-norfloxacin, we wondered whether
this compound had any antibacterial activity. Consequently, we
determined the MICs and MBCs of Leu-norfloxacin for E. coli,
S. aureus, and B. melitensis. Whereas norfloxacin was active
against these bacteria in vitro, Leu-norfloxacin was inactive
under the same conditions (Table 2).
In vivo assays. To test these molecules under conditions
resembling the clinical situation as closely as possible, we com-
pared the antibacterial activities of native norfloxacin and the
different macromolecular conjugates of norfloxacin in mice
infected with M. bovis BCG. PBS was also tested as a negative
control, and isoniazid was tested as a positive control. The
results, expressed as the number of CFU per lung, are pre-
sented in Fig. 8. As expected, isoniazid was active against M.
bovis BCG, whereas PBS was not. Despite its in vitro activity,
native norfloxacin was not active in vivo, probably because it is
rapidly filtered by the kidneys (2). Only the conjugate contain-
ing both an ␣ bond and mannose was as active as isoniazid
against M. bovis BCG. The other conjugates, which contained
either an amide bond with or without mannose or an ␣ bond
without mannose, were all inactive.
TABLE 2. MICs and MBCs of norfloxacin and Leu-norfloxacin for
E. coli, S. aureus, and B. melitensis
Norfloxacin
Leu-norfloxacin
Bacterium
MICa
MBCa
MICa
MBCa
E. coli K-12
S. aureus
0.078
0.625
0.625
0.156
1.25
2.5
10
10
Ͼ10
Ͼ10
10
FIG. 5. In vitro release of norfloxacin from macromolecular con-
jugates as a function of time. Open triangles, amide bond; open
squares, ␣ bond, pH 7.4; open circles, ␣ bond, pH 5.5; closed circles, ␣
bond, pH 5.5, in the presence of cathepsin B.
B. melitensis
2.5
a The results are expressed in milligrams of equivalent norfloxacin literϪ1
.

3440
ROSEEUW ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 8. In vivo anti-M. bovis BCG activities of isoniazid (INH),
PBS, norfloxacin, and the four macromolecular conjugates of norfloxa-
cin. These conjugates were mannosylated (Man) or not and contained
either an ␣ bond or an amide bond. The antimicrobial activities,
expressed as the number of CFU per organ, were measured in the
lungs of mice (n ϭ 5). Error bars indicate standard deviations. ءءء, a
highly significant difference (P Ͻ 0.001) compared to the results for the
control (PBS-treated) group. No significant differences were observed
for the other groups.
FIG. 6. In vitro anti-M. bovis BCG activities of norfloxacin (dia-
monds) and its nonmannosylated macromolecular conjugates contain-
ing an ␣ bond (inverted triangles) or an amide bond (triangles) as a
function of the molar concentration of native or conjugated norfloxa-
cin present in the medium. Bacterial multiplication was assessed by
measuring the incorporation of [3H]uracil by M. bovis.
that can be targeted to macrophages. Members of our group
have previously developed several macromolecular prodrugs
that include norfloxacin as the antibiotic (4). To enable it to be
released preferentially in the lysosomes, the antibiotic was
linked to the carrier through one of two tetrapeptides, Gly-
Phe-Ala-Leu or Gly-Phe-Leu-Gly, both of which may be
cleaved by lysosomal proteases such as cathepsin B (11). In
these compounds, an amide bond linked the piperazine ring of
norfloxacin to the C-terminal amino acid (i.e., leucine or gly-
cine). As this amide bond is not destroyed by cathepsin B in
vitro, Leu-norfloxacin and Gly-norfloxacin, respectively, are
released (4). These derivatives, as well as Lys-norfloxacin, had
no activity against E. coli, S. aureus, and B. melitensis (this study
and unpublished results). Another norfloxacin derivative,
4-bromoethyloxy-carbonyl-norfloxacin, displayed only low lev-
els of activity against E. coli. This residual activity is due to
chemical hydrolysis at pH 7.4, which leads to the subsequent
release of native norfloxacin at proportions of 7% after 1 day
and 18% after 3 days (7). All these observations demonstrate
that the release of native norfloxacin is essential for antibac-
terial activity.
DISCUSSION
The medical importance of killing intracellular bacteria has
led to the development of different macromolecular prodrugs
As it is critical that the antibiotic be released in its active
form, we linked norfloxacin to the carrier through an NOC
bond to the C-terminal glycine (called the ␣ bond) of Gly-Phe-
Gly-Gly: the piperazine ring of norfloxacin substitutes for an ␣
hydrogen atom of the methylene group of the glycine residue
to form an ␣-substituted glycine, as suggested by Nichifor and
Schacht (12). The drug forms the lateral chain of the ␣-sub-
stituted glycine. When the atom (of the drug forming the
lateral chain) bound to glycine is N, O, or S, the ␣-substituted
glycine is unstable unless its amino group is engaged in an
amide bond. Under the conditions of our study, the amino
group of the glycine derivative was linked to another glycine
residue, its COOH was blocked by a methanol residue, and it
bore norfloxacin linked through the secondary amine of its
piperazine ring. In the presence of cathepsin B, the C-terminal
FIG. 7. In vitro anti-M. bovis BCG activities of isoniazid (closed
squares), norfloxacin (diamonds), and the nonmannosylated macro-
molecular conjugate of norfloxacin containing an ␣ bond (inverted
triangles) as a function of the molar concentration of active drug (i.e.,
isoniazid or norfloxacin) in the medium. Bacterial multiplication was
assessed by measuring the incorporation of [3H]uracil by M. bovis.

VOL. 47, 2003
TARGETED MACROMOLECULAR PRODRUGS OF NORFLOXACIN
3441
amide bond of Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe was hy-
drolyzed, releasing the unstable ␣-substituted glycine, which
spontaneously decomposed to release native norfloxacin.
In vitro, about 1% norfloxacin was released from the tet-
rapeptide Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe per hour at
the blood pH. This slow norfloxacin release explains its in vitro
activity against M. bovis. Gly-Phe-Gly-Gly(␣-norfloxacin)-OMe is
also susceptible to proteolysis by cathepsin B, which can occur
in the lysosome. In the lysosomal environment, the proteolytic
release of norfloxacin from the linking arm may be more rapid
than that in vitro, as several different proteases with different
specificities are present. This peptide can be used for the ly-
sosome-controlled release of norfloxacin because it is sepa-
rated from its temporary carrier after uptake of the macromo-
lecular prodrug by the macrophages. A release occurring
mainly in the phagolysosome, i.e., in close contact with the
pathogenic bacteria, should avoid the renal elimination of the
antibiotic before it has time to act on the bacteria.
Among the four conjugates tested, only the conjugate con-
taining both mannose as a homing device and an ␣ bond was
active in vivo against M. bovis BCG. Several conclusions can be
drawn from these results.
First, the fact that native norfloxacin did not exert an anti-
bacterial effect in vivo demonstrates the importance of and the
interest in drug targeting. This antibiotic, which was active in
vitro and inactive in vivo, became active in vivo when it was
carried by a targeted macromolecule.
sults indicate that antibiotic was released close to the bacteria,
thus supporting the observations of Clemens and Horwitz (3).
Finally, as the conjugate containing an amide bond had no
antimicrobial activity, it can be concluded that the carrier had
no antimicrobial effect by itself.
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