ORAL FORMULATIONS COMPRISING TIGECYCLINE
This application claims priority to U.S. Application No. 60/753,035, filed
December 22, 2005, which is hereby incorporated by reference.
[001] In one embodiment, this invention relates to oral formulations
comprising tigecycline.
[002] Tigecycline is a glycylcycline antibiotic, i.e., a t-butylglycyl
substituted naphthacenecarboxamide free base, and an analog of the
semisynthetic tetracycline, minocycline.

[003] Tetracyclines such as chlortetracycline hydrochloride (Aureomycin)
and oxytetracycline (Terramycin) are safe and have been used therapeutically as
broad-spectrum antibiotics since 1948. However, the emergence of resistance to
these antibiotics had limited their continued widespread usage. Tigecycline was
thus developed as an agent to potentially restore therapeutic utility to tetracyclines
by overcoming tetracycline resistance mechanisms. Tigecycline may also provide
activity against emerging multi-drug resistant pathogens. Glycylcyclines, including
tigecycline, are active against many antibiotic-resistant gram-positive pathogenic
bacteria, such as methicillin-resistant Staphylococcus aureus, penicillin-resistant
Streptococcus pneumoniae, and vancomycin-resistant enterococci (Weiss et al.,
1995; Fraise et al., 1995), Tigecycline is also active against bacterial strains
carrying the two major forms of tetracycline resistance, efflux and ribosomal
protection (Schnappinger and Hillen, 1995).
[004] Minocycline is currently available in oral and IV forms. Although an
intravenous formulation of tigecycline has been prepared, simple oral immediate
release prototypes containing tigecycline have resulted in poor bioavailability in

animals. (Petersen et al., Antimicrobial Agents and Chemotherapy, April 1999,
Vol. 43, No. 4 p. 738-744.)
[005] Tigecycline is very soluble in water with solubility greater than 295
mg/mL over the entire pH range of 1 to 14. However, cell monolayer permeability
studies of tigecycline (1 mM in ethanol and buffer, pH 6 to 6.4) show a low value
of 0.4 nm s-1 suggesting a low Gl permeability, which is consistent with the low
oral bioavailability found in animals.
[006] Accordingly, there remains a need to develop an oral formulation of
tigecycline.
BRIEF DESCRIPTION OF THE DRAWINGS
[007] FIG. 1 is a plot of percent release of tigecycline (y-axis) versus time
(x-axis, min);
[008] FIG. 2 shows the analytical performance of tigecycline in monkey
plasma, low QC (quality control) - 300 ng/mL as a plot of tigecycline plasma
concentration (y-axis) vs. curve number (x-axis);
[009] FIG. 3 shows the analytical performance of tigecycline in monkey
plasma, mid QC A-663 ng/mL as a plot of tigecycline plasma concentration (y-
axis) vs. curve number (x-axis);
[010] FIG. 4 shows the analytical performance of tigecycline in monkey
plasma, mid QC B-556 ng/mL as a plot of tigecycline plasma concentration (y-
axis) vs. curve number (x-axis);
[011] FIG. 5 shows the analytical performance of tigecycline in monkey
plasma, high QC - 3000 ng/mL as a plot of tigecycline plasma concentration (y-
axis) vs. curve number (x-axis);
[012] FIG. 6 is a plot of plasma concentration (y-axis) vs. time (x-axis)
profile of tigecycline in monkeys after a single intravenous dose of 5 mg/kg;
[013] FIG. 7 is a plot of tigecycline plasma concentration (y-axis) vs.
curve number (x-axis), showing the analytical performance of tigecycline assay in
monkey plasma: low QC (quality control) - 30 ng/mL;
[014] FIG. 8 is a plot of tigecycline plasma concentration (y-axis) vs.
curve number (x-axis), showing the analytical performance of tigecycline assay in
monkey plasma: middle QC - 300 ng/mL;

[015] FIG. 9 is a plot of tigecycline plasma concentration (y-axis) vs.
curve number (x-axis), showing the analytical performance of tigecycline assay in
monkey plasma: high QC - 800 ng/mL; and
[016] FIG. 10 is a plot of plasma concentration of tigecycline (ng/ml, y-
axis) vs. time (h, x-axis) after a single oral dose (1.00 mg encapsulated
microparticulate capsule) in fasted male cynomolgus monkey.
[017] One embodiment of the present invention provides a
pharmaceutical composition comprising tigecycline having at least one enteric
coating. In one embodiment, the composition is in oral dosage form. The enteric
coated tigecycline compositions may further comprise one or more of the further
ingredients described herein.
[018] In one embodiment, "having an enteric coating" refers to
surrounding a bulk of tigecycline. In another embodiment, the enteric coating
surrounds substantially each Tigecycline particle. "Coating" can comprise either a
coating or subcoating. "Coating," or "surrounds" as used herein, may range, for
example, from at least partially coating or surrounding up to and including a
complete coating or surrounding. In one embodiment, coating or surrounding
refers to substantially coating, such as 90%, 95%, and 99% coating by weight. In
one embodiment, the enteric coating may be sufficiently uniform to confer physical
stability to the tigecycline, e.g., by preventing degradation by any method
disclosed herein.
[0-19] In one embodiment, an "enteric coating" can allow at least a
substantial portion of a formulation to pass through the stomach and disintegrate
in the intestines. Exemplary materials for the preparation of enteric coatings
include, but are not limited to, hydroxypropylmethylcellulose,
hydroxyethylcellulose, methylhydroxyethylceliulose, sodium
carboxymethylcellulose, hydroxypropylcellulose, polyvinyl pyrrolidone,
dimethylaminoethyl methacrylatemethylacrylate acid ester copolymer, anionic
acrylic resins such as methacrylic acid/methyl acrylate copotymer and methacrylic
acid/ethyl acrylate copolymer, ethylacrylate-methylmethacrylate copolymer,
hydroxypropylmethylcellulose acetate succinate (HPMCAS),
hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate
(CAP), carboxymethylcellulose acetate phthalate (CMCAP), shellac,
methylcellulose, and ethylcellulose, and blends and copolymers thereof.

[020] In one embodiment, the enteric coating may be formed by methods
known in the art for forming polymeric films.
[021] In one embodiment, the composition further comprises a seal coat.
In one embodiment, the seal coat is positioned underneath the enteric coat. In
another embodiment, the composition can contain at least one additional seal coat
that overcoats the enteric coat, which in turn overcoats a first seal coat. In one
embodiment, the seal coat comprises any material suitable for forming enteric
coatings, such as hydroxypropyl cellulose, polyvinyl pyrrolidone, sodium
carboxymethy(cellulose, and hypromellose, or any other enteric coating material
disclosed herein.
[022] In one embodiment, the at least one enteric coating can protect
tigecycline from substantial degradation. Tigecycline may have at least two
degradation mechanisms. At low pH, epimerization of the dimethylamino group at
4-position has been identified as a major degradation route. At pH higher than
7.4, the degradation mechanism shifts to oxidation, as the phenolic groups can
become deprotonated. Tigecycline can, for example, be stabilized in both solid
and solution states by eliminating oxygen. Once oxygen is eliminated, the pH of
optimum stability shifts from 4.5 to 8 where epimerization is at its minimum.
[023] In one embodiment, the enteric coating allows delivery of the oral
formulation to the gastrointestinal (Gl) tract for selective release into the
gastrointestinal tract such as the lower gastrointestinal tract. The gastrointestinal
tract includes the upper and lower Gl tract. The upper Gl tract includes the
stomach and esophagus. In one embodiment, "lower gastrointestinal tract" as
used herein refers to the ileum and large intestine. "lleum" as used herein refers
to a third part of the small intestine that continues to the duodenum and jejunum.
"Large intestine" as used herein comprises the cecum, colon, and rectum.
"Cecum" refers to a blind sack (cul-de-sac) starting from the large intestine and in
one end of which the ileum opens.
[024] In one embodiment, the oral formulation does not release a
substantial amount of tigecycline in the stomach and a substantial release occurs
when the formulation reaches the gastrointestinal tract, such as the lower Gl tract.
[025] In one embodiment, the composition further comprises at least one
chelating agent. Calcium binds to tetracyclines, which reduce its water solubility.
There may be a 30 to 40% loss of tigecycline due to precipitation of the calcium

complex at pH 7.4. Thus, calcium binding and subsequent precipitation of the
calcium/tigecycline salt may be at least partially responsible for low oral
bioavailability. Exemplary chelating agents include ethylenediaminetetraacetic
acid (EDTA), O,O'-bis(2-aminoethyl.)ethyleneglycol-N,N,N',N'-tetraacetic acid
(EGTA), citrates, and tartrates.
[026] In one embodiment, the composition further comprises at least one
base. In one embodiment, the at least one base provides the composition with a
microenvironment having a pH ranging from 4 to 8.5 when released, such as a pH
ranging from 7.8 to 8.5 when released. In one embodiment, the pH of the
microenvironment refers to the pH of the area immediately surrounding the
composition. In another embodiment, the microenvironment refers to the area
inside the seal coat. Exemplary bases include, but are not limited to, phosphates,
such as at least one sodium phosphate, carbonates such as sodium and
potassium carbonate, bicarbonates, such as sodium and potassium bicarbonate,
citrates, such as sodium citrate, and tartrates.
[027] Additionally, in some embodiments, buffer species can negatively
affect the stability of tigecycline. In one embodiment, the at least one base may
be capable of countering the effects of such buffer species.
[028] In one embodiment, the composition further comprises at least one
biopolymer. For example, in embodiments where the composition is used to treat
infections in the Gl tract, such as the inner or lower Gl tract, the at least one
biopolymer can act as an adhesive to the inner Gl tract and therefore allow for
enhanced absorption of tigecycline. Exemplary biopolymers include, but are not
limited to, hypromellose and xanthan gum, and carbomer.
[029] "Pharmaceutical composition" as used herein refers to a medicinal
composition. The pharmaceutical composition may contain at least one
pharmaceutically acceptable carrier.
[030] In one embodiment, the composition further comprises at least one
inert, pharmaceutically acceptable-excipient or carrier. "Pharmaceutically
acceptable excipient" as used herein refers to pharmaceutical carriers or vehicles
suitable for administration of tigecycline including any such carriers known to
those skilled in the art to be suitable for oral administration.
[031] Suitable excipients include, for example, (a) fillers or extenders
such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders

such as cellulose and cellulose derivatives (such as
hydroxypropylmethylcellulose, hydroxypropylcellulose, and
carboxymethylcellulose), alginates, gelatin, polyvinylpyrrolidone, sucrose, and
acacia; (c) humectants such as glycerol; (d) disintegrating agents such as sodium
starch glycolate croscarmellose agar-agar, calciurn carbontate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding
agents such as paraffin; (f) absorption accelerators such as quaternary
ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol
monostearate, fatty acid esters of sorbitan, poloxamers, and polyethylene glycols;
(h) absorbents such as kaolin and bentonite clay; (i) lubricants such as talc,
calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof; and (j) glidants (antiadherents) such as talc, and
silicone dioxide. Other suitable excipients include, for example, sodium citrate or
dicalcium phosphate. The dosage forms may also comprise buffering agents.
[032] Oral formulations may also employ fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as well as high
molecular weight polyethylene glycols.
[033] The pharmaceutical compositions may optionally contain
opacifying agents and colorants. They may also be in a form capable of
controlled or sustained release. Examples of embedding compositions that can
be used for such purposes include polymeric substances and waxes.
[034] Where the-cornposition is a suspension containing powdered
tigecycline, the suspension can further comprise, for example, from about 0.05%
to 5% of suspending agent by weight, syrups containing, for example, from about
10% to 50% of sugar by weight, and elixirs containing, for example, from about
20% to 50% of ethanol by weight.
[035] The pharmaceutical compositions disclosed herein may contain, for
example, an amount ranging from about 25% to about 90% of the active
ingredient relative to the total weight of the composition, or from about 5% and
60% by weight.
[036] The tigecycline can be provided as a pharmaceutically acceptable
salt. The terms "pharmaceutically acceptable salt" can refer to acid addition salts
or base addition salts of the compounds in the present disclosure. A
pharmaceutically acceptable salt is any salt which retains the activity of the parent

compound and does not impart any deleterious or undesirable effect on the
subject to whom it is administered and in the context in which it is administered.
Pharmaceutically acceptable salts include metal complexes and salts of both
inorganic and organic acids. Pharmaceutically acceptable salts include metal
salts such as aluminum, calcium, iron, magnesium, manganese and complex
salts. Pharmaceutically acceptable salts include acid salts such as acetic,
aspartic, alkylsulfonic, arylsulfonic, axetil, benzenesulfonic, benzoic, bicarbonic,
bisulfuric, bitartaric, butyric, calcium edetate, camsylic, carbonic, chlorobenzoic,
cilexetil, citric, edetic, edisylic, estolic, esyl, esylic, formic, fumaric, gluceptic,
gluconic, glutamic, glycolic, glycolylarsanilic, hexamic, hexylresorcinoic,
hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic,
lactic, lactobionic, maleic, malic, malonic, mandelic, methanesulfonic, methylnitric,
methylsulfuric, mucic, muconic, napsylic, nitric, oxalic, p-nitromethanesulfonic,
pamoic, pantothenic, phosphoric, monohydrogen phosphoric, dihydrogen
phosphoric, phthalic, polygalactouronic, propionic, salicylic, stearic, succinic,
sulfamic, sulfanilic, sulfonic, sulfuric, tannic, tartaric, teoclic, toluenesulfonic, and
the like. Pharmaceutically acceptable salts may be derived from amino acids,
including but not limited to cysteine. Other acceptable salts may be found, for
example, in Stahl et al., Pharmaceutical Salts: Properties, Selection, and Use,
Wiley-VCH; 1st edition (June 15, 2002).
[037] Another embodiment provides a method of preparing a
pharmaceutical composition comprising coating a tigecycline with at least one
enteric coating. The coating can be performed using any known process in the
art, such as by introducing the tigecycline into a fluid bed processor (or other
coating device, such as a pan coater) containing the enteric coating material.
Prior to its introduction into the coating device, the tigecycline can be combined
with one or more of at least one base/buffer, at least one chelating agent, at least
one biopolymer, and other ingredients suitable for the oral formulation.
[038] Another embodiment provides a method of treating at least one
bacterial infection, comprising:
orally administering to a subject in need thereof a pharmaceutical
composition comprising a therapeutically effective amount of tigecycline having at
least one enteric coating.

[039] Another embodiment provides a method of treating antibiotic
associated pseudomembranous colitis caused by C. difficile and enterocolitis
caused by S. aureus and associated methicillin resistant strains comprising:
orally administering to a subject in need thereof a pharmaceutical
composition comprising a the rapeutically effective amount of tigecycline having at
least one enteric coating.
[040] In one embodiment, "therapeutically effective amount" refers to that
amount of a compound that results in prevention or amelioration of symptoms in a
patient or a desired biological outcome, e.g., improved clinical signs, delayed
onset of disease, reduced/elevated levels of lymphocytes and/or antibodies, etc.
The effective amount can be determined by one of ordinary skill in the art. The
selected dosage level can depend upon the severity of the condition being
treated, and the condition and prior medical history of the patient being treated.
However, it is within the skill of the art to start doses of the compound at levels
lower than required to achieve the desired therapeutic effect and to gradually
increase the dosage until the desired effect is achieved.
[041] In one embodiment, the subject treated can be a mammal, such as
a human. In one embodiment, the subject is suspected of having a bacterial
infection, e.g., shows at least one symptom associated with the infection. In
another embodiment, the subject is one susceptible to having the bacterial
infection, for example, a subject genetically disposed to having the disease.
[042] "Treating" as used herein refers to both therapeutic treatment and
prophylactic/preventative measures. Those in need of treatment may include
individuals already having a particular medical disease as well as those at risk for
the disease (i.e., those who are likely to ultimately acquire the disorder). A
therapeutic method results in the prevention or amelioration of symptoms or an
otherwise desired biological-outcome and may-be evaluated by improved clinical
signs, delayed onset of disease, reduced/elevated levels of lymphocytes and/or
antibodies, etc.
[043] In one embodiment, the administering is performed with a nasal-
gastric tube.
[044] Actual dosage levels of tigecycline in the pharmaceutical
compositions of this invention may be varied so as to obtain the therapeutically

effective amount necessary to achieve the desired therapeutic response for a
particular patient.
[045] Generally dosage levels of about 0.1 ug/kg to about 50 mg/kg,
such as a level ranging from about 5 to about 20 mg of active compound per
kilogram of body weight per day, can be administered topically, orally or
intravenously to a mammalian patient. Other dosage levels range from about 1
g/kg to about 20 mg/kg, from about 1 g/kg to about 10 mg/kg, from about 1
g/kg to about 1 mg/kg, from 10 g/kg to 1 mg/kg, from 10 g/kg to 100 g/kg,
from 100 g to 1 mg/kg, and from about 500 g/kg to about 5 mg/kg per day. If
desired, the effective daily dose may be divided into multiple doses for purposes
of administration, e.g., two to four separate doses per day. In one embodiment,
the pharmaceutical composition can be administered once or twice per day.
[046] In one embodiment, the tigecycline is multi-particulate. As used
herein, "multi-particulate tigecycline" refers to a collection of tigecycline particles.
In one embodiment, the multi-particulate tigecycline has a mean particle size
ranging from 0.3 mm to 1.5 mm. The multi-particulate tigecycline can be provided
as a powder, or provided as a capsule encased within a shell, or any other dosage
form as described herein.
[047] In one embodiment, dosage forms for oral administration include,
but are not limited to, capsules, tablets, pills, powders (e.g., dispersible powders,
suspensions containing such powders), dragees, granules, and lyophilized cakes
and powders. Such forms may include forms that dissolve or disintegrate quickly
in the oral environment. In another embodiment, the oral dosage form slows the
dissolution of the drug immediately following oral administration and allows a
substantial portion of the dissolution to occur in the Gl tract, such as the lower Gl
tract. In one embodiment, the dosage form (e.g., powders, cakes) is provided in
vials or other suitable containers.
[048] In one embodiment, the pharmaceutical composition comprises a
compressed tablet containing tigecycline in an amount ranging from 100 mg to
300 mg.
[049] In one embodiment, the pharmaceutical composition comprises
enteric coated multi-particulate pellets incorporated into a hard gelatin capsule,
and each pellet comprising tigecycline and microcrystalline cellulose, and a
combination of one or more of the following: at least one base/buffer (e.g., at

least one sodium phosphate), at least one chelating agent (e.g., EDTA), and at
least one biopolymer (e.g., xanthan gum).
[050] In one embodiment, the pharmaceutical composition comprises an
enteric coated tablet comprising tigecycline and microcrystalline cellulose, and
further comprises one or more of the following: at least one base/buffer (e.g., at
least one sodium phosphate), at least one chelating agent (e.g., EDTA), and at
least one biopolymer (e.g., xanthan gum).
[051] In one embodiment, the pharmaceutical composition comprises
multi-particulate pellets incorporated into an enteric coated soft gelatin capsule,
and each pellet comprising tigecycline and microcrystalline cellulose, and one or
more of the following: at least one base/buffer (e.g., at least one sodium
phosphate), at least one chelating agent (e.g., EDTA), and at least one
biopolymer (e.g., xanthan gum).
[052] In one embodiment, the pharmaceutical composition comprises an
enteric coated soft liquid gel capsule, and further comprising a non-aqueous
solution of tigecycline, and one or more of the following: at least one base/buffer
(e.g., at least one sodium phosphate), at least one chelating agent (e.g., EDTA),
and at least one biopolymer (e.g., xanthan gum).
[053] In one embodiment, the pharmaceutical composition comprises a
capsule or bi-layer tablet comprising both an immediate release portion and an
extended release portion. In one embodiment, "extended release" involves
release of substantially all of the tigecycline over a time period of at least 4 hours,
such as a time period of at least 6 hours, at least 12 hours, at least 24 hours, or at
least 48 hours.
[054] In one embodiment, the pharmaceutical composition may be used
as a treatment against drug-resistant bacteria. For example, it may be active
against methicillin-resistant Staphylococcus aureus, penicillin-resistant
Streptococcus pneumoniae, vancomycin-resistant enterococci (D.J. Beidenbach
et. al., Diagnostic Microbiology and Infectious Disease 40:173-177 (2001); H.W.
Boucher et. al., Antimicrobial Agents & Chemotherapy 44:2225-2229 (2000); P.A.
Bradford Clin. Microbiol. Newslett. 26:163-168 (2004); D. Milatovic et. al.,
Antimicrob. Agents Chemother. 47:400-404 (2003); R. Patel et. al., Diagnostic
Microbiology and Infectious Disease 38:177-179 (2000); P.J. Petersen et. al.,
Antimicrob. Agents Chemother. 46:2595-2601 (2002); and P.J. Petersen et. al.,

Antimicrob Agents Chemother. 43:738-744(1999), and against organisms
carrying either of the two major forms of tetracycline resistance: efflux and
ribosomal protection (C. Betriu et. al., Antimicrob. Agents Chemother. 48:323-325
(2004); T. Hirata et. al. Antimicrob. Agents Chemother. 48:2179-2184 (2004); and
E.J. Petersen et al., Antimicrob Agents Chemother 43: 738-744(1999).
[055] In one embodiment, the pharmaceutical composition may be used
in the treatment of many bacterial infections, such as complicated intra-abdominal
infections (clAI), complicated skin and skin structure infections (cSSSl),
Community Acquired Pneumonia (CAP), and Hospital Acquired Pneumonia (HAP)
indications, which may be caused by gram- negative and gram-positive
pathogens, anaerobes, and both methicillin- susceptible and methicillin-resistant
strains of Staphylococcus aureus (MSSA and MRSA). Additionally, the
pharmaceutical composition may be used to treat or control bacterial infections in
warm-blooded animals caused by bacteria having the TetM and TetK resistant
determinants. Also, the pharmaceutical composition may be used to treat bone
and joint infections, catheter-related Neutropenia, obstetrics and gyne'cological
infections, or to treat other resistant pathogens, such as VRE, ESBL, enterics,
rapid growing mycobacteria, and the like.
[056] In one embodiment, the pharmaceutical composition may be used
in the treatment of bacterial infection in the gastrointestinal tract, such as the lower
gastrointestinal tract.
[057] In one embodiment, the anaerobe is Clostridium difficile.
EXAMPLES
Example 1
[058] In this Example, the dissolution behavior of enteric coated
tigecycline granules in capsules was investigated in a solution of 0.1 N HCl, then in
phosphate buffer pH 6.8 at 37°C. These conditions mimic the gastric system
(0.1 N) and the lower intestinal tract (pH 6.8).
[059] The formulation used is described in Example 3, below.
[060] Gelatin capsules of enteric coated granules of 100 mg tigecycline
were added to three separate vessels (Capsules 1, 2, and 3). The capsules were
dissolved with a USP Apparatus 2 (paddles) at 100 rpm in 750 mL of 0.1 N HCI at

37°C. The dissolution was allowed to occur for 2 h, followed by addition of 250
mL of 0.2M Na3PO4. The pH of this mixture was adjusted to 6.8. Table I below
lists the dissolution data.
Table I. Percent release of gelatin capsules of enteric coated 100 mg
tigecvcline granules

[061] FIG. 1 is a plot of the data of Table I of percent release (x-axis)
versus time (min). The ratio of AUC to mg/ml is according to the equation y =
16279x - 58.773.
[062] This Example demonstrates that the formulation releases
substantially most of the tigecycline at higher pH, e.g., after 2 hours.
Example 2
[063] This Example demonstrates the oral bioavailability of tigecycline in
cynomolgus monkeys when administered as an oral formulation (gavage). The

pharmacokinetics of tigecycline after single oral and intravenous administration
are also presented in this Example.
[064] Male monkeys were first administered an oral (gavage) dose of 15
mg/kg of tigecyciine and then an intravenous dose of 5 mg/kg of tigecycline after a
one-week wash-out period.
MATERIALS AND METHODS
Study Design
[065] Four male cynomolgus monkeys were used in the study. In a first
dosing period, each monkey was administered a single 15 mg/kg oral (gavage)
dose of tigecycline in 0.9% saline. The dosing volume was 10 mL/kg. Blood
samples (2 mL per sample) were obtained prior to dosing (0 hr) and at 0.5, 1, 2, 4,
6, 8, 12, 24, 32 and 48 hr after the oral dose. After a one-week washout period,
each monkey was administered a single 5 mg/kg intravenous dose of tigecycline
in 0.9% saline. Blood samples (2 mL) were obtained pre-dose (0 hr) and at 5
mm., 0.5, 1, 2, 4, 6, 8, 12, 24, 32 and 48 hr post-dose. Blood samples were
collected using a stainless steel needle and vacutainer tube containing sodium
heparin as the anticoagulant. Blood samples were placed on ice after collection
and centrifuged at approximately 4°C. Plasma samples was separated, frozen
and stored at approximately -70°C prior to analysis.
Quantitation of Tigecycline in Monkey Plasma
[066] Tigecycline concentrations were determined using an HPLC
method that was previously validated in rat and dog plasma, although this method
was modified to be used in monkey plasma. In this method, tigecycline in 0.2 mL
of monkey plasma samples was extracted by protein precipitation with acetonitrile
and the precipitated proteins were separated by centrifugation. The supernatant
was evaporated and the extract was reconstituted in 0.05N HGI for HPLC
analysis. Regression analysis was performed on the calibration curve using a
quadratic fit with a weighting factor of 1/(concentration)2. By using 0.2 mL of
monkey plasma sample, the assay limit of quantitation (LOQ) was 100 ng/mL and
the curve range was between 100 and 6400 ng/mL.

Pharmacokinetic Calculations
[067] Pharmacokinetic parameters were calculated using the
pharmacokinetics analysis program WinNonlin, version 2.1 (Scientific Consulting
Inc.) from the individual animal concentration vs. time profiles. This program
analyzes data using a model-independent approach and the standard methods
described by Gibaldi and Perrier (Gibaldi M, Perrier D., Pharmacokinetics, 2nd ed.,
Marcel Dekker, Inc., NY, 1982). For the purpose of this analysis, no attempt was
made to back extrapolate the concentration immediately after the IV bolus dose,
rather the concentration at 0 hr (Co, immediately after dosing) was assumed to be
equal to the first measured concentration (at 5 minutes, C5min). To determine the
mean plasma drug concentrations, all values below the lower limit of quantitation
(LOQ = 100 ng/mL) were treated as zero. The terminal half-life (t1/2) was
determined by 0.693/., where  is the terminal rate constant and is determined by
a log-linear fitting of the terminal portion of the concentration-time curve. AUC0-4
was calculated by AUC0-t + Ct/, where AUC0-t was the AUC from time 0 to t, the
last quantifiable time point and Ct was the last quantifiable concentration. The
area under the plasma concentration-time curve from time 0 to t (AUC0-t) was
calculated using the linear trapezoidal method. Systemic clearance (CLT) after
the iv dose was calculated using the formula of Dose/AUC0-4. The volume of
distribution at steady-state (Vdss) was calculated using the formula of MRTiv x CLT,
where MRTiv, is the mean residence time after iv dosing and equals AUMC0-
4/AUC0-4. For the oral dose, Cmax and tmax values were obtained by inspection of
the concentration vs. time curves. Due to the paucity of quantifiable
concentrations after oral administration, the AUC0-4 could not be calculated.
Analytical Performance of the HPLC Method for Tigecycline in Monkey Plasma
[068] Five analytical runs were performed for the analysis of samples.
The back-calculated values of the calibration curves are presented in Table II.
The CV of tigecycline calibration standards were between 2.1 and 6.3% and the
bias values ranged from -5.4 to 3.8%.

Table II. Analytical Performance of Tigecycline Assay in Monkey Plasma:
Back-Calculated Values of Tigecycline Calibration Standards

NA: Not applicable
[069] The calibration curve parameters are shown in Table III.

Table HI. Analytical Performance of Tigecycline Assay in Monkey Plasma:
Calibration Curve Parameters

[070] Regression analysis was performed with the following equation:
y = ax2 + bx + c
—where:
a = 2nd Order regression line constant.
b = 1st Order regression line constant.
c = Intercept.
y = Internal standard peak height ratio of tigecycline.
x = tigecycline concentration (ng/mL).
[071] In all analytical runs, the coefficients of determination (R2) were
>0.99. In all analytical runs, two replicates of low, mid-range and high QC
samples were analyzed along with study samples. The low QC and the high QC
have nominal concentrations of 300 and 3000 ng/mL, respectively. For the mid
range QC, the target nominal concentration was 900 ng/mL. Two separate
batches of mid-range QC were prepared and both had concentrations below the

target (ca. 600 ng/mL). The target concentrations of the mid-range QC batches
were determined by analyzing four (batch A) or eight (batch B) replicates of each
mid-range QC batch. Mid-range QC batch A (determined concentration of 663
ng/mL) was analyzed with curves 1 and 2. Mid-range QC batch B (determined
concentration of 556 ng/mL) was analyzed with curves 3, 4 and 6. The results of
QC samples from all analytical runs are shown in Table IV.
Table IV. Analytical Performance of Tigecycline Assay in Monkey Plasma:
Results of QC Samples

NA: Not applicable; this QC batch was not analyzed with this run.

[072] The CV of QC samples were between 5.9 and 13.1% and the
biases were between -1.0 and 7.7%. The QC results are also depicted in QC
charts and they are presented in FIGs. 2 to 5.
Pharmacokinetics of Tigecycline in Cynomolgus Monkeys
[073] The concentrations of tigecycline after a single 15 mg/kg oral dose
in monkeys are presented in Table V.
Table V. Plasma Concentrations (ng/mL) of Tigecycline in Monkeys After a
Single Oral (gavage) Pose of 15 mq/kq

[074] The concentrations of tigecycline after a single 5 mg/kg iv dose are
shown in Table VI.

Table VI. Plasma Concentrations (ng/mL) of Tigecycline in Monkeys After a
Sinqle Intravenous Pose of 5 mg/kg

[075] Plasma concentrations vs. time profiles after a single iv dose of
tigecycline in monkeys are depicted in FIG. 6. Pharmacokinetic parameters from
individual animals are tabulated in Table VII.

Table VII. Individual and Mean (± SD) Pharmacokinetic Parameters of
Tiqecycline in Monkeys After a Single Oral (gavage) Dose of 15 mg/kg or
After a Single Intravenous Dose of 5 mg/kg

a Cmax = C5min. after the iv dose.
b t = 2 hr for AUC determination.
c t = 1 hr for AUC determination.
NA: Not applicable.
nc: AUCO-4 or t1/2 value not calculated due to insufficient data in the apparent terminal phase.
[076] After a singie 15 mg/kg oral (gavage) dose, tigecycline was
detected in samples up to 2 hours post-dose. The mean (± SD) Cmax value was

163 ± 27.1 ng/mL and the tmax values were between 1 and 2 hours. Due to the
paucity of quantifiable concentrations in the terminal phase of the concentration
vs. time curves after oral dosing, AUC0-4, and t1/2 values were not estimated after
the oral dose. Also, due to the limited number of time points with quantifiable
tigecycline concentration and the partial AUC values estimated, absolute
bioavailability of tigecycline after oral dosing could not be determined.
[077] A 0.5% blood bioavailability is suitable for treating Gl tract
infections since the desired site of action is in the Gl tract and not in the blood.
Thus, a 0.5% blood bioavailability can translate to approximately 99%
bioavailability in the Gl tract.
[078] After a single 5 mg/kg intravenous dose in monkeys, the plasma
concentrations of tigecycline declined polyexponentially. The mean t1/2 value
estimated from the terminal phase of the plasma concentration vs. time curves
was 14.1 ± 3.4 hours, that was similar to the MRTiv of 12.8 ± 5.4 hours. The mean
(± SD) AUC0-4,. value of tigecycline was 18267 ± 3030 ng hr/mL. The mean
tigecycline CIT was 0.280 ± 0.053 L/kg/hr and the mean Vdss was 3.47 ± 1.09 L/kg.
Discussion
[079] The results of this study showed that the blood bioavailability of
tigecycline was low after oral administration. When treating GI tract infections, low
blood bioavailability is desired because the drug is kept within the stomach for
local action against the organisms in the Gl tract. The absolute bioavailability
could not be estimated after a single 15 mg/kg oral dose due to insufficient data in
the terminal phase for the estimation of AUC0-4 values. After a single iv dose in
monkeys, the plasma concentrations of tigecycline declined polyexponentially.
The terminal half-lives estimated from the terminal phase of the plasma
concentration vs. time curves were between 11.4 and 19.1 (mean 14.1) hours and .
were similar to the MRTiv (mean 12.8 hours). The systemic clearance (CIT) of
GAR-93 6 in monkeys was relatively low (mean 0.280 L/kg/hr) but similar to that in
dogs (ca. 0.26 L/kg/hr after a single 5 mg/kg dose). The steady-state volume of
distribution (Vdss) of tigecycline in monkeys was large (3.47 L/kg) and in excess of
the volume of total body water in this species (see Davies B, Morris T.
"Physiological parameters in laboratory animals and humans.," Pharm. Res. 1993;

10:1093-95), suggesti0ng that tigecycline should be distributed to various tissues
and organs.
Example 3
[080] This Example demonstrates the oral bioavailability in fasted male
cynomolgus monkeys from an encapsulated microparticulate (100 mg) formulation
administered as a single enteric coated oral formulation. Tigecycline plasma
concentrations were determined for the formulation type by an LC/MS/MS
method.
Materials and Methods
Formulation
[081] The tigecycline formulation was a 100 mg, encapsulated multi-
particulate formulation having the components listed in Table VIII below:

aPotency of tigecycline is adjusted against microcrystalline cellulose (MCC)
[082] The enteric coating comprised a Seal Coat, YS-1 -7006, and
Enteric Coat (Acryl-EZE). The final potency for enteric coated tigecycline was 209
mg/g. Each 100 mg capsule contained 478.5 mg enteric coated granules.

Experimental Design and Sample Collection
[083] The bioavailability of tigecycline was investigated with four male
cynomolgus monkeys, each having body weights ranging from 5.5 to 7.1 kg. The
monkeys were housed in Bioresources vivarium with free access to water and
food. The four monkeys received the oral formulation described above (1 x 100
mg multi-particulate capsule). The formulation was administered with 10 mL
water. All monkeys were fasted overnight prior to dosing (with free access to
water) and were fed 4 hours after dose administration.
[084] Blood samples were drawn from the saphenous vein at 0
(predose), 0.5, 1, 2, 3, 4, 8,12 and 24 hours after dosing. Approximately 3 mL of
blood were drawn into Vacutainer® tubes containing sodium heparin as the
anticoagulant. Plasma was separated in a refrigerated centrifuge and stored at
700C. Plasma samples were delivered to the assay site packed on dry ice.
[085] Plasma tigecycline concentrations were determined by an
LC/MS/MS method described above. Based on a 0.5 mL sample volume, the
method has a limit of quantitation of 10 ng/mL.
Determination of Tigecvcline Concentrations in Monkey Plasma
[086] Tigecycline concentrations were determined by an LC/MS/MS
method. Using 0.50 mL of sodium heparin monkey plasma, the lower limit of
quantitation (LLOQ) was 10.0 ng/mL and the assay range was 10.0 to
1000 ng/mL. To monitor assay performance, all analytical runs were analyzed
with low, mid-range, and high concentration (30, 300, and 800 ng/mL nominal
concentrations) quality control samples (QCs) in quintuplets.
Analytical Performance of Tigecycline LC/MS/MS Assay in Monkey Plasma
[087] There was one analytical run for the quantitation of tigecycline in
monkey plasma samples from this study. The back-calculated values of
tigecycline calibration standards prepared in monkey plasma and the calibration
curve regression constants are shown in Table IX.

Table IX. Analytical Performance of Tigecycline Assay in Monkey Plasma:
Back-Calculated Concentrations of Calibration Standards and Calibration
Curve Regression Constants
(A) Back-Calculated Concentrations of Tigecycline Calibration Standards in
Monkey Plasma

aA linear regression method was used with 1/concentration2 as the weighting factor.
[088] Linear regression was performed using a weighting factor of
1/(concentration)2. The mean biases of back-calculated calibration standards
ranged from -11.6% to 13.0%. The R2 value of the calibration curve was 0.9895.
[089] Results of tigecycline quality control (QC) samples prepared in
monkey plasma and analyzed with the study samples are summarized in Table X.


[090] The CV of the QC samples ranged from 1.9% to 6.1% and the
mean biases ranged from -14.3% to -2.7%. The QC results are also depicted
graphically in FIGs. 7 to 9.
Plasma Concentrations of Tigecycline in Monkeys
[091] Tigecycline plasma concentrations (ng/mL) in fasted monkeys after
a single oral dose (100 mg capsule) of tigecycline from an encapsulated
microparticulate formulation are presented in Table XI and shown graphically in
FIG. 10.


SAN: Study animal number
Plasma Concentration-Time Data Analysis
[092] Noncompartmental analysis of the individual monkey plasma
tigecycline concentration-time profiles was performed using WinNonlin, Model
200. Area under the plasma tigecycline concentration-time curves (AUC) were
calculated by log/linear trapezoid rule. The peak plasma tigecycline
concentrations (Cmax) and the time to reach Cmax (tmax) were noted directly from
the plasma tigecycline concentration-time profiles.
[093] The AUC (ng hr/mL, mean ± SD) value for the formulation was
2830 ± 1111. The Cmax value (ng/mL, mean ± SD) for the formulation was 225 ±
92.4.
Pharmacokinetics
[094] The individual and mean monkey pharmacokinetic parameters are
reported in Table XII.



[095] Table XIIl compares the mean pharmacokinetic parameters and
the absolute and relative bioavailability of tigecycline in the encapsulated multi-
particulate formulation to the 0.9% saline tigecycline solution administered IV and
orally (gavage), as described in Example 2 above.

Table XIII. Comparison of Pharmacokinetic Parameters [Mean (n=4)] in Male
Cvnomolqus Monkeys After A Single Dose Administration of Tigecycline

See Example 2
[096] The AUC (nghr/mL, mean ± SD) value for the formulation was
2830 ±1111. The Cmax values (ng/mL, mean ± SD) for the formulation was 225 ±
92.4.
[097] A bioavailability study of a tigecycline formulation has been
conducted in cynomolgus monkeys to assess the bioavailability of an enhanced
encapsulated microparticulate oral dosage formulation.
[098] The results of this study showed that the absolute bioavailability of
tigecycline in the blood was 5% after oral administration. The capsule formulation
(16 mg/kg) demonstrated significantly higher oral exposure (AUC) values as
compared to previous studies conducted by preclinical development at 15 mg/kg.
[099] When treating bacterial infections, a blood bioavailability of at least
5% can be suitable. For treating Gl tract infections, a 5% blood bioavailability can
translate to 95% availability in the Gl tract.

Example 4
[0100] This Example describes a dry powder layering process for the
preparation of an oral formulation. Table XIV lists the formulation ingredients.
Table XtV

[0101 ] In this example the tigecycline, lactose, sodium phosphate and
EDTA were blended together and fed through a screw feed into a fluid bed rotor
granulator containing sucrose or microcrystalline spheroids. A 5-10% binder
solution of hypromellose was sprayed simultaneously into the spinning bed of
spheroids while the tigecycline blend was slowly added. After the desired quantity
of tigecycline blend was added to the spheres, they were dried and discharged for
enteric coating. Enteric coating was applied via a fluid bed processor using
polymethacrylates. Other enteric polymers normally used in industry can also be
used.
[0102] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification and examples be
considered as exemplary only, with a true scope and spirit of the invention being
indicated by the following claims.

[0103] Unless otherwise indicated, all numbers expressing quantities of
ingredients, reaction conditions, and so forth used in the specification and claims
are to be understood as being modified in all instances by the term "about."
Accordingly, unless indicated to the contrary, the numerical parameters set forth in
the following specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the present
invention.

Claims:
1. A pharmaceutics! composition comprising tigecycline having at least one enteric
coating.
2. The composition according to claim 1, wherein the at least one enteric coating is
chosen from dimethylaminoethyl methacrylatemethylacrylate acid ester copolymer,
anionic acrylic resins such as methacrylic acid/methyl acrylate copolyrner and
methacrylic acid/ethyl acrylate copolymer, ethylacrylate-methylmethacrylate copolymer,
hydroxypropylmethylcellulose acetate succinate (HPMCAS),
hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP),
carboxymethylcellulose acetate phthalate (CMCAP), hydroxypropylmethylcellulose,
hydroxyethylcellulose, methylhydroxyethylcellulose, sodium carboxymethylcellulose,
hydroxypropylcellulose, polyvinyl pyrrolidone, shellac, methylcellulose, and
ethylcellulose, and blends and copolymers thereof.
3. The composition according to claim 1 or 2, wherein the composition is in an oral
dosage form chosen from capsules, tablets, pills, powders, granules, and lyophilized
cakes and powders.
4. The composition according to any one of claims 1 to 3, wherein the tigecycline is
multi-particulate.
5. The composition according to claim 4, wherein the multi-particulate tigecycline has a
mean particle size ranging from 0.3 mm to 1.5 mm.
6. A pharmaceutical composition comprising an enteric coated tablet, or enteric coated
multi-particulate pellets which are incorporated into hard or soft gelatin capsules,
wherein each tablet or pellet comprises tigecycline and microcrystalline cellulose, and at
least one component chosen from at least one base, at least one chelating agent, and at
least one biopolymer.

7. A pharmaceutical composition comprising an enteric coated soft liquid gel capsule,
and further comprising a non-aqueous solution of tigecycline and at least one base, at
least one chelating agent, and at least one biopolymer.
8. The composition according to any one of the preceding claims, further comprising at
least one base chosen from phosphates, carbonates, bicarbonates, citrates, and
tartrates.
9. The composition according to claim 8, wherein the at least one base is chosen from
sodium phosphates, sodium carbonate, sodium bicarbonate, and sodium citrate.

10. The composition according to any one of the preceding claims, further comprising at
least one chelating agent.
11. The composition according to any one of claims 6, 7, 8, or 10, wherein the at least
one chelating agent is chosen from EDTA, EGTA, citrates, and tartrates.
12. The composition according to any one of the preceding claims, further comprising at
least one biopolymer.
13. The composition according to any one of claims 6, 7, 8, or 12, wherein the at least
one biopolymer is chosen from hypromellose, xanthan gum, and carbomer.
14. A method of treating at least one bacterial infection chosen from complicated intra-
abdominal infections (clAI), complicated skin and skin structure infections (cSSSI),
Community Acquired Pneumonia (CAP), Hospital Acquired Pneumonia (HAP)
indications, bacterial infections caused by bacteria having the TetM and TetK resistant
determinants, bone and joint infections, catheter-related Neutropenia, obstetrics and
gynecological infections, and bacterial infections caused by VRE, ESBL, enterics, and
rapid growing mycobacteria, comprising: orally administering to a subject in need thereof
a pharmaceutical composition comprising a therapeutically effective amount of
tigecycline having at least one enteric coating.

15. The method according to claim 14, wherein the tigecycline is multi-particulate
tigecycline.

Disclosed herein are pharmaceutical compositions comprising tigecycline for oral administration. The composition
can comprise tigecycline having at least one enteric coating.