WO 2006/099258 PCT/US2006/008827
TIGECYCLINE COMPOSITIONS AND METHODS OF PREPARATION
This application claims priority from copending provisional application
Serial Number 60/661,030 filed March 14, 2005 the entire disclosure of which is
hereby incorporated by reference.
The present invention relates to improved tigecycline compositions and
methods for making such compositions. The inventive compositions have
improved stability in both solid and solution states. The inventive compositions
comprise tigecycline, a suitable carbohydrate, and an acid or buffer. The
combination of the suitable carbohydrate and the acid or buffer reduces
tigecycline degradation as explained below. The present invention provides
advantages over the prior art by providing for stable tigecycline compositions and
methods for making such compositions that achieve stability against both
oxidative degradation and eplmerization. These compositions are, therefore,
more stable when dissolved, lyophilized, reconstituted, and/or diluted than
compositions of tigecycline not made according to the invention.
Tigecycline is a known antibiotic in the tetracycline family and a chemical
analog of minocycline. It may be used as a treatment against drug-resistant
bacteria, and it has been shown to work where other antibiotics have failed. For
example, it is 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); PJ. Petersen
et. al., Antimicrob. Agents Chemother. 46:2595-2601 (2002); and P.J. Petersen et.
a!., 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
P.J. Petersen et. a!., Antimicrob. Agents Chemother. 43:738-744(1999).
1
WO 2006/099258 PCT/US2006/008827
Tigecyciine has historically been administered intravenously because it
exhibits generally poor bioavailability when given orally. Intravenous solutions
have largely been prepared immediately prior to use, e.g., administration to a
patient, from lyophilized powders because tigecyciine degrades in solution
principally via oxidation. It would be preferable as well as desirable to have an
intravenous formulation of tigecyciine that did not require immediate use and
could remain stable in solution for up to 24 hours.
Tigecyciine is currently manufactured as a lyophilized powder. Due to the
propensity for tigecyciine to degrade, these powders are prepared under low-
oxygen and low-temperature conditions in order to minimize degradation. Such
processing is expensive because it requires special equipment and handling.
The typical process for preparing these powder compositions involves
dissolving tigecyciine in water (compounding) and lyophilizing (freeze-drying) the
solution to dryness to form solid cakes of amorphous tigecyciine. These cakes
are then loaded under nitrogen into stoppered glass vials and shipped to end
users such as hospital pharmacies. Prior to being administered to patients, the
cakes are reconstituted, often in 0.9% saline, to a concentration of, for example,
about 10 mg/mL. At this concentration, tigecyciine degrades rapidly in solution
and therefore, must be used without delay. Thus, these reconstituted solutions
are immediately diluted (also known as admixing) to about 1 mg/mL with saline or
other pharmaceutically acceptable diluents into intravenous bags for patient
delivery.
In this diluted state, tigecyciine is ready for intravenous delivery to a
patient. At a concentration of 1 mg/mL, however, tigecyciine should be used
within 6 hours of dilution. Because intravenous infusions may take several hours,
hospital personnel must act quickly so that from the time admixture begins to the
time the tigecyciine dose has been administered to a patient, not more then 6
hours have elapsed. It would be more preferred to provide hospital staff with the
flexibility and advantages that come with longer admixture and reconstitution
times so that, for instance, a hospital pharmacist could prepare a solution the day
before it is needed to be administered to a patient.
2

WO 2006/099258 PCT/US2006/008827
Tigecycline has such a short admixture time and the reconstitution time is
essentially zero because in solution, tigecycline oxidation is relatively rapid.
Under current manufacturing, storage, and administration conditions, the most
prevalent form of degradation is via oxidation. The reason oxidation is the most
prevalent form of degradation in previous formulations relates to the chemical
structure of tigecycline. It possesses a phenol moiety, and it is well known in the
art of organic chemistry that phenols are particularly prone to oxidation. When
tigecycline is dissolved in water prior to lyophilization, the pH is slightly basic
(about 7.8). This is higher than the pKa of the phenolic group on tigecycline.
Thus, in both water and saline solutions, the phenolic group becomes
deprotonated and more susceptible to reaction with oxygen which is why
tigecycline compounding and lyophilization occur under a nitrogen blanket.
Accordingly, care to avoid unnecessary exposure to oxygen must be taken by
hospital staff during reconstitution and dilution.
If the pH of the tigecycline solution were less than the pKa of the phenolic
group on tigecycline, then oxidation would occur, but to a lesser extent. Indeed, it
has been observed that tigecycline oxidative degradation does decrease when the
pH is lowered. At low pH, however, another degradative process occurs,
epimerization. At lower pHs, epimerization emerges as the most predominant
degradation pathway.
Tigecycline differs structurally from its epimer in only one respect.

WO 2006/099258 PCT/US2006/008827
In tigecycline, the N-dimethyl group at the 4 carbon is cis to the adjacent hydrogen
as shown above in formula I, whereas in the epimer, formula II, they are trans to
one another in the manner indicated. Although the tigecycline epimer. is believed
to be non-toxic, it lacks the anti-bacterial efficacy of tigecycline and is, therefore,
an undesirable degradation product.
In the lyophilized state, tigecycline follows the same degradation pathways
as in solution, but the rate of degradation is slower. Thus, when tigecycline is
lyophilized in water such that the pH is about 7.8, the resulting iyophilized cake
exhibits oxidative degradation, albeit at a slower rate than in solution. Similarly,
when tigecycline is lyophilized in an acidic solution, the primary degradation
pathway is epimerization and it also occurs at a slower rate than in solution.
Epimerization is a known degradation pathway in tetracyclines generally,
although the rate of degradation may vary depending upon the tetracyciine.
Comparatively, the epimerization rate of tigecycline is particularly fast. The
tetracyciine literature reports several methods scientists have used to try and
minimize epimer formation in tetracyclines. In some methods, the formation of
calcium, magnesium, zinc or aluminum metal salts with tetracyclines limit epimer
formation when done at basic pHs in non-aqueous solutions. (Gordon, P.N,
Stephens Jr, C.R., Noseworthy, M. M., Teare, F.W., U.K. Patent No. 901,107). In
other methods, (Tobkes, U.S. Patent No. 4,038,315) the formation of a metal
complex is performed at acidic pH and a stable solid form of the drug is
subsequently prepared.
Other methods for reducing epimer formation include maintaining pHs of
greater than about 6.0 during processing; avoiding contact with conjugates of
weak acids such as formates, acetates, phosphates, or boronates; and avoiding
contact with moisture including water-based solutions. With regard to moisture
protection, Noseworthy and Spiegel (U.S. Patent No. 3,026,248) and Nash and
Haeger, (U.S. Patent No. 3,219,529) have proposed formulating tetracyciine
analogs in non-aqueous vehicles to improve drug stability. However, most of the
vehicles included in these inventions are more appropriate for topical than
parenteral use. Tetracyciine epimerization is also known to be temperature
dependent so production and storage of tetracyclines at low temperatures can
4

WO 2006/099258 PCT/US2006/008827
also reduce the rate of epimer formation (Yuen, P.H., Sokoloski, T.D., J. Pharm.
Sci. 66: 1648-1650,1977; Pawelczyk, E., Matlak, B, Pol. J. Pharmacol. Pharm. 34:
409-421,1982). Several of these methods have been attempted with tigecycline
but none have succeeded in reducing both epimer formation and oxidative
degradation while not introducing additional degradants. Metal complexation, for
example, was found to have little effect on either epimer formation or degradation
generally at basic pH.
Although the use of phosphate, acetate, and citrate buffers improve
solution state stability, they seem to accelerate degradation of tigecycline in the
lyophilized state. Even without a buffer, however, epimerization is a more serious
problem with tigecycline than with other tetracyclines such as minocycline.
Others of these methods similarly failed to reduce both epimerization and
oxidative degradation. Although it was found that maintaining a pH of greater than
about 6.0 helps reduce epimer formation, as noted above, such conditions lead to
greater oxygen sensitivity. With respect to non-aqueous vehicles, although water
is known to accelerate tigecycline degradation, it would be impractical to prepare
an intravenous medication using such vehicles.
Whereas it has been determined that processing at temperatures lower
than room temperature, such as below about 10°C, reduces the tigecycline
degradation rate, such processing is expensive and it would be advantageous to
use a composition that did not require expensive refrigeration during processing.
Chinese patent application CN 1390550A discloses that minocyciine could
be combined with an acid to increase the stability toward the oxidative
degradation. It further discloses the use of a caking agent, such as mannitol.
This reference says nothing about tigecycline nor does it suggest that
carbohydrates could be used to reduce either oxidation or epimerization for
minocycline in reduced pH environments. Indeed, minocycline can be formulated
as a hydrochloride salt in intravenous products without significant epimerization.
In tigecycline hydrochloride salts, however, significant epimerization occurs.
Thus, minocycline and tigecycline possess different epimerization properties.
In another experiment, minocycline was lyophilized at a pH of about 5.0
and the lyophilized cake was stored for 20 days at 40°C and 75% relative
5

WO 2006/099258 PCT/US2006/008827
humidity. At the end of the 20 days, the cake was analyzed by HPLC. The
epimer of minocycline was measured to be present at a level of 2.65% by mass.
By comparison, when tigecycline was lyophilized at a pH of about 5.0 and the
sample stored under the same conditions, but for only 4 days followed by HPLC
analysis, the tigecycline epimer was measured to be at a leve! of 5.40%, over
twice as much even though tigecycline was only stressed for 1/5th as long'as
minocycline. Thus, tigecycline epimerizes much more readily than minocycline,
and epimerization is a much more significant problem with tigecycline than it is for
minocycline.
The present invention addresses the various problems and disadvantages
of the prior art by providing for stable compositions of tigecycline in solid and
solution form. By lyophilizing an aqueous solution containing tigecycline and a
suitable carbohydrate at an acidic pH, we have prepared tigecycline compositions •
that are more stable against both oxidative degradation and epimerization than
existing compositions. Because the pH is acidic, oxidative degradation has been
minimized. Furthermore, it has been determined that suitable carbohydrates act
to stabilize-tigecycline-againstepimerformation-at acidic pHs
Compositions of the invention are more stable in the lyophilized state than
the existing compositions and do not require low-temperature or low-oxygen ■
processing conditions. Such compositions are also expected to possess
reconstitution and admixture stability times greater than that of the existing
compositions. For example, one embodiment of the invention is stable for 6 hours
after reconstitution and stable for an additional 18 hours after admixture. These
extended stability times make tigecycline much easier to use in a hospital
environment by providing needed flexibility to hospital staff when treating patients.
Solid-state compositions of the invention comprise tigecycline, a suitable
carbohydrate, and an acid or buffer.
Suitable carbohydrates are those carbohydrates capable of reducing
epimer formation in at least one solid form prepared in at least one pH
environment when compared to a tigecycline solid form prepared at the same pH
environment lacking suitable carbohydrates. In one embodiment, the pH
environment ranges from about 3.0 to about 7.0, such as pHs ranging from about
6

WO 2006/099258 PCT/US2006/008827
4.0 to about 5.0, or from about 4.2 to about 4.8. In one embodiment, the at least
one solid form is chosen from powders and lyophilized cakes of tigecycline.
Examples of suitable carbohydrates include the anhydrous, hydrated, and
solvated forms of compounds such as lactose, mannose, sucrose, and glucose.
Suitable carbohydrates include mono and disaccharides e.g. an aldose •
monosaccharide or a disaccharide, preferably a disaccharide such as lactose and
sucrose. Lactose is most preferred. Accordingly, suitable carbohydrates may
include different solid forms. For example, by iactose we include the different
solid forms of lactose such as anhydrous lactose, lactose monohydrate or any
other hydrated or solvated form of lactose. Lactose and sucrose are
disaccharides. It is therefore expected that disaccharides as a class will work
according to the invention.
The compositions of the invention include solutions, such as those
prepared prior to lyophilization, containing tigecycline, a suitable carbohydrate,
and an acid or buffer. In some embodiments of the invention, the solutions may
be stored for several hours prior to lyophilization in order to provide greater
-manufacturing flexibility. Compositions-ofthe invention further include lyophilized
powders or cakes containing tigecycline, a suitable carbohydrate, and an acid or
buffer.
In some embodiments of the invention, the suitable carbohydrate used is
lactose monohydrate and the molar ratio of tigecycline to lactose monohydrate in
the lyophilized powder or cake is between about 1:0.2 to about 1:5. Some
embodiments have tigecycline to lactose monohydrate molar ratios of between
about 1:1.6 to about 1:3.3.
Compositions of the invention also include solutions made from the
lyophilized powder or cake by, for example, reconstitution with saline or other
pharmaceutically acceptable diluents. Compositions of the invention further
include solutions resulting from diluting those reconstituted solutions with
pharmaceutically acceptable diluents for use in intravenous bags.
Any carbohydrate capable of reducing epimer formation in the invention is
a suitable carbohydrate and this invention is not limited to compositions employing
those carbohydrates specifically identified.
7

WO 2006/099258 PCT/US2006/008827
It is expected that derivatives of sugars, for example, may work according
to the invention to reduce epimer formation. Thus, to the extent that derivatives of
sugars, such as sugar alcohols, glucoseamines, and alkyl esters alone or in
combination reduce epimer formation according to the invention, they are suitable
carbohydrates. Likewise, other suitable carbohydrates may include higher
saccharides such as polysaccharides; complex carbohydrates such as hetastarch,
dextran; and celluloses such as hydroxypropylmethyl cellulose and hydroxypropyl
cellulose. It is further expected that combinations of carbohydrates, including
monosaccharides and trisaccharides, will be suitable carbohydrates and work to
reduce epimer formation according to the invention.
Acids and buffers of the invention include any pharmaceuticaliy acceptable
acid or buffer capable of adjusting the pH of a tigecycline/suitable carbohydrate
solution to between about 3.0 to about 7.0, about 4.0 to about 5.0, or about 4.2 to .
about 4.8. Examples of such acids include, but are not limited to, hydrochloric •
acid, including 1.0 N HCl, gentisic acid, lactic acid, citric acid, acetic acid, and
phosphoric acid. Examples of suitable buffers include succinates.
Compounds of the invention may be prepared via a number of acceptable
methods. The methods described below are exemplary and not meant to limit the
invention.
In one method of the invention, tigecycline is dissolved in water to form a
solution. The pH of the solution is subsequently lowered by addition of an acid or
buffer. A suitable carbohydrate is then dissolved in the solution and the solution is
lyophilized to dryness to form a lyophilized powder or cake.
Tigecycline may be blended with a suitable carbohydrate and dissolved in
water. After the pH of the solution is adjusted so that it is acidic, the solution may
then be lyophilized to dryness to form a lyophilized powder or cake.
Lyophilization of solutions of the invention may be accomplished by any
pharmaceuticaliy acceptable means. Once lyophilized, compositions of the
invention may be stored under an inert gas, such as nitrogen, to further slow the
degradation process, but, unlike the current tigecycline composition, such low
oxygen environments are not necessary for the invention.
8

WO 2006/099258 PCT/US2006/008827
When tigecycline is combined with a suitable carbohydrate, any solid-state
form of tigecycline that is sufficiently soluble in water may be used. Such solid-
state forms include crystalline tigecycline polymorphs, amorphous forms, and
salts.
Additionally, when preparing tigecycline solutions of the invention for
lyophilization, one adds sufficient acid or buffer to the aqueous solution containing
tigecycline to obtain a pH from about 3.0 and about 7.0 including from about 4.0 to
about 5.0 and from about 4.2 to about 4.8.
The compositions of the invention may be prepared for single-dosage use.
In this embodiment, the solutions of the invention are lyophilized in individual vials,
such as 20 ml vials. Upon lyophilization, the vials are stoppered with any
pharmaceutically acceptable stopper. The stoppered viais are then shipped for
use.
When needed, the vials can be reconstituted by adding sufficient diluent to
achieve the desired concentration of tigecycline. The concentration of
reconstituted solutions may be easily determined by those of ordinary skilled in
the art Any pharmaceutically acceptable diluent may be used. Examples of such
diluents include water, saline, such as 0.9% saline, Lactated Ringer's Injection
solution and dextrose solutions including 5% dextrose (D5W).
Reconstituted solutions of the invention may then be stored in a
reconstituted state, unlike current compositions, prior to admixture. Admixture can
occur, for example, in an intravenous bag. To prepare an admixture, sufficient .
reconstituted solution is mixed in an intravenous bag containing a
pharmaceutically acceptable diluent such as saline solution or dextrose solution
such as D5W. The concentration of admixtures may be easily determined by
those of ordinary skill in the art. Admixture times for compositions of the invention
can be much longer than those of the existing composition. Once admixed, the
tigecycline solution is ready for patient administration. The admixture may be
administered alone or together with another pharmaceutical agent or composition.
The following six examples illustrate various embodiments of the invention
and are not intended to limit the invention in any way. Each example details
several experiments where tigecyciine was dissolved with a carbohydrate in
9

WO 2006/099258 PCT/US2006/008827
aqueous acidic solution, lyophilized, and analyzed for degradation products by
HPLC. The HPLC conditions for each example were essentially the same. The
tables accompanying the examples reflect the results of the HPLC data which
show the oxidative degradation products identified in the tables as relative
retention times (RRT) 0.50/MW 601 and RRT 0.55/MW 583, the epimer (RRT
0.74/MW 585), and the total amount of tigecycline present under a variety of
conditions (identified as "Tigecycline" in the tables"), in many instances, after the
solutions were lyophilized, they were placed under accelerated stability conditions
of 40°C and 75% relative humidity. These conditions are industry standards used
for simulating the effect of long-term storage under normal shelf conditions. •
In example 1, solutions of tigecycline, lactose, and 1.0 N HCl were
lyophilized and the resulting cakes were placed in stability chambers at 40°C and
75% relative humidity for 25 days. At the end of the 25 days, the cakes were
analyzed by HPLC to identify degradation products.
A similar experiment is detailed in Example 2a. There, the lyophilized
cakes were analyzed by HPLC after being stored for 39 days at 40°C and 75%
-relative-humidity Sample cakes from two of the experiments were-reconstituted
in D5W (5% dextrose) and samples from the remaining cakes were reconstituted
in saline immediately prior to HPLC analysis.
In experiment 2b, after the lyophilized cakes were stressed as per the
conditions in example 2a, several of the cakes were reconstituted in 0.9% saline
and kept in solution for 6 hours. Others were reconstituted in dextrose. At the
end of the 6 hour period, some of these solution samples, as identified in table 2b,
were tested by HPLC.
Example 2c illustrates a stability test on admixed solutions. In these
solutions, the reconstituted solutions of example 2b were held for 6 hours at about
10 mg/mL and then diluted to about 1 mg/mL, the typical intravenous
concentration for tigecycline, and held for 18 hours prior to analysis by HPLC
(table 2c).
In example 3, gentisic acid, rather than hydrochloric acid, was used to
reduce the pH of the pre-lyophilized solutions of tigecycline. Once lyophilized, the
10

WO 2006/099258 PCT/US2006/008827
cakes were stressed at 45°C and 75% relative humidity for 48 days and then
analyzed by HPLC.
The samples of example 4 show the effects of changing from lactose to
other carbohydrates on epimer formation and tigecycline recovery when making
the pre-lyophilized tigecycline solutions. In each of examples 4a, 4b, and 4c, the
indicated solutions were prepared and lyophilized. Each cake was stressed
according to the parameters provided in examples 4a-4c, taken into solution, and
analyzed by HPLC.
Hold time, the time in between compounding and lyophilization, and order
of tigecycline and lactose addition were studied as factors in epimer formation and
tigecycline recovery in example 5. Once the cakes were lyophilized, they were
stressed at 40°C and 75% relative humidity for 48 days prior to HPLC analysis.
Summaries of the HPLC data appear in table 5.
The ratio of lactose to tigecycline was varied in the experiments in example
6. When preparing the solutions to be lyophilized, varying ratios of lactose to
tigecycline were employed. The mass ratios are reported in the first column of
Table 6 . The so1utions,which each had a pH of about 5.0, were subsequently
lyophilized to dryness and the resulting cakes were stressed at 40°C and 75%
relative humidity for 20 days and analyzed by HPLC.
Example 1
Tigecycline (1880 mg) was dissolved in 75ml of Milli-Q water to form, a bulk
solution. An aliquot from this bulk solution containing approximately 100 mg of
tigecycline was dissolved into a 20 ml vial containing 200 mg of lactose
monohydrate. Another aliquot of the bulk solution containing approximately 100
mg of tigecycline was placed into an empty 20 ml sample vial. No pH adjustment
was made to either of these two solutions. The solutions were subsequently
lyophilized to dryness.
The pH of the remaining bulk solution was lowered to about 6.0 with the
addition of 1.0N HCl. Once a pH of about 6.0 was obtained, an aliquot from the
bulk solution containing about 100 mg of tigecycline was dissolved into a 20 ml
sample vial containing about 200 mg of lactose monohydrate and the resulting
11

WO 2006/099258 PCT/US2006/008827
solution was lyophilized to dryness. The remaining bulk solution was treated with .
1 .0N HCl until a pH of about 5.5 was obtained at which point 100 mg of .tigecycline
from the bulk solution was transferred into a 20 ml vial containing 200 mg of
lactose monohydrate. After dissolution, the solution was lyophilized to dryness.
Similarly, 20 ml sample vials containing solutions of about 100 mg of tigecycline
and about 200 mg of lactose were prepared at pHs of about 5.0 and about 4.5.
Another solution sample was prepared at about pH 4.5 without any lactose. In
each case, the solutions were subsequently lyophilized to dryness. All
lyophilizations were done on solutions frozen at -70°C by dry ice with acetone.
The lyophilized samples were placed in a 40°C/75%RH chamber for 25
days. Afterwards, the samples were analyzed by HPLC and a summary of the
results appears below in table 1, which reflects the major degradation products for
each cake that was tested. The sum total of the 6 major degradation products
listed in the tables does not equal 100% because not all degradation products are
listed in the table. Of the 7 cakes tested in example 1, 5 were compositions of the
invention and the first two (tigecycline alone without pH adjustment and tigecycline
plus lactose without pH adjustment) where controlos
The advantages of the compositions of the invention are apparent from this
example. For instance, in the composition prepared without lactose at a pH of
about 4.5, only 74.10% tigecycline was detected whereas the epimer was present
in an amount of 23.51%. By comparison, the pH 4.5 sample with lactose'
contained only 2.53% epimer and had a tigecycline content of 97.17%.
12

WO 2006/099258 PCT/US2006/008827
Table 1
Epimer
Sample ID RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556 585
Tigecycline only (no
pH adjustment) 0.57 2.15 6.50- .2.50 1.72 80.59
Tigecyciine +lactose
(no pH adjustment) 0.61 0.48 1.05 0.71 1.05 91.95
pH6.0 + lactose 0.04 0.15 2.56 0.04 0.12 96.83
pH5.5 + lactose 0.01 0.11 2.54 0.01 0.04 97.07
pH5.0 + lactose 0.01 0.04 2.43 ND 0.02 97.27
pH 4.5 (no lactose) 0.11 0.21 23.51 0.14 0.16 74.10
pH 4.5 + lactose - - o.o1 0.05" "2.53 ND ' 0.01 97.17
ND = Not detected; MW is molecular weight; RRT means relative retention time to
tigecycline peak.
Example 2
2a. Tigecycline (1700mg) was dissolved in 85ml of Milli-Q water to form a bulk
solution. Solutions containing about 100 mg of tigecycline and about 200 mg of
lactose monohydrate were prepared at pHs of about 5.2, 5.0, 4.8, and 3.0 in the
same manner that tigecycline/lactose/HCl solutions were prepared in example 1.
A solution of tigecycline and lactose at pH of about 4.5 was prepared by adding
1.0 N NaOH to the bulk solution at pH 3.0 followed by dissolving an aliquot of bulk
solution containing about 100 mg of tigecycline into a 20 ml vial containing about
200 mg of lactose monohydrate. All samples were lyophilized (frozen at -50°C by
freeze dryers from AdVantage/Virtis) to dryness. The lyophiiized samples were
placed in a 40°C/75%RH chamber for 39 days and sub-sampled and analyzed by
HPLC. The data are shown in table 2a.
13

WO 2006/099258 PCT/US2006/008827
2b. At the end of 39 days, the lyophilized cakes of Example 2a were
reconstituted with 0.9% NaCl to a concentration of 10 mg/ml of tigecycline and
kept at room temperature for 6 hours. Separate aliquots of the solutions at pH of
about 5.0 and about 4.5 were reconstituted with 5% Dextrose, instead of saline, to
a concentration of about 10mg/ml and kept at room temperature for 6 hours;
Each of the solutions was then analyzed by HPLC, and the results are shown in
Table 2b.
The data show that the compositions of the invention protect against
epimer formation in reconstituted solutions for 6 hours. Indeed, the maximum
epimer content of any one of these examples was only 2.45%, whereas the
minimum tigecycline content was 97.1 %. In one embodiment, where the pH was
about 4.5 and the diluent was saline, at the end of the 6 hour reconstitution period,
only 1.60% of epimer was present. In that embodiment, the amount of tigecycline
was measured to be 98.15%, which, in some applications, may be of sufficient
purity for hospital use.
2c. Admixture solutions of tigecycline (at 1 mg/ml) were made by diluting the
reconstituted solution (from example 2b) with 0.9% NaCl or 5% Dextrose
depending upon which diluent was used for reconstitution. The solutions were
then kept at room temperature for 18 hours and analyzed by HPLC. The results
are summarized in Table 2c.
The sample at about pH 4.5 with lactose and without dextrose had its
epimer concentration increase from 1.60% to only 1.80% on. going from
reconstitution to admixture whereas the overall tigecycline content decreased only
slightly for that sample from 98.15% to 97.97%. These results on the about pH
4.5 sample illustrate that that sample is sufficiently stable after the lyophilized
cake is stored under accelerated stability conditions for 39 days followed by 6
hours of reconstitution and 18 hours of admixture.
14

WO 2006/099258 PCT/US2006/008827
Table 2a
Epimer
Sample ID RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556. 585
pH 5.2+ lactose 0.01 0.08 2.21 ND ND 97.58
pH 5.0+ lactose 0.01 0.07 2.20 ND 0.01 97.57
pH 5.0+ lactose in
5%dextrose 0.01 0.08 2.21 ND 0.01 97.38
pH 4.8+ lactose 0.01 0.02 2.15 ND ND 97.63
pH 4.5+ lactose 0.01 0.03 1.37 ND 0.01 98.42
pH 4.5+ lactose in
5%dextrose 0.01 0.02 1.35 ND ND 98.23
pH 3.0+ lactose 0.01 0.02 1.34 ND ND 98.49

Table 2b '
Epimer
Sample ID RRT 0.5 0.55 0.74 1.25 1.67 Tigecycfine
MW 601 583 585 528 556 585
pH 5.2+ lactose 0.01 0.12 2.31 0.01 0.04 97.37
pH 5.0+ lactose 0.01 0.10 2.37 ND 0.03 97.33
pH 5.0+ lactose in
5%dextrose 0.01 0.10 2.45 0.01 0.03 97.10
pH 4.8+ lactose 0.01 0.09 2.32 ND 0.02 97.41
pH 4.5+ lactose 0.01 0.09 1.60 0.01 0.02 98.15
pH 4.5+ lactose in
5%dextrose 0.01 0.08 1.65' ND 0.01 97.96
pH 3.0+ lactose 0.01 0.06 2.10 ND ND 97.70
15

WO 2006/099258 PCT/US2006/008827
Table 2c
Epimer
Sample ID RRT 0.5 0.55 . 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556 585
pH 5,2+ lactose 0.01 0.05 2.49 0.01 0.09 97.11
pH 5.0+ lactose 0.01 0.06 2.57 0.01 0.06 97.09
pH 5.0+ lactose in
5%dextrose 0.02 0.05 2.80 0.01 0.06 96.66
pH 4.8+ lactose 0.02 0.04 2.52 0.01 0.04 97.19
pH 4.5+ lactose 0.01 0.03 1.80 ND 0.03 97.97
pH 4.5+ lactose in
5%dextrose 0.02. 0.02 2.02 ND 0.02 97.56
pH 3.0+ lactose 0.01 0.04 2.72 ND ND 97.13
Example 3
Tigecyciine (700mg) was dissolved in 28ml of Milli-Q water to form a bulk
solution. An aliquot of the bulk solution containing about 100 mg of tigecycline
was loaded into a 20 ml vial as control sample. Solution samples of tigecycline,
lactose, and an acid were prepared at pHs of about 5.8, 5.1, and 4.5 according to
the methods of example 1 except that gentisic acid was used to lower the pH of
the bulk solution rather than 1.0 N HCl. An additional two samples of tigecycline
solutions without lactose were prepared, one at a pH of about 5.1 and another at a
pH of about 4.5. All of the solutions were frozen at -70°C (by dry ice with
acetone) and lyophilized to dryness. The lyophilized samples were placed in a
40°C /75%RH chamber for 48 days and analyzed by HPLC. The data are
summarized in Table 3 and show that this composition works according to the
invention to reduce degradation.
16

WO 2006/099258 PCT/US2006/008827
Table 3
Epimer
Sample ID RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556 585
Control 0.37 2.17 7.37 1.50 1.47 81.13
pH 4.5 no lactose 0.02 0.05 28.11 0.04 0.02 71.37
pH 4.5+lactose 0.01 0.02 6.32 ND ND 93.42
pH 5.1 no lactose 0.05 0.10 20.90 0.10 0.08 77.87
pH5.1+lactose 0.01 0.02 3.94 ND 0.02 95.82
pH 5.8 no lactose 0.04 0.13 17.38 0.21 0.21 81.31
Example 4
4a. Tigecycline (1600mg) was dissolved in 64ml-of Milli-Q water to form a bulk
solution and two samples from the solution, each containing about 100 mg of
tigecycline, were loaded into two separate sample 20 ml sample vials containing
160 mg of lactose monohydrate and 160 mg mannitol respectively. A third
sample containing about 100 mg of tigecycline from the bulk solution was loaded
into a blank 20 ml vial. The pH of the remainder of the bulk solution was
sequentially adjusted with 1.0N HCI to about 7.0, 6.5, and 6.0 as per the
procedure outlined in example 1. Sample solutions each containing about 100
mg tigecycline were loaded into 20 ml vials containing 160 mg of lactose
monohydrate, 160 mg of mannitol, or neither at each pH value. The resulting
solutions were lyophilized (frozen at-70°C by dry ice with acetone) to dryness.
The lyophilized samples were placed in a 40°C oven for 70 hours and then
analyzed by HPLC. The data are summarized in table 4a.
4b. Tigecycline (1800mg) was dissolved in 72ml of Milli-Q water to form a bulk
solution. Aliquots from the bulk solution containing about 100 mg of tigecycline
were loaded into three separate 20 ml vials containing about 200 mg of lactose
monohydrate, fructose, and sucrose respectively. The pH of the bulk solution was
sequentially adjusted with 1.0N HC! to about 6.0 and 5.4 according to the
17

WO 2006/099258 PCT/US2006/008827
procedure outlined in example 1. At each pH value, aliquots of solution containing
about 100 mg of tigecycline were taken into 20 ml vials containing 200mg of one
of the following carbohydrates:
lactose monohydrate, fructose, or sucrose and dissolved. Solutions without
carbohydrates were also prepared at each pH value. The solutions were
lyophilized (frozen at -70°C by dry ice with acetone) to dryness. The lyophilized
samples were placed in a 40°C oven for 89 hours and analyzed by HPLC. The
results are summarized in Table 4b.
4c. Tigecycline (1000mg) was dissolved in 50ml of Mil!i-Q water to form a bulk
solution. The pH of the bulk solution was adjusted with 1.0N.HCI to about 5.0.
Four aliquots of bulk solution, each containing about 100 mg of tigecycline, were
ioaded into 20 m! vials containing about 200 mg of glucose, mannose, ribose, and
xylose respectively and dissolved. A fifth aliquot of bulk solution containing about
100 mg of tigecycline was loaded into a 20 ml.vial containing about.125mg of
threose and dissolved. All five solutions were lyophilized (frozen at -50°C by
freeze dryers from AdVantage/Virtis) to dryness. The lyophilized samples were
placed in a 25°C/ 60%RH chamber for 42 days and analyzed by HPLC. The
results are summarized in table 4c. Data in tables 4a-4c are meant to illustrate
the effect of suitable carbohydrates such as lactose on the invention.

WO 2006/099258 PCT/US2006/008827

Table 4a t
Epimer
Sample ID RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556 585
Tigecycline only 0.03 0.07 1.08 ND 0.07 98.51
pH 7.0 0.03 0.06 1.15 0.02 0.09 98.35
pH6.5 0.03 0.06 1.73 0.02 0.09 97.78
pH6.Q 0.02 0.06 2.69 0.02 0.08 96.82
Tigecycline +Iactose 0.03 0.10 0.89 ND 0.07 98.33
pH 7.0 +lactose 0.03 0.08 0.94 ND 0.06 • 98.45
pH 6.5+lactose 0.02 0.05 0.91 ND NA 98.50
pH 6.0 +lactose ND 0.04 0.90 ND NA 98.54
Tigecycline +mannitol 0.05 0.13 ■1.40 ND 0.14 97.69
pH 7.0 +mannitol 0.05 0.11 1.80 ND 0.12 97.45
pH 6.5+ mannito! 0.03 0.08 2.28 ND 0.08 96.98 .
pH 6.0 + mannitol 0.02 0.06 2.56 ND 0.07 96.82
19

WO 2006/099258 PCT/US2006/008827
Table 4b
Epimer
Sample ID RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556 585
Tigecycline only 0.04 0.12 1.06 0.04 0.12 98.39
pH 6.0 0.03 0.09 2.72 0.03 0.08 96.90
pH 6.0+ lactose 0.01 0.04 0.97 ND 0.03 98.76
pH 5.4+ lactose 0.01 0.06 1.01 0.01 0.03 98.71
pH 6.0+ fructose 0.04 0.09 17.70 0.02 0.02 81.92
pH 6.0+ sucrose 0.01 0.08 1.38 0.02 0.03 98.32
Table 4c
Epimer
Sample ID RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556 585.
pH 5,0+ glucose 0.01 0.06 1.02 ND 0.01 98.81
pH 5.0+ mannose 0.01 0.06 1.23 ND ND 98.60
pH 5.0+ ribose 0.44 0.02 33.30 ND 0.01. 65.94
pH 5.0+ xylose 0.02 0.09 18.05 ND ND 81.68
pH 5.0+ threose 0.91 3.41 7.00 0.07 0.79 22.85
Example 5
5a. Tigecycline (1000 mg) was dissolved in 40ml of Milli-Q water to form a
bulk solution. The pH of the bulk solution was adjusted with 1.0N HCI to about
5.0. At that pH, two aliquots of the bulk solution, each containing about 100 mg
tigecycline, were loaded separately into two 20 ml vials each containing about
200mg lactose monohydrate. One sample was frozen immediately at -70°C (by
20

WO 2006/099258 PCT/US2006/008827
dry ice with acetone), and the other sample was kept at room temperature for 5
hours before freezing. Frozen samples were subsequently lyophilized to
dryness. The lyophilized samples were placed in a 40°C/75%RH chamber for 48
days and analyzed by HPLC. The results are summarized in Table 5 as the "A"
samples.
5b. Lactose monohydrate (750 mg) was dissolved in 15ml of Milli-Q water.
Tigecycline (375mg) was added to this solution and the pH was adjusted to about
5.0 with 1.0N HCI. At this pH, two aliquots from the solution, each containing
about 100 mg of tigecycline and about 200 mg of lactose monohydrate, were
loaded into two 20 ml vials respectively. The solution in one sample vial was
frozen immediately at-70°C (by dry ice with acetone). The solution in the other
sample was kept at room temperature for 5 hours before freezing. Frozen
samples were lyophilized to dryness. The lyophilized samples were placed in a
40°C/75%RH chamber for 48 days and analyzed by HPLC. The results are
summarized in Table 5 as the "B" samples. The "A" and "B" data illustrate
compositions of the invention reducing degradation products.
Table 5
Epimer
Sample ID RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556 585
A (lactose dissolved
in tigecycline) 0.01 0.02 3.18 0.01 0.02 96.57
B (tigecycline
dissolved
in lactose) 0.00 0.02 3.32 ND 0.01 96.43
A left in RT for 5 hrs
before freeze 0.01 0.03 5.67 0.02 0.02 94.03
B left in RT for 5 hrs
before freeze 0.01 0.02 3.82 ND 0.02 95.86
21

WO 2006/099258 PCT/US2006/008827
Example 6
6a. Tigecycline (1700mg ) was dissolved in 85ml of Milli-Q water to form a bulk
solution. The pH of the bulk solution was adjusted to about 5.0 with 1.0N HCl.
Four aliquots of the bulk solution, each containing about 100 mg tigecycline, were
loaded separately into four 20 ml vials containing about 50,100, 200, and-300mg
of lactose monohydrate respectively. Once the lactose completely dissolved, the
samples were lyophilized (frozen at -50°C by freeze dryers from AdVantage/Virtis)
to dryness. The iyophilized samples were piaced in a 40°C 75%RH chamber for 4
days and analyzed by HPLC. The results are summarized in Table 6a and give
examples of compositions of the invention.
6b. Tigecycline (400mg) was dissolved in 20ml of Milli-Q water to form a bulk
solution. The pH of the bulk solution was adjusted to about 5.0 with 1.0N HCl.
Three aliquots of the bulk solution, each containing about 100 mg tigecycline, were
loaded separately into three 20 ml vials containing 15, 31, and 62 mg lactose
monohydrate respectively. Upon dissolution, the samples were lyophilized (frozen
at -50°C by freeze dryers from AdVantage/Virtis) to dryness. The lyophilized
samples were placed in a 40°C/75%RH chamber for 20 days and analyzed by
HPLC. The results are summarized in Table 6b and show compositions of the
invention.
22
WO 2006/099258 PCT/US2006/008827
Table 6a
Epimer
Sample ID
(molar ratio) RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583 585 528 556 585
pH 5.0+ lactose
50 mg (1:0781) ND 0.04 1.01 ND ND 98.53 ■
pH 5.0+ lactose
100 mg (1:1.62) ND 0.04 0.82 ND ND 98.73
pH 5.0+ lactose
200 mg (1:3.25) ND 0.04 0.82 ND ND 98.69
pH 5.0+ lactose
300 mg (1:4.87) ND 0.04 0.87 ND ND 98.64

Table 6t )
Epimer
Sample ID (molar ratio) RRT 0.5 0.55 0.74 1.25 1.67 Tigecycline
MW 601 583.. .. 585 528 556 585
pH 5.0 no lactose 0.03 0.07 5.40 0.02 0.07 94.19
pH 5.0+ lactose
15mg(1:0.24) 0.02 0.04 3.83 0.01 0.05 95.87
pH 5.0+ lactose
31 mg(1:0.50) 0.01 0.03 3.02 ND 0.03 96.72
pH 5.0+ lactose
62 mg (1:1.00) 0.01 0.03 2.18 ND 0.02 97.61
23

WO 2006/099258 PCT/US2006/008827
WHAT IS CLAIMED IS:
1. A composition comprising tigecycline, at least one suitable carbohydrate,'
and an acid or buffer.
2. The composition of claim 1 wherein one suitable carbohydrate is chosen
from lactose, mannose, sucrose, and glucose.
3. The composition of claim 2 wherein the suitable carbohydrate is lactose.
4. The composition according to any one of claims 1 to 3 wherein the
composition is lyophilized.
5. The composition according to any one of claims 1 to 4 further comprising a
pharmaceutically acceptable diluent.
6. The composition of claim 5 wherein the pharmaceutically acceptable
diiuent is a saline, Lactated Ringer's Injection solution or dextrose solution.
7. The composition of claim 5 wherein the pH of the composition is between
about 3.0 and about 7.0.
8. The composition of claim 7 wherein the pH of the composition, is between
about 4.0 and about 5.0
9. The composition of claim 8 wherein the pH of the composition is between
about 4.2 and about 4.8.
10. The composition of any one of claims 1 to 9 wherein the acid is 1.0 N HGl.
11. The composition of any one of claims 1 to 9 wherein the acid is gentisic
acid.
12. A process for preparing a tigecycline composition comprising combining at
least one carbohydrate suitable for reducing epimerization with tigecycline and
water to form a solution; reducing the pH of the solution with an acid or buffer to
reduce oxidative degradation; and lyophilizing the solution to dryness.
13. The process of claim 12 wherein one carbohydrate suitable for reducing
epimerization is selected from lactose, mannose, sucrose, and glucose.
14. The process of claim 13 wherein the carbohydrate suitable for reducing
epimerization is lactose.
15. The process of claim 13 further comprising combining the composition
obtained by lyophilizing the solution to dryness with a saline, Lactated Ringer's
Injection solution or dextrose solution.
24

WO 2006/099258 PCT/US2006/008827
16. The process of any one of claims 12 to 15 wherein the acid or buffer
reduces the pH of the solution to between about 3.0 and about 7.0.
17. The process of claim 16 wherein the pH of the solution is reduced to
between about 4.0 and about 5.0.
18. The process of claim 17 wherein the pH of the solution is reduced to
between about 4.2 and about 4.8.
19. A process as claimed in any one of claims 12 to 18 wherein the acid is 1.0
N HCl.
20. A process as claimed in any one of claims 12 to 18 wherein the acid is
gentisic acid.
21. A pharmaceutical composition comprising tigecycline, a suitable
carbohydrate, and an acid or buffer.
22. The composition of claim 21 wherein the suitable carbohydrate is a
disaccharide or an aldose monosaccharide.
23. A process for preparing a tigecycline pharmaceutical composition .
comprising combining a suitable carbohydrate with tigecycline and water to form a
solution; reducing the pH of the solution with an acid or buffer; lyophiiizing the
solution to dryness.
24. The process of claim 23 wherein the suitable carbohydrate is a
disaccharide or an aldose monosaccharide.
25

The present invention relates to novel tigecycline compositions with improved stability in both solid and solution
states and processes for making these compositions. These compositions comprise tigecycline, a suitable carbohydrate, and an acid
or buffer.