Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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-3 TEST MATERIALS
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1~ The present invention relates to antibiotic
'6 susceptibility test materials and to processes for
17 their preparation.
i ~
9 Antibiotic sensitivity testing of micro-organisms,
~0 especially of bacteria, is a routine procedure in
1 clinical laboratories. One convenient form of testing
-2 is the paper disc sensitiviy test in which a small
~3 paper disc impregnated with antibiotic is placed on
~4 the surface of agar in a petri dish, which agar has
~5 been inoculated with the bacteria to be tested. The
~6 petri dish is then incubated to allow bacterial growth
~7 to take place, during which incubation the antibiotic
~8 diffuses from the paper in the agar and sets up a
~'9 concentration gradient. Provided the amount of
~0 antibiotic on the disc is suitable the concentration
~1 gradient will be such that at some point from the disc
the antibiotic concentration in the agar will
~3 correspond to the sensitivity of the bacteria under
test, giving a zone of inhibition around the disc. The
~5 size o this zone of inhi~ition can be used as an index
~6 of the sensitiviy o the bacteria under test to the
37 antibiotic used. These known paper discs are normally
38 prepared by wetting the disc with an aqueous solution
39 of the antibiotic and allowing the disc to dry.
~0 .,
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31 - 2 -
3~
02 U~Ko Patent No. 1500994 discloses that sensitivity
03 discs may be prepared by contacting p~per discs with a
04 suspension of a crystalline ~-lactam antibiotic in a
05 non-solvent therefor and then evaporating the
06 non-solvent.
07
08 It has been found that the s~sceptibility discs
09 prepared by the process of U.K. Patent No. 1500994 lose
activity on handling owing to material abrading from
11 the surface of the disc.
12
13 A process has now been discovered by which
14 sensitivity discs having both useful mechanical and
chemical stability may be prepared.
16
17 Accordingly the present invention provides a
18 ~-lactam-antibiotic susceptibility test material
19 comprising an absorbent material on which a solid solution
of ~-lactam antibiotic in an organic carrier is
~1 uniformly deposited.
~2
~3 The preferred absorbent material is an absorbent
~4 paper. In general paper suitable for use in the
~5 preparation of conventional paper sensitiviy discs is
~6 suitable for use according to the present invention.
~7 The paper may be cut, or otherwise formed into discs of
~8 generally similar shape and size to known sensitivity
~ discs~ For example a suitable size is about 6 mm
3n diameter. Convenien~ly commercially available
31 ~ pre-formed discs may be used, for example Whatmann AA
~2 discs (available form W. and R. Balston Limited, Spring-
3~ field Mill, Maidstone, Kent)or S&S 740-E (available from
3~ Schleicher and Schuell, Keene, New Hampshirej.
3~ Suitable ~lactam antibiotics for ~se in the test
36 materials of the present invention are those having the
37 partical structure (I):
~ TrQJc ~ k
01 ~" ~ 3 ~ ~21~8
~2
G3
~4 _
~5
~J6 ~ _
07 (I)
~8
!~9
More suitably the ~-lactam antibiotic is a
11 bicyclic compound s~ch as, for example a penicillin,
i2 penicillin derivativet cephalosporinr cephalosporin
13 derivative, carbapenem, or clavulanic acid or a
'4 derivative thereof.
.~.5
:'.6 The ~-lactam antibiotic may be present alone or in
.l7 combination with other antibiotlcs and/or a ~-lactamase
.18 inhibitor.
.19
:~0 Suitable penicillins for incl~sion in the test
~1 . materials of this invention include benzylpenicillin,
~2 phenoxymethylpenicillin, carbenicillin, azidocillin,
~3 propicillin, ampicillin, amoxycillin, epicilllin,
24 ticarcillin, cyclacillin, pirbenicillin, azlocillin,
~5 mezlocillin, sulbenicillin, pipericillin, and other
~6 well known penicillins includiny pro-drugs thereof such
27 as their in vivo hydrolysble esters s~ch as the
28 acetoxymethyl, pivaloyloxymethyl, ~-ethoxycarbonyloxy-
~9 ethyl or phthalidyl esters of amp.icillin, benzyl-
~n penicillin or amoxycillin, and aldehyde or ketone
~1 adducts of penicillins containing a 6~-aminoacetamide
32 side chain (such as hetacillin, metampicillin and
~3 analogous derivat.ives of amoxycillin) or ~-esters of
~4 carbenicillin or ticarcillin such as their phenyl or
indanyl ~-esters.
36
37 Suitable cephalosporins for inclusio~ in the test
38 materials of this i~vention include, for example,
39 cefat~-izine, cephaloridine, cephalothin, cefa~olin,
cephalexin, cephacetrile, ~eph~pirin, cephaman~ole
41 nafate, cephradine, 4-hydroxycephalexin, cefaparole,
.
Z~3~
:2 cephaloglycin, cefoperazonel and other well known
03 cephaloa~orins or pro-dr~gs thereof.
~4
~)5 Such compo~nds are frequently used in ~he form of
')6 a salt of hydrate or the like~
~)7
0~ In a preferred embodiment of the present invention
~9 the ~-lactam antibiotic is a penicillin or
cephalosporin as hereinbefore described in combination
11 with a compound of formula III) or a pharmaceutically
'.~ acceptable salt or ester thereof:
:!3
. ~
`IS H
- 0~ ,CH2R
I3 1 ~H
9 ~ ~ _ ~
2 o o d ` Co2H
21 H
22 (II)
23
24
~5 wherein Rl is hydroxyl, substituted hydroxyl, thiol,
26 suhstituted thiol, amino, mono or di-hydrocarbyl amino,
77 or mono or di-acylamino.
~8
29 In these formulations the ratio of ~-lactam
~0 antibiotic to clavulanic acid derivative will normally
~1 be in the range 10:1 to 1:10 (w/w), for example 5:1 to
~3
34 In a particularly preferred embodiment of the
present invention the ~-lactam antibiotic is a
36 penicillin or cephalosporin having a side chain (joined
37 at the 6- or 7- position respectively) of formula
38 (III):
~1 5 _ ~2~30~
Q2
03 D
~5 R2-CH-CO- (III)
G6
07 NH2
G8
Gg
wherein R2 is phenyll ~-hydroxyphenyl or
1I cydohexadienyl, or a salt thereof in combination with
1~ clavulanic acid or a salt thereof. Suitable salts
11 include the alkali metal salts such as, for example the
i~l lithium, sodium, or potassium salts.
~6 Suitable penicillins having a side chain of
:.7 formula (III) include amoxycillin and ampicillin, and
18 salts thereof such as the sodium and potassium salts.
: 9
~0 Suitable organic carriers for use in the p~esent
Jl invention will be bacteriologically inert and
~2 compatible with the ~-lactam antibiotic and abs~L-bent
~3 material. The organic carrier is suitable hydrophilic
and non-hygroscopic.
~5
~6 Conveniently the carrier will be water soluble,
~7 thereby enabling ready release of ~-lactam antibiotic
~rom the absorbent material on contact with test medium
such as, for example, agar.
~n
3.l Examples of suitable organic carriers include
~2 certain organic synthetic polymers such as polyvinyl
33 pyrrolidone and polyvinyl pyrrolidone/polyvinyl acetate
34 copolymers; certain organic semi-synthetic polymers
such as methyl and hydroxypropylmethylcellulose, and
36 sodium carboxymethylcellulose, and other cell~lose
37 polymers; certain saccharides such as sucrose and
38 lactose; and mannitol.
39
~Z IL~8
01 - 6 -
02 Particularly suitable organic polymers include
03 polyvinyl pyrrolidone (such as the material available
04 ~ from BASF under the trade ~ ~ollidon (25)~ and
C5 polyvinyl pyrrolidone/polyvinyl acetate copolymers
06 (such as the material available from BASF under the
07 trade name Kollidon VA64~.
G8
09 The ratio of ~-lactam antibiotic to organic
carrier may be varied as desired provided there remains
11 sufficient carrier to form the solid solution. For
1~ example, a suitable solid solution may contain 0.1 to 25
l3 by weight ~-lactam antibiotic, more suitable 0.1 to 10%
14 ~-lactam antibiotic.
16 The invention also provides a process for the
~7 preparation of the ~-lactam antibiotic susceptibility
18 test material which process comprises contacting an
19 absorbent material and a solution of ~-lactam
~0 antibioticand an organic carrier.
~1
~2 The absorbent material is contacted with the
23 solution in conventional manner. For example drops of
~4 the solution may be placed on individual discs of
absorbent material or on a larger scale, sheets of
26 absorbent material may be dipped into the solution.
~7 The absorbent material is then dried under mild
~8 conditions and if necessary the absorbent material is
~9 then cut or otherwise formed into discs.
~n
31 Depending on the solubilities of the reagent and
32 carrier, the necessary solution may be pLepared in
33 aqueous, organic or mixed solvents. The reagent and
34 the carrier will be dissolved in the solvent in the
relative proporti~ns desired in the test materials to
36 be prepared form that solution. Examples of suitable
37 solvents include inert volatile solvents such as, for
3~ example, Cl_4alkanols such as methanol and e~hanol r
- ~2~03~8
01 _ 7 _
02 acetonitrile, dicnloromethane, chloroform and the
03 like. Where possible methanol will be used as the
04 solvent.
05
06 The solution will suitably contain 0~25 to 10%
07 organic carrier.
08
09 The solutions used in the process of this
invention must contain the concentration of ~-lactam
11 antibiotic required to give the desired amount of
12 ~-lactam antibiotic per unit area of absorbent
13 material. The solutions are suitably prepared by
14 dissolving the correct amount of ~-lactam antibiotic in
the chosen solvent. In the cases where a mixture of
16 ~-lactam antibiotics is used or where a ~-lactamase
17 inhibitor is present the solutions are suitably
18 prepared by dissolving the correct amount of each
19 ~ ingredient in the chosen solvent, or by mixing together
separate solutions of the correct concentrations of
21 each of the ingredients in the solvent. The organic
22 carrier may be dissolved in the solvent before or after
23 dissolving ~he ~-lactam antibiotic or the organic
24 carrier may be dissolved in a separate portion of
solvent and then mixed with the ~-lactam antibiotic
26 ~ solutionO
27
28 Afte~ the a~sorbent material has been contacted
29 with the solution the material is dried under mild
conditions, for example by placing the material in a
31 desiccator, a free~e drying apparatus or in a flow of
32 air or inert gas. The choice of drying technique will
33 depend upun the physico-chemical properties of the
34 ~ sol-~ent system selected.
36 The test materials of this invention sh~uld
37 suitably be stored in a sea1ed container, more suitably
38 in the presence of a desiccant~
39
`
30151
01 - 8 -
02 The test materials may be used ~o provide a known
03 micro-dosage quantity of !3-lactam antibiotic for use in
04 the testing of microorganisms using conventional
05 methods, such as, for example the paper disc
~6 sensitivity test as hereinbefore described.
07
08 The following examples illustrate the present
C9 invention.
~ O
9--
EXAMPLE 1
Each disk to contain: ~moxicillin, 20mg and Clavulanic Acid, lOmg.
Amoxicillin Trihydrate 647 mg
Potassium Clavulanate 373 mg
P.V.P. (Plasdone C~15 GAF~ 5 y
Anhydrous Methanol 500 ml
Dissolve the amoxicillin trihydrate in the methanol with continuous
mixing. Add and dissolve the P.V.P. Add and dissolve the potassium
clavulanate. Dip 4" x 4" sheets of S&S 740-E filter paper. When
thoroughly wet, pass through free-floating rubber rollers to re-
move the excess solution. Place on a small mesh wire screen and
place in a forced air oven at 55C and dry for 30 minutes.
Remove from drier, punch out 6.35-mm disks and package with a
desiccant.
EXAMPLE 2
.
Ticarcillin, Monosodium 2.62 g
Potassium Clavulanate 583 mg
PVP (Plasdone C-15 GAF) 5.0 g
Anhydrous Methanol 500 ml
Add and disolve in the methanol in the following ordex: PVP, ticarcillin,
and potassium clavulanate. Mix well for 3-5 minutes and dip sheets of
filter paper (S&S 740-E on equivalent) into the solution. Pass the
moistened sheets through free-floating rubber rollers to remove excess
solution. Place sheets on fine mesh screen wire and dry in forced air
oven at 55C for 20 - 30 minutes.
Punch out 6.35-mm disks from the sheets and package in glass vials
contai~ing desiccant and which have air-tight closu~es.
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