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I
OPHTHALMIC USE OF SODIUM CEPHALOTHIN:AN IN VIVO COMPARISON
THESIS
Presented to the Graduate Council ofNorth Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF ARTS
by
Gerald R. Carney, B.A.
August, 1979
Carney, Gerald R., Ophthalmic Use of Sodium Cephalothin: An In Vivo
Comparison. Master of Science (Biology), August, 1979, 44 pp., 13 tables
2 illustrations, bibliography, 43 titles.
A rabbit keratoconjunctivities model was used to evaluate ophthalmic
formulations containing 1 percent sodium cephalothin in silicon oil, a
1 percent sodium cephalothin aqueous solution, and a 0.3 percent gentamicin
sulfate solution. Rabit eyes were inoculated intracorneally with Pseudomonas
aeruginosa, Staphylococcus aureus, or Streptococcus pneumoniae, After
topical treatment, none of the antibiotic formulations were effective in the
P. aeruginosa model; all three showed good activity against S. aureus, and
against S. pneumoniae, the caphalothin formulations were more effective
than gentamicin.
In a related stability study, the cephalothin potency of the silicon
formulation was maintained for 16 weeks at 4, 25, and 450 C
These studies suggest that sodium cephalothin can be formulated as
an effective and stable ophthalmic dosage form,
C) 1979
GERALD RAY CARNEY
ALL RIGHTS RESERVED
UniversityMicrofilms
International300 N. ZEEB ROAD, ANN ARBOR, MI 48106
TABLE OF CONTENTS
Page
LIST OF TABLES . . . . . . ........ . . . . . . ivLIST OF ILLUSTRATIONS . ........... v
CHAPTER
I. INTRODUCTION...... ........... .
Sodium CephalothinGentamicin SulfateSodium Cephalothin StabilityPurpose
II. METHODS AND MATERIALS . . . . . . . . . . . . . . 11
MicroorganismsAntibiotic FormulationsIntracorneal InoculationSlit-Lamp ExaminationsCephalothin/Silicon Stability Assay
III. RESULTS . .. ...... . . . . .. . . . . . ... * 19
Defining "Severity Threshold" LevelsPseudomonas aeruginosa ModelStaphylococcus aureus ModelStreptococcus pneumoniae ModelVehicle and Negative ControlsCephalothin/Silicon Stability
IV. DISCUSSION.....-.-................ .9..*.....31
V. SUMMARY AND CONCLUSIONS . - - -* . . . . . . . . 36
LITERATURE CITED - - - - - - - - -. . . . . . . . . ......... 39
iii
LIST OF TABLES
Table Page
I. In Vitro Susceptibilities of Important OcularPathogens to Cephalothin . . . . . . . . . . . . . . 4
II. In Vitro Susceptibilities of Important OcularPathogens to Cephalothin and Gentamicin . - - . . . 8
III. Criteria for Analysis of 24 Hour Slit-Lamp Examina-tions - Defining "Severity Threshold" Levels . . . . 19
IV. Criteria for Analysis of 72 Hour Slit-Lamp Examina-tions - Defining "Severity Threshold" Levels . . . . 20
V. Evaluation of Slit-Lamp Data for Pseudomonasaeruginosa Infections at 24 Hours - - - . . . . . . . 21
VI. Evaluation of Slit-Lamp Data for Pseudomonasaeruginosa Infections at 72 Hours -0-......... . 22
VII. Evaluation of Slit-Lamp Data for Staphylococcusaureus Infections at 24 Hours - - . - - . . . . . . . 24
VIII. Evaluation of Slit-Lamp Data for Staphylococcusaureus Infections at 72 Hours - - - - - . . . . . * 25
IX. Evaluation of Slit-Lamp Data for Streptococcuspneumoniae Infections at 24 Hours . - - . . . . . . . 27
X. Evaluation of Slit-Lamp Data for Streptococcuspneumoniae Infections at 72 Hours . - . - . . . . . 28
XI. Evaluation of Slit-Lamp Data for Vehicle and NegativeControls.... ..........-.-.-.-...-.... . . . . 29
XII. Cephalothin/Silicon Stability Assay Data . . . . . 30
XIII. Summary of Results.......-.-.......-.............36
iv
LIST OF ILLUSTRATIONS
Figure Page
1. Sodium Cephalothin Molecular Structure and Sites ofEnzymatic Activity . . - -* * - - - - - * * . . . 5
2. Molecular Structure of Gentamicin - Components C1 ,C2V C l o0 0 0 0 * 9 * 0 0
V
CHAPTER I
INTRODUCTION
Staphylococcus aureus and Streptococcus pneumoniae arethe most important bacterial pathogens affecting the humaneye. They are the etiologic agent in a wide variety ofinfections of the cornea, conjunctiva, eyelids, and intra-ocular tissues. When combined, S. aureus and S. pneumoniaeaccount for 86 percent of all corneal infections, 82 percentof all conjunctival infections, and even higher percentages
in orbital cellulitis infections (23). Pseudomonas aeruginosais the third most important ocular pathogen. Although
P. aeruginosa is not the causative agent in a high percentageof ophthalmic infections, its effect on ocular tissues
is often devastating and the infections are difficult to
treat with antibiotics.
In recent years ophthalmologists have become concernedwith the high rate of resistance of bacteria to antibiotics
used for the treatment of ocular infections. Weinstein
(43) reported that bacterial resistance to chloramphenicol
in vivo is a problem of increasing importance with gram-positive and gram-negative microorganisms stating that morethan 50 percent of staphylococci in some hospital environmentsare resistant. Aminoglycoside antibiotics such as gentamicin
1
2
and tobramycin have good activity against staphylococci
and pseudomonas but are considered only moderately effective
against the streptococci (3, 22, 24). Kim et al. (19)
determined the quantitative antibiotic sensitivities of
108 strains of S. aureus isolated from eye cultures. Of
the nine antibiotics tested, sodium cephalothin was the
most effective. The minimal inhibitory concentrations
(MIC's) and minimal bactericidal concentrations (MBC's)
of gentamicin sulfate were very similar to those of cepha-
lothin. Erythromycin, sodium ampicillin, penicillin, tetra-
cycline, sodium methicillin, and disodium carbenicillin
were moderately good. Chloramphenicol had the lowest activity
when tested on these S. aureus strains.
Sodium cephalothin has excellent activity against S.
aureus and S. pneumoniae, but P. aeruginosa is quite resis-
tant (3, 19, 22, 24, 40). The spectrum of activity of
cephalothin against gram-positive bacteria makes it a very
attractive antibiotic for ophthalmic use (35, 36). At
present primary indications for the use of cephalothin
are limited to ophthalmic infections such as bacterial
endophthalmitis (27, 36) and orbital cellulitis patients
who are allergic to penicillin. Cephalosporins have been
restricted to intravenous (32, 34) and subconjunctival
(5, 28) injection because of their rapid degradation in
aqueous media (14). For this reason, cephalosporins are
3
not used as topical solutions (eye drops). This is a
report of the use of a nonaqueous sodium cephalothin suspen-
sion as an effective topical ophthalmic antibiotic.
Sodium Cephalothin
Sodium cephalothin (Keflin*) is a broad spectrum anti-
biotic marketed by Eli Lilly and Co. (Indianapolis, IN.,
U.S.A.) for parenteral administration. It is a semi-synthe-
tic antibiotic derived from cephalosporin C, a natural
antibiotic produced by a strain of the mold Cephalosporium
acremonium (2, 22). Cephalothin is generally bactericidal
for 0-hemolytic and other streptococci, staphylococci
(coagulase positive and coagulase negative, as well as
penicillinase producing strains). It is also effective
against Haemophilus, Klebsiella, and Proteus organisms.
Species and strains of Proteus which produce indole and
Pseudomonas are generally resistant. Griffith and Black
(16) found that the MIC's for cephalothin against S. aureus
strains both sensitive and resistant to penicillin were
< 1.0 pg/ml for approximately 80 percent of the strains
tested. At least 80 percent of the 'S. pneumoniae strains
tested were sensitive to concentrations of < 0.4 pg/ml
and all Pseudomonas were judged resistant with MIC's of
> 50.0 pAg/ml (Table I).
4
TABLE I
IN VITRO SUSCEPTIBILITIES OF IMPORTANT OCULAR PATHOGENS TO CEPHALOTHINa
Total Minimal Inhibito LConcentration -u/ )Organism Strains <0.04 0.1010.27 . 1.0 2.015.O~10.0125.0 >50.0
Pseudomonas sp. 10 10Staphylococcusb 17 5 1 3 6 1 1
aureus SStaphylococcusc 26 1 1 8 9 6 1
aureus (RStreptococcus 10 1 3 4 1 1
pneumoniae
a From Griffith (16).b (S) - Sensitive to penicillin.
C (R) - Resistant to penicillin.
Cephalothin is similar in molecular structure to the
penicillins but instead of having the five-membered
thiazolidine ring, it has a six-membered dihydrothiazine
ring fused to a g-lactam ring (27). This structure is more
resistant to hydrolysis by s-lactamase than penicillin (2).
The mechanism of action for cephalothin is, like penicillin,
associated with inhibition of cell wall synthesis. The
cephalosporin lactam structure attaches to and inactivates
the bacterial enzyme transpeptidase. Cephalothin blocks
closure of glycine bridges in the final steps of bacterial
cell wall synthesis.
There are several enzymes that will inactive cephalo-
thin. Hydrolysis by acyl esterase, amidase, and 8-lactamase
effectively cleaves the cephalothin molecule and renders
5
it harmless to bacteria (Fig. 1). Betalactamase, penicil-.linase, and cephalosporinase are produced within, or onthe bacterial cell wall, and are probably modified trans-.peptidases (36).
S H H HH C -C -4- C- .C '
2 1 H2 S0 2H3C -- C - 0 - -- .. .
3 ItH C ''o0 2 0
NaOC0aCAmidase0 + H20
Acyl Esterase L+ H20
'~L CLfdmse
+ H20
Fig. 1. Sodium cephalothin molecular structure andsites of enzymatic activity [modified from Records (36)].
The cephalosporins as a class are relatively nontoxic.However, there is evidence of nephrotoxicity, encephalopathy(29), and Coombs positive hemolytic anemia (22) linked tocephalothin. Antibodies to cephalothin have been found inpatients known to be allergic to penicillin. Allergicreactions in patients with histories of penicillin allergyoccurred in 5-8 percent of the patient population studiedby Meyers (27). Cephalothin is often administered to
6
patients with known penicillin sensitivity and although
cross sensitivity has not been a major clinical problem,
cephalothin must be administered with caution to these
individuals (36).
Clinical results of combination antibiotic therapy
(cephalothin-tobramycin, cephalothin-carbenicillin and
cephaloridine-gentamicin) have been reported by Klastersky
and Fernandes (12, 20, 21). These studies showed that
the antimicrobial regimens were similarly effective and
that they resulted in favorable clinical responses but
that the administration of the cephalothin-tobramycin combi-nation was associated with a significant frequency of nephro-toxicity. Meyers (27) and Weinstein (43) report that nephro-
toxicity has also been found following the administration
of cephalothin and gentamicin. Meyers (27) adds that a
more recent analysis of this problem revealed no increase
in nephrotoxicity when the two agents were combined and
that animal experimentation has not substantiated the
reports of increased kidney toxicity with the two agents.
In fact, a protective effect was observed when cephalothin
was administered prior to gentamicin and tobramycin therapy
(4, 27).
When the activities of cephalothin and gentamicin against
S. aureus, S. pneumoniae, and P. aeruginosa are examined
as in Table II, the advantages of a cephalothin/gentamicin
combination be come apparent. These MIC's are reported
7
from independent sources (3, 7, 8, 22, 24) and show thepotential complimentary activity of the two agents.
Gentamicin Sulfate
Gentamicin sulfate was chosen as the in vivo test anti-biotic to compare against sodium cephalothin. This amino-glycoside is widely used in ophthalmology and is marketed bySchering Corporation (Kenilworth, NJ, U.S.A.) as Garamycine.Gentamicin is a broad spectrum antibiotic derived from
Micromonospora purpurea, an actinomycete. It is recognized
as the most important of the aminoglycosides in that 95percent of P. aeruginosa isolates are inhibited by < 10 pg/mland practically all penicillin-sensitive strains of S.aureus are inhibited at < 5 pg/ml. Also, Escherichia coli,Klebsiella, and Enterobacter are highly sensitive whileS. pneumoniae, H. influenzae, and Proteus are moderately
sensitive. Like other aminoglycosides, gentamicin actsdirectly on bacterial ribosomes where it inhibits proteinsynthesis and decreases the fidelity of genetic code trans-lation. Prevention of amino acid polymerization after
formation of initial complexes appears to be the major
result of gentamicin activity (11, 15).
Gentamicin is composed of three closely related componentsdesignated gentamicins C1, C2 , and Cla (Fig. 2). The threecomponents have nearly identical antimicrobial activity.
8
TABLE II
IN VITRO SUSCEPTIBILITIES OF IMPORTANT OCULAR PATHOGENS TOCEPHALOTHIN AND GENTAMICIN
Organism Minima-.Inhiitor.Concentration u /mlCephalothin GentamicinPseudomonas aeruginosa (R) Resistanta,b,c 2-16 > Rab
1.0 - 8.0b0.5 - 16.0
Staphylococcus aureus 0.05 - 0.2a 0.2 - 4.0a......e5. .0.125 -m 2.o d
Streptococcus pneumoniae 0.06 - P.12b 16.0 320.39 18.0- 2O8.0 - 16.0da From Barker(3).
b From Kucer (22).c From Lorian (24).d From Cagle (7, 8).
Sodium Cephalothin Stability
Sodium cephalothin hydrolyzes rapidly and therefore
is not stable in aqueous solutions. Galleli et al. (14)
studied the stability of several antibiotics in normalsaline solution and found that cephalothin decreased 50
percent in antimicrobial activity when stored at 25 C for
seven days and 75 percent by the 14th day. The manufacturer
claims that sodium cephalothin in aqueous solution has satis-factory potency for four days if refrigerated.
With an understanding of the antimicrobial activity andaqueous instability of cephalothin, it seemed reasonable toassume that a nonaqueous cephalothin preparation could be
9
used successfully. There are several nonaqueous liquids
that could serve as a vehicle for an ophthalmic cephalothin
formulation. Mineral oil and silicon fluid were considered
H
OH H H 2
H HN 0
H H0H 0 1
R'NHCH OH
I HRH
H
Gentamicin R R'
C1 CH3 CH3C2 CH3 HC H Hla
Fig. 2. Molecular structure of gentamicin compon-ents C1, C2 , and Cla [from Weinstein (43)].
the best choices because both contain little or no water
and are probably nonirritating to the eye. Dow 360 silicon
fluid at a viscosity of 100 cps (Dow Chemical Co., DE, U.S.A.)
was selected over mineral oil for initial experiments when
it was determined that silicon fluid contained the lesser
amount of water.
A 1 percent suspension of cephalothin was formulated in sil-icon and placed in plastic containers. After 2 days at 25 Cthe cephalothin was drastically altered as determined by a
10
distinct color change from white to a dark brown. Thechange in color was assumed to be caused by atmospheric
moisture penetrating the plastic containers used for storage.Other suspensions were prepared in amber glass bottles
with moisture resistant plastic screw caps. These suspen-sions did not show visual signs of physical deterioration
after fourteen days at 25 C. After this was established
it was decided to initiate in vivo antibacterial tests
along with stability assays of cephalothin in silicon fluid.
Purpose
The intent of this paper is to describe and analyze
experiments designed to evaluate the in vivo antimicrobial
effectiveness of a 1 percent cephalothin suspension insilicon fluid and to determine the antimicrobial stability
of the formulation.
The experiments were designed to compare the in vivoeffectiveness of a 1 percent cephalothin suspension in
silicon to that of a 0.3 percent aqueous gentamicin sulfate
solution and a 1 percent aqueous cephalothin solution againstP. aeruginosa, S. aureus, and S. pneumoniae keratitis infec-tions, to determine the ocular toxicity associated with
topical application of silicon fluid in comparison to acommercial antibiotic medium, and finally, to determine
the antimicrobial stability of the 1 percent cephalothin
suspension at 4, 25, and 45 C for a period of sixteen weeks.
CHAPTER II
METHODS AND MATERIALS
Microorganisms
Clinical isolates obtained from the Alcon Laboratories
Microbiology Culture Collection were used in this study:
Pseudomonas aeruginosa (SH-3108)Staphylococcus aureus (BS-4)Streptococcus pneumoniae ATCC 6301 (type 1)
Cultures of each organism were maintained by cryopreserva-
tion (liquid nitrogen -196 C). S. aureus and P. aeruginosa
were thawed and transferred to Mueller Hinton (MH) agar
plates. Streptococcus pneumoniae was streaked onto MH agar
plates containing 5 percent defibrinated rabbit blood.
These cultures were incubated for 48 h and isolated colonies
streaked onto agar slants of MH and MH + 5 percent defibri-
nated rabbit blood. After 18 h incubation at 32-35 C, each
organism was harvested by washing the slants with 3.0 ml
of sterile MH broth. The broth suspensions were standardized
to a light transmission of 50 percent with a Spectronic 20
spectrophotometer (Bausch and Lomb, Rochester, NY, U.S.A.)
at 580 nm. These standardized MH broth suspensions were
diluted 1:10 in MH broth and 0.01 ml used to inoculate the
eyes of experimental animals. Serial 1:10 dilutions were
also made and pour plate counts performed to determine the
number of viable cells in the animal inoculum.
11
12
Antibiotic Formulations
Three antibiotic formulations were tested: two cephalo-
thin preparations and one gentamicin.
1. 1 percent cephalothin . . ig sodium cephalothin (Lot#1NW83A, in 100 ml Dow Fluid360 (Dow Chemical Co., DE,U.S.A.)
2. 1 percent cephalothin . . ig sodium cephalothin in 100 mlGaramycin* vehicle (aqueoussolution)
3. 0.3 percent gentamicin . .Garamycin Ophthalmic Solution(Garamycin*) (Lot #7AMS39, Schering Corpo-ration, Kenilworth, NJ, U.S.A.)
Garamycin was chosen as the reference antibiotic solution
because it is a successful commercial ophthalmic preparation
possessing broad spectrum antimicrobial activity. Also,
this laboratory has performed numerous in vivo and in vitro
comparisons (6, 7, 8, 38) using gentamicin as a reference
compound. The cephalothin in Garamycin vehicle formula-
tion was included to compare the efficacy of the nonaqueous
silicon vehicle to a vehicle of known merit.
Intracorneal Inoculation
New Zealand white rabbits of both sexes weighing between
1.5 and 2.5 kg were used in this study. Any animal posses-
sing ocular pathology as determined by slit-lamp biomicroscopy
was rejected from the study. The rabbits were tranquilized
by intramuscular injection of 1.0 ml of Thorazine* (Smith
Kline, and French Laboratories, Philadelphia, PA., U.S.A.).
13
After tranquilization each rabbit eye was topically anes-
thetized with 2 drops of Alcaine* (Alcon Laboratories, Ft.
Worth, TX, U.S.A.) and proptosed.
The corneal stroma of the proptosed eye was inoculated
with approximately 106 colony forming units of a specific
bacterium by injecting 0.01 ml of the prepared bacterial
suspension from a Hamilton Microliter* syringe (Hamilton
Co., Inc., Whittier, CA., U.S.A.) fitted with a 30 gauge
inoculating needle., A circular opacity of approximately
2 mm was produced at the site of injection.
This in vivo study involved 85 rabbits. The animals
were divided into 17 experimental groups of 5 rabbits each.
Antibiotic dosing began one hour after inoculation and con-
tinued for nine treatments. At each one hour interval 0.1 mlof a specific formulation was deposited in the cul-de-sac
of each treated eye.
Experimental Groups
1. Group 1 - S. aureus Positive Controls. Each cornea was
injected with approximately 106 organisms and remained
untreated for the duration of the experiment.
2. Group 2 - S. aureus was injected intracorneally, as in
Group 1 and dosed with the 1 percent cephalothin in
silicon formulation.
3. Group 3 - S. aureus was injected intracorneally, as
in Group 1 and dosed with the 1 percent cephalothin
in Garamycin vehicle.
4. Group 4 - S. aureus was injected intracorneally, as
in Group 1 and dosed with the 0.3 percent Garamycin.
5. Group 5 - P. aeruginosa Positive Controls. Each corneawas injected with approximately 106 organisms and remain-ed untreated for the duration of the experiment.
6. Group 6 - P. aeruginosa was injected intracorneally,
as in Group 5 and dosed with the 1 percent cephalothin
in silicon formulation.
7. Group 7 - P. aeruginosa was injected intracorneally,
as in Group 5 and dosed with the 1 percent cephalothin
in Garamycin vehicle.
8. Group 8 - P. aeruginosa was injected intracorneally,
as in Group 5 and dosed with the 0.3 percent Garamycin.9. Group 9 - S. pneumoniae Positive Controls. Each cornea
was injected with approximately 106 organisms and remain-ed untreated for the duration of the experiment.
10. Group 10 - S. pneumoniae was injected intracorneally,
as in Group 9 and dosed with the 1 percent cephalothin
in silicon formulation.
11. Group 11 - S. pneumoniae was injected intracorneally,
as in Group 9 and dosed with the 1 percent cephalothin
in Garamycin vehicle.
12. Group 12 - S. pneumoniae was injected intracorneally,
as in Group 9 and dosed with the 0.3 percent Garamycin.13. Group 13 - Sham Controls. The inclusion of this group
assessed ocular changes, if any, associated with injection
14
15
of Thorazine*, proptosing, and injection of 0.01 ml
of sterile MH broth. The animals of this group received
no organism inoculation nor antibiotic dosage.
14. Group 14 - Silicon Controls. Each cornea was dosed
with 0.1 ml of sterile Dow 360 silicon hourly for 9
hours. These eyes were not inoculated with bacteria.
15. Group 15 - Garamycin Vehicle Controls. Each cornea
was dosed with 0.1 ml of sterile Garamycin vehicle
hourly for 9 hours. These eyes were not inoculated
with bacteria.
16. Group 16 - Negative Controls. These animals were not
anesthesized, proptosed, inoculated, or dosed.
17. Group 17 - S. aureus/Silicon Controls. S. aureus was
injected intracorneally, as in Group 1 and dosed with
sterile Dow 360 silicon.
Slit-Lamp Examinations
All test rabbits were examined by slit-lamp biomicro-
scopy 24 hours before corneal inoculation. Both eyes of
every rabbit were examined for evidence of conjunctivitis,
swelling, and discharge (Draize score), flare, iritis, and
corneal involvement (intensity and opacity).
Each animal was again examined 24 and 72 hours aftercorneal inoculation. The presence of ocular pathology was
determined within slit-lamp biomicroscope parameters (26).
The treatment group to which an animal belonged was not
16
made known to the investigator performing the biomicroscope
examinations (observer blind study).
The microscopic slit-lamp examination of the rabbit
eyes measures the following ocular reactions:
Parameter Score Range Ocular Tissue
Draize Congestion 0-3 ConjunctivaDraize Swelling 0-4 ConjunctivaDraize Discharge 0-3 Conjunctiva
Total Draize Score 0-10 ConjunctivaLight Reflex 0-2 IrisFlare 0-3 Aqueous HumorIritis 0-4 IrisCorneal Opacity 0-4 CorneaCorneal-Area Involved 0-4 Cornea
Each of the above parameters is an important indicator
of eye irritation, inflammation, or infection. Therefore
there is a point in each parameter range which is biologi-cally significant and, when exceeded, indicates that severeocular problems exist. This point is called the "SeverityThreshold." Values lower than this indicate only minimalocular involvement or irritation; values greater than this"Severity Threshold" indicate severe ocular problems or
serious damage to the eye (38).
The scores achieved in the Positive Control group, whichis infected but not treated, should represent the most severeor serious ocular injury obtainable in the animal model.Therefore, the scores for the animal eyes in the PositiveControl group are used to determine the "Severity Thresholds".
17
The "Severity Thresholds" represent 1/2 the mean slit-lamp
values for each Positive Control group. There are different
"Severity Thresholds" for each infection model since the
severity of infection depends upon the virulence and patho-
genicity of the infecting bacterial strain (38).
In Tables III and IV, are given the "Severity Threshold"
values obtained at 24 and 72 h post inoculation for the
three animal infection models. Animal groups having meanscores greater than the values given in the table are judgedas having serious ocular inflammatory problems (+) whereas
those groups with mean scores less than these values haveonly minimal (-), if any, ocular problems.
The percent incidence is calculated for each inoculated
test group and represents the total number of individual
judgments which exceed the "Severity Thresholds"/total number
of individual threshold judgements in the test group.
Cephalothin/Silicon Stability Assay
Sodium cephalothin 1 percent suspensions were prepared
by mixing 1 g of antibiotic powder to 100 ml silicon fluid.
Aliquots of these suspensions were dispensed into 15 ml
amber bottles with plastic screw caps. These supplies weredesignated "cephalothin/silicon stability samples". Storagetemperatures were 4, 25, and 45 C. The samples were assayedfor sodium cephalothin potency at 0, 4, 11, and 16 weeks.
18
The methodology for determination of the antimicrobial
potency of sodium cephalothin in aqueous solutions is
described in the United States Code of Federal Regulations
(10). This standard agar diffusion assay method was altered
to facilitate the assay of 1 percent cephalothin/silicon
stability suspensions. The following ether extraction pro-
cedure (9) was used. One ml of a nonaqueous 1 percent
cephalothin suspension was placed into a 125 ml separatory
funnel and the 1 ml volumetric pipet used for transfer rinsed
twice with pH 6.0 phosphate buffer. The rinse was added
to the separatory funnel (the same rinse procedure was used
for the cephalothin standard). Fifty ml of peroxide-free
ether was then added to the separatory funnel and the
sample/ether suspension shaken until homogenous. Twenty-
five ml of pH 6.0 phosphate buffer was then added, the
separatory funnel was shaken well and the buffer (aqueous)
and ether phases were allowed to separate. The buffer layer
was removed and the extraction repeated six times collecting
the cephalothin extract in a 200 ml volumetric flask. The
resulting concentration of sodium cephalothin = 50 pg/ml.
A 1:50 dilution was made of the 50 pg/ml cephalothin solution
and this 1 jig/ml final concentration then used to fill
appropriate agar diffusion assay cylinders.
CHAPTER III
RESULTS
Defining "Severity Threshold" Levels
Tables III and IV give the "Severity Threshold" levels
for the three animal infection models at 24 and 72 h post
inoculation. Animal groups having mean scores greater than
the values given in the tables are judged to have serious
ocular inflammatory problems (+), whereas those groups with
mean scores less than these values have only minimal (-),
if any, ocular problems.
TABLE III
CRITERIA FOR ANALYSIS OF 24 HOUR SLIT-LAMP EXAMINATIONS
SeverityThresholdsaDraize Light Corneal CornealScores Reflex Flare Iritis Opacity Area
Group Mean Valuesb
Pseudomonas aeruginosa 4.0 0.7 1.2 1.6 1.5 1.6
Staphylococcus aureus 4.4 1.0 1.5 2.0 1.7 2.0
Streptococcus pneumoniae 1.6 0.3 0.8 1.0 0.9 0.9
b Based on Slit-Lamp observations at 24 hours postinoculation.Based on 1/2 Mean Values of Positive Controls.
19
20
TABLE IV
CRITERIA FOR ANALYSIS OF 72 HOUR SLIT-LAMP EXAMINATIONS
Severit ThresholdsaDraize Light Corneal CornealScores Reflex Flare Iritis Opacity Area
Group Mean Valuesb
Pseudomonas aeruginosa 4.0 0.9 1.4 1.8 1.8 1.8
Staphylococcus aureus 4.5 1.0 1.5 2.0 2.0 2.0
Streptococcus pneumoniae 4.0 0.8 1.4 1.9 1.8 1.9
a Based on Slit-Lamp observations at 72 hours postinoculation.b Based on 1/2 Mean Values of Positive Controls.
Pseudomonas aeruginosa Model
The mean scores and severity judgments based on 24 h
and 72 h slit-lamp data are given in Tables V and VI,
respectively. The percent Incidence (Total No. of indi-
vidual judgments which exceed the "Severity Thresholds"/
Total No. of individual judgments in test group) was 83
percent at the 24 h readings and 90 percent at 72 h for
the Positive Control Group. At 72 h, all the eyes of the
three antibiotic formulation groups were judged severely
infected. Neither cephalothin formulation was effective
24 or 72 h after P. aeruginosa inoculation. Garamycin was
moderately effective (50 percent Incidence of severity)
at 24 h post inoculation.
7
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23
Staphylococcus aureus Model
The mean scores and severity judgments for this model
are given in Tables VII and VIII. Both cephalothin formu-
lations were effective against this S. aureus strain at
24 and 72 h post inoculation. At 24 h Garamycin was not
as effective as the two cephalothin formulations. The per-
cent incidence values show that Garamycin proved to be
almost equivalent to the cephalothins at 72 h. Although
the percent incidence for the Garamycin group was 12 percent
compared to 17 percent for the Cephalothin/Garamycin Vehicle
group at 72 h, the mean severity judgment values for
Garamycin were significantly greater than those for either
cephalothin test group.
24
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26
Streptococcus pneumoniae Model
Against this gram-positive organism, the cephalothin
formulations were again more active than the 0.3 percent
Garamycin. Mean scores and severity judgments from TablesIX and X show that at 24 and 72 h the percent Incidence
for the Positive Control Group was 60 percent and 94 percent,
respectively. Garamycin was less active than the cephalo-
thins at 24 and 72 h. The mean severity judgment values
were greater for the Garamycin treatment group than for
either cephalothin group.
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29
Vehicle and Negative Controls
Compared to the Negative Control eyes (Group 16),
neither the Silicon or Garamycin Vehicle Controls (Groups
14 and 15) showed significant ocular pathology. Table XI
shows that the Negative and Garamycin Vehicle Controls had
almost identical slit-lamp mean score values whereas the
Silicon Control eyes had slight conjunctiva involvement
(Draize Score). The eyes inoculated with S. aureus and
treated with Dow 360 silicon (Group 17) were severely infec-
ted at 24 h (compared with S. aureus Positive Control Slit-
lamp values).
TABLE XI
EVALUATION OF SLIT-LAMP DATA FOR VEHICLE a AND NEGATIVE CONTROLS
Mean Values
Test Draizec Light Corneal CornealTest Group Period Score Reflex Flare Iritis Opacity AreaNegative Controls 24 h 0.3 0.0 0.0 0.0 0.1 0.1(Group 16) 72 h 0.1 0.0 0.0 0.0 0.0 0.0Silicon Controls 24 h 1.2 0.0 0.0 0.0 0.0 0.0(Group 14) 72 h 0.5 0.0 0.0 0.0 0.0 0.0Garamycin Vehicle 24 h 0.2 0.0 0.0 0.0 0.0 0.0Controls (Group 15) 72 h 0.1 0.0 0.0 0.0 0.0 0.0S. aureus/Silicon 24 h 10.0 2.0 3.0 4.0 4.0 4.0Controls (Group 17)
a Vehicle Controls Eyes of Groups 14 and 15 were dosed with sterileDow 360 silicon or Garamycin vehicle, respectively. These eyeswere not inoculated with bacteria. Eyes of Group 17 were inocu-lated with S. aureus and dosed with sterile Dow 360 silicon.Negative Controls - Eyes were not anesthesized, proptosed, inocu-lated, or dosed.
c Draize Score - Measure of conjunctival pathology. Range 0-10.
30
Cephalothin/Silicon Stability
Table XII describes the potency assay results for the
cephalothin in silicon stability samples. Each cephalothin
stability sample retained its initial potency after a totalof 16 weeks at storage temperatures of 4, 25, and 45 C.
Assay difficulties were experienced because of the viscosity
of the silicon formulation. The samples had to be mechan-
ically shaken for approximately 2 minutes to resuspend the
cephalothin. Laboratory experience with the potency assay
of nonaqueous sodium cephalothin formulations has shown
the assay variability range to be + 5 percent. It is
assumed that the low potencies reported at 4 weeks storage
were because of inadequate resuspension and not to anti-
biotic potency loss.
The visual appearance of the samples remained unchanged.
TABLE XII
CEPHALOTHIN/SILICON a STABILITY ASSAY DATA
Assayed Potency (% of Standardb)Time (weeks) 4 C 25 C 45 C
Initial984 85 86 8611 101 95 10016 103 103 103
a 1 g sodium cephalothin to 100 ml silicon fluid (1% suspen-sion)
b Standard-sodium cephalothin aqueous suspension.
CHAPTER IV
DISCUSSION
The data obtained from these experiments demonstrate
the effectiveness of a 1 percent suspension of cephalothin
in silicon as a nonaqueous topical ophthalmic formulation.
The suspension was equal to the 1 percent cephalothin in
aqueous solution in preventing S. aureus and S. pneumoniae
keratitis infections and more effective than Garamycin in
these models. The average incidence of severe infection
for the two gram-positive organisms at 24 and 72 h post
inoculation in the Cephalothin/Silicon treatment group was
12.7 percent, in the Cephalothin/Garamycin Vehicle group
it was 12.2 percent, and in the Garamycin treatment group
it was 32.7 percent. Both cephalothin formulations were
ineffective against P. aeruginosa whereas the gentamicin
solution did show antimicrobial activity against this gram-
negative pathogen. These results are consistent with pub-
lished in vitro susceptibility data (Tables I and II).
Approximately 106 organisms were injected into each
test rabbit cornea. This inoculating dose was higher than
needed. In this investigator's experience, 104 organisms
per dose produce adequate keratitis infections. The 106inoculating dose attributed to the high incidence of infection
31
32
for the P. aeruginosa model. Therefore, the 72 h datafor this model was essentially lost due to the rapid
invasive capabilities of P. aeruqinosa.
The cephalothin in silicon formulation was easily
managed during the animal dosing periods. The formulationwas readily resuspended and dispensed from the 1 ml dosingsyringes. The color and consistency remained constantthroughout the 9 h treatment period. All the animals tol-erated the silicon formulation well. There was a siliconresidue about the eyes of rabbits treated with the cepha-lothin in silicon suspension indicating the possibility
of increased contact time with this formulation.
There was some concern as to whether the cephalothinwould be in some way 'bound' or otherwise retained in thesilicon fluid at the corneal surface. This apprehension
of non-bioavailability can now be discounted in that thecephalothin in silicon and cephalothin in aqueous vehicleformulations were equivalent in their antimicrobial activity.
Ocular pathology was not evident when either the Siliconor Garamycin Vehicle Controls were administered to the non-inoculated rabbit eyes. Although not considered significant,the Silicon Control eyes did show slight conjunctival
involvement when compared to the Negative Controls.
The visual appearance of each cephalothin in siliconstability sample remained unchanged but the cephalothindid fall out of suspension with time. This settling caused
assay problems because the suspensions became increasinglymore difficult to resuspend. It was necessary to mechanicallyshake the samples for extended periods before uniformitywas evident. The addition of a dispersing agent e.g.anhydrous lanolin or tween 80 and/or formulating with alower cephalothin concentration may be required to correctthis problem. The Dow 360 silicon used for this study wasat a viscosity of 100 cps. Another approach to solvingthe resuspension problem is to suspend in a less viscoussilicon (a Dow 360 silicon of 20 cps is available).
Cephalothin in silicon fluid was stable for sixteenweeks at 4, 25, and 45 C. With essentially no loss inpotency at 45 C for 16 weeks it is assumed that the cepha-lothin in silicon formulation has a room temperature (25 C)stability of 32 weeks or twice the elevated temperaturestorage time. These estimations of potency derived fromelevated temperature studies are common in the pharmaceuticalindustry (17) and are useful; but, empirically, it can onlybe said that the 1 percent cephalothin in silicon has astability of 16 weeks as determined by the stability dataat 25 C obtained in this study.
The bacterial keratitis, toxicity, and stability datagenerated by this study encourage this investigator toexamine the possible use of nonaqueous cephalothin formula-tions. At the 18th Interscience Conference on AntimicrobialAgents and Chemotherapy (1978) several papers were presented
33
which described new cephalosporins demonstrating anti-pseudomonal activity (1, 18, 25, 30, 33, 39, 41, 42). Onesuch cephalosporin (HR756) was reported to have MIC'sagainst P. aeruginosa in the range of 6.2 - 12.5 pg/ml (41).This cephalosporin would surely be an important ophthalmicantibiotic with its inherent Staphylococcus and Streptococcusactivity combined with the additional Pseudomonas activity.This antibiotic may become even more noteworthy in lightof reported Pseudomonas resistance with repeated gentamicin
usage.
The results of this in vivo comparison suggest a numberof studies that should be performed to fully realize thepotential of nonaqueous antibiotic formulations. Becauseof the slight conjunctival involvement seen with the SiliconControls, longer in vivo toxicity studies should be performedwith the silicon vehicle. Also, the lowest effective con-centration of cephalothin in silicon should be determined.The 1 percent concentration tested in this study is assumedto be higher than the effective dose. It would be desirableto reduce the cephalothin concentration to 0.3 percent.Both gentamicin and tobramycin ophthalmic formulations areat this concentration and in vitro data suggests that cepha-lothin would be effective at this lower concentration (3, 22).
Finally, although combination antibiotic therapy isoften discouraged because of possible bacterial resistanceand/or additive toxicity complications, a cephalothin in
34
35
gentamicin combination ophthalmic formulation might proveto be extremely effective. An in vivo study involving S.aureus, S. pneumoniae, and P. aeruginosa could answer manyquestions regarding synergistic effects with the two anti-biotics.
CHAPTER V
SUMMARY AND CONCLUSION
TABLE XIII
SUMMARY OF RESULTSa
Percent IncidencebTest Group P. eruginosa . aureus S. pneumoniaePOSITIVE 24 h 83 100 60CONTROLS 72 h 90 100 94CEPHALOTHIN/ 24 h 88 3SILICON 72 h 100 7 2CEPHALOTHIN/ 24 h 88 10GARAMYCIN 72 h 100 17 7VEHICLE
GARAMYCIN 24 h 50 53 3172 h 100 12 35
a Results from Tables V, VI, VII, VIII, IX, and X.b % Incidence: Total No. of individual slit-lamp judgments whichexceeded the "Severity Thresholds"/Total No. of individual thresh-old judgments in test group.
The 1 percent cephalothin in silicon and the 1 percentcephalothin in aqueous vehicle were both more effectivethan Garamycin against S. aureus and S. pneumoniae keratitisinfections when topically applied to the rabbit eye. Com-parisons of the percent incidence of ocular infections inTable XIII show that the activity of the 1 percent cephalothin
36
was not reduced when formulated in the nonaqueous silicon.Garamycin was more effective in preventing P. aeruginosa
infections. Neither cephalothin formulation had activityin this keratitis model.
The in vitro antimicrobial potency assay of the 1 percentcephalothin in silicon formulation was accomplished by modi-fying the methodology published in the U.S. Federal Registerto include an ether extraction procedure. By this assaymethod it was determined that the cephalothin in siliconformulation did not decrease in antimicrobial potency for16 weeks at 4, 25, and 45 C. From the elevated temperaturestability study it is assumed that the cephalothin in siliconformulation has a room temperature (25 C) stability of atleast 32 weeks. This would be a significant increase fromthe present 2-3 day stability of aqueous cephalothin pre-
parations.
Two potential formulation problems became apparent duringthe study. The cephalothin in silicon formulation becameincreasingly more difficult to resuspend with time; therefore,a dispersing agent, a lower cephalothin concentration, and/ora less viscous silicon medium will be required to correctthis problem. Also, although the silicon vehicle did notcause significant ocular pathology after 9 hourly doseswith 0.1 ml, it is noteworthy that the mean draize scorevalues were higher in this group than in the GaramycinVehicle Control group.
37
38
This study presents data indicating that sodium cepha-lothin can be formulated into an ophthalmic dosage formwhich is both effective and stable. It may be that thenonaqueous vehicle concept can be expanded to include cepha-losporins with antipseudomonal activity, combinationcephalosporin/aminoglycoside formulations, or other anti-biotics which are unstable in aqueous medium.
LITERATURE CITED
1. Acar, J.F., J.M. Husson. 1978. HR 756 Pharmacokinetics,
preliminary therapeutic results in severe human infec-tions. U. Pierre et Marie Curie, Paris. 18th ICCACmeeting, presentation No. 82.
2. Barber, M., P. Waterworth, 1964. Penicillinase - resis-tant penicillins and cephalosporins. Brit. med. J.2:344.
3. Barker, B.M., F. Prescott. 1973. Antimicrobial agentsin medicine. Blackwell Scientific Publications, Oxford.
4. Bloch, R., F. Luft. 1979. Protection from gentamicinnephrotoxicity by cephalothin and carbenicillin.
Antimicrob. Agents Chem. 15:4649.
5. Boyle, G.L., R. Abel. 1972. Intraocular penetrationof sodium cephalothin in man after subconjunctival
injection. Am. J. Oph thal. 74:868-874.
6. Cagle, G.D., G.R. Carney, 1977. StatrolO vs. Neosporin*:An in vivo comparison. Alcon Laboratories Microbiology
Technical Report 012:7330:76/77.
7. Cagle, G.D., G.R. Carney, B.A. Schlech. 1976. BB-K122III. In Vitro evaluation against Streptococcus species.Alcon Laboratories Microbiology Technical Report
0225:7330:75/76.
39
8. Cagle, G.D., B.A. Schlech, G.R. Carney. 1976. BB-K122 II. In Vitro evaluation against Staphylococcus
aureus. Alcon Laboratories Microbiology Technical
Report 024:7330:75/76.
9. Carney, G.R. 1978. Cephalothin sodium agar diffusionassay. Alcon Laboratories Microbiology Procedure No.
341.06.
10. Code of Federal Regulations. 1977. Cephalothin agardiffusion potency assay procedure. 21CFR Parts 300 to499 revised as of April, 1977, 436.105, 436.103b1,436.101(1), 436.200(1), 436.106, 430.6(b)(34). Pub-lisher: Office of the Federal Register National Archivesand Records Service General Services Administration.
11. Corcoran, J.W., F.E. Hahn. 1975. Antibiotics III.Mechanism of action of antimicrobial and antitumor
agents. Springer-Verlag, New York.
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