TOBRAMYCIN

Alekha K. Dash

Department of Pharmaceutical Sciences

School of Pharmacy and Allied Health Professions

Creighton University

Omaha, NE 68 178

ANALYTICAL PROFILES OF DRUG SUBSTANCES 579

AND EXCIPIENTS-VOLUME 24
580 ALEKHA K. DASH

TABLE OF CONTENTS

1. History and Therapeutic Properties

2. Description
2.1 Nomenclature
2.1.1 Chemical Name
2.1.2 Generic Name
2.1.3 Trade Names
2.1.4 CAS Registry Number
2.2 Formula and Molecular Weight
2.3 Elemental Composition
2.4 Appearance, Color and Order
2.5 Pharmaceutical Dosage Forms

3. Synthesis

4. Physical Properties
4.1 Infrared Spectrum
4.2 H Nuclear Magnetic Resonance Spectrum
4.3 3C Nuclear Magnetic Resonance Spectrum
4.4 Ultraviolet Spectrum
4.5 Mass Spectrum
4.6 Thermal Behavior
4.7 Melting Point
4.8 Solubility
4.9 X-Ray Powder Diffraction Patterns
4.10 Dissociation Constants
 TOBRAMYCIN 581

5. Methods of Analysis
5.1 Identification Tests
5.2 Spectrophotometric
5.3 Chromatographic
5.3.1 Thin Layer Chromatography
5.3.2 High Pressure Liquid Chromatography
5.3.3 Gas Chromatography
5.4 Biological
5.4.1 Microbiological Assay
5.4.2 Radioimmuno Assay
5.4.3 RadioenzymaticAssay
5.4.4 Fluorescence Polarization Immunoassay
5.4.5 Fluorescence Immunoassay

6. Stability, Degradation and Incompatibility

7. Pharmacokinetics

8. Toxicity

9. Acknowledgments

10. References
582 ALEKHA K. DASH

1. History and Therapeutic properties

In 1957, investigators at Lilly Research Laboratories first isolated a

streptomyces species from soil samples collected in Hermosillo (Sonora,
Mexico). Seven antibiotic factors were isolated from Streptomyces
tenebrarius (ATCC 17920) [ 11, and factor 6 was subsequently designated
as tobramycin [2]. Tobramycin is an aminoglycoside antibiotic, exhibits
bactericidal activity against a broad spectrum of bacteria, and is only active
on actively multiplying bacteria. It inhibits protein synthesis, possibly on
the 30s subunits of the bacterial ribosomes [3]. Indications of this drug
include sepsis, urinary tract infections, infections of the skin, soft tissue
infections, respiratory tract infections, erc. It is especially useful in
treatment of infections due to Pseudomonas and indolepositive Proteus.

2. Description

2.1 Nomenclature

2.1.1 Chemical Name

0-3-amino-3 -deoxy-a-D-glucopyranosy1-(1-6)-0- [2,6-diamino-

1-4)]-2-deoxy-D-
2,3,6-trideoxy-a-D-ribo-hexopyranosyl-(
streptamine.

0-3-amino-3-deoxy-a-D-glucopyranosyl-( 1-4)-0-[2,6-diamino-
1-6)]-2-deoxy-L-
2,3,6-trideoxy-a-D-ribo-hexopyranosyl-(
streptamine.

2.1.2 Generic Name

Tobramycin

2.1.3 Trade Names

Distobram, Gernebcin, Obramycin, Nebcin, Tobradistin,

Tobralex, Tobramaxin, and Tobrex.
 TOBRAMYCIN 583

2.1.4 CAS Registry Number

32986-56-4

2.2 Formula and Molecular Weight

Free base C I 8H37N509 MW=467.45

Sulfate salt (C18H37N509)2 5 H2S04 MW = 1425.4

2.3 Elemental Composition

The theoretical elemental composition of tobramycin, based on the

molecular formula C18H37N509, is: C 46.24% , H 7.98%, N 14.98%,
0 30.80% [4]. Elemental analysis of tobramycin monohydrate has
been reported by Koch et al. [5] as:
Calculated, C: 44.52%; H: 8.10%; N: 14.43%.
Found, C: 44.39%; H: 8.15%; N: 14.07%.

2.4 Appearance, Color and Odor

Tobramycin is obtained as a crystalline, white to off-white, hygroscopic

powder.
5x4 ALEKHA K. DASH

2.5 Pharmaceutical Dosage Forms

Tobramycin is available in the following dosage forms: tobramycin

injection, tobramycin sulfate injection, tobramycin ophthalmic ointment and
tobramycin ophthalmic solution.

3. Synthesis

Takagi et al. [6] have synthesized tobramycin from kanamycin B.

Kanamycin B was converted to penta-N-ethoxycarbonyl-4",6"-0-
cyclohexylidene-2"-benzoylkanamycinB. This compound (1 mol
equivalent) was then treated with excess of p-toluenesulfonyl chloride (5
mol equivalent) in pyridine at 25°C overnight to give the 3'-O-tosyl
derivative as the major product. Iodination of 3'-O-tosyl derivative (mp
149-150°C) was achieved after reacting with sodium iodide in
dimethylformamide (4.9 g NaI in 10 mL of DMF) at 100°C to produce a
unstable 3I-iodide derivative. This unstable derivative was subsequently
hydrogenated with Raney nickel and hydrogen in dioxane to give 2"-0-
benzoyl-4",6"-O-cyclohexylidene-3'-deoxy-penta-N-
ethoxycarbonylkanamycin (mp 2485250°C). This compound was
successively treated with hot 4N barium hydroxide and 50% v/v acetic acid
at 80OC to give crude 3'-deoxykanamycin B. This material was purified by
chromatography and recrystallized as a monohydrate.

4. Physical Properties

4.1. Infrared Spectrum

The infrared spectrum of tobramycin is shown in Figure 1. The spectrum

was obtained in potassium bromide disk (0.5% w/w) using a FTIR (model
1600,Perkin-Elmer spectrophotometer. Assignments of the characteristic
bands in the spectrum are listed in Table 1 [7].
c
.d
586 ALEKHA K. DASH

Table 1

Infrared Spectral Assignments for Tobramycin

Energy (cm-1) Assignment

3400 - 3200 N-H stretching (s, br)

0-H stretching (s, br)

2910 Aliphatic C-H stretching (m)

1588 N-H bending (s)

1461 CH2 scissoring (m)

1377, 1349 0-H inplane bending vibration (m)

1032 C-N stretching ( s )

C - 0 stretching (s)

(br) = Broad
(m) = Medium intensity
(s) = Strong intensity
 TOBRAMYCIN 587

4.2 'H Nuclear Magnetic Resonance Spectrum

The 200-MHz proton nuclear magnetic resonance spectrum of tobramycin

was obtained on a Bruker AM 200 NMR spectrometer, and is shown in
Figure 2. The spectrum was recorded at an ambient temperature.
Deuterated water (D20) was used as the solvent, and tetramethylsilane was
the reference standard [7].] The chemical shifts, multiplicities and peak
assignments of characteristic protons are given in Table 2, and these were
found to be close to the reported values [5].

4.3 13C Nuclear Magnetic Resonance Spectrum

The 15.08 MHz 13C Nuclear Magnetic Resonance spectrum of 0.3-0.5 M

aqueous solution of tobramycin were recorded in aqueous solution,
containing 5% dioxane as an internal standard. The nmr instrument
consisted of a Varian Associates DP-60 magnet working at 14 kG with an
external 19F lock. The samples were spun in 13-mm 0.d. tubes [8].
Chemical shifts and structural assignments are outlined in Table 3, and are
based on the number system given above.
Figure 2. Proton Magnetic Resonance Spectrum of Tobramycin free
base in D7O.
 TOBRAMYCIN 589

Table 2

N M R Spectral Assignments for Tobramycin

Chemical Shift Multiplicities Number of Assignment
@Pm> protons

6.25 d 1 Anomeric Protons

6.05 d 1 (1' and 1")

4.15-4.85 m 10 Protons on carbon

bonded to hydroxyl
group or ether linkage

3.85-4.15 m 6 Protons on carbon

bonded to amino
groups

2.9-3.17 m 2 (es) Methylene group in

a six membered ring

2.55 9 1 (ax) Methylene group in

a six membered ring

2.25 9 1 (ax> Methylene group in

a six membered ring

d = doublet eq = equatorial
m = multiplet ax = axial
q = quartet
590 ALEKHA K.DASH

Table 3

C NMR Spectral Assignments for Tobramycin [81

Chemical Shift Assignments

@Pm>

99.2 c-1'
49.5 c-2'
34.7 c-3'
65.9 c-4'
73.1 c-5'
41.5 C-6'
50.2 c-1
35.5 c-2
49.0 c-3
86.0 c-4
74.4 c-5
87.8 C-6
99.1 c-1"
71.6 c-2"
54.2 c-3"
69.2 c-4"
71.9 c-5"
60.2 C-6"
 TOBRAMYCIN 59 1

4.4 Ultraviolet Spectrum

-
Owing to its saturated ring system and lack of suitable chromophores,
tobramycin does not exhibit any significant absorption between 230 and 360
[91.

4.5 Mass Spectrum

A Finnigan INCOS-SOB quadrupole mass spectrometer linked to a Hewlett-

Packard gas chromatograph using electron impact at an electron energy of
70 eV and a source temperature of 180°C was used to study the mass
spectrum and fragmentation behavior of tobramycin. Unfortunately, no
useful mass spectra were obtained, as had been reported [lo].

4.6 Thermal Behavior

The Differential Scanning Calorimetry (DSC) thermogram of tobramycin

base is shown in Figure 3. The sample was heated from 30 - 25OoC in a
nonhermetically crimped aluminum pan at a rate of 1O"C/min on a DuPont
model 950 thermal analysis system. The first endothermic peak was
attributed to compound dehydration, and was followed by the melting of the
metastable form at 164OC. The metastable forms recrystallizes to the stable
form as evidenced by the exotherm at 197SoC, and finally melts at 217OC
[111.

Thermogravimetric (TG) analysis of the base was carried out on a DuPont

model 95 1 thermogravimetric analyzer, and the resulting TG and
differential TG thermograms are shown in Figure 4. It was concluded from
this work that the commercially available sample consisted of the
monohydrate phase containing some absorbed water [l 11.

4.7 Melting Point

The metastable form was observed to melt at approximately 164OC, while

the stable form melts at approximately 2 17OC [111.
 113.69-C

t63.91°C

216.78-C

I 1
I I
50 100 150 200 PI
Temperature ('13

Figure 3. Differentialscanning calorimetry thermogram of

Tobramycin free base.
 0.W

-
0.06
100

-u
0.04 5
bJ
C
98- 0
-..

VI
:
w
W 0.02 ;
.LI
a.
u
0

96-

0.00

-0.02

Figure 4. (a) Thermogravimetricanalysis and (b) differential

thermogravimetricanalysis thermograms of Tobramycin free
base.
594 ALEKHA K. DASH

4.8 Solubility

'Tobramycin is freely soluble in water (1 in 1.5 parts), very slightly soluble

in ethanol (1 in 2000 parts), and practically insoluble in chloroform and
ether [9,12]. A 10% (w/v) solution of tobramycin in water has a pH of 9-1 1
[13].

4.9 X-Ray Powder Diffraction Pattern

The powder pattern data of tobramycin base was obtained using a wide
angle X-Ray diffractometer (model D500, Siemens). The powder
diffraction patterns of the two polymorphs are shown in Figure 5a and 5b.
The calculated d-spacings for the diffraction patterns are provided in Table
4 [14].

4.10 Dissociation Constants

In one publication, three pKa values were reported for tobramycin as 6.7,
8.3. and 9.9 [15]. However, in another work four pKa values (6.2,7.4, 7.6,
and 8.6) were reported by Raymond and Born [ 161.

5. Methods of Analysis

5.1 Identification [13]

Tobramycin is identified by a thin layer chromatographic method, and the

exact details of this procedure are described in the subsequent TLC
discussion (section 5.3.1).
 TOBRAMYCIN 595

6.0 8.0 10 12 14 $6 I8 20 22 24

28, degrees

Figure 5. Powder x-ray diffiaction patterns of; (a) commercially

available Tobramycin free base (Form I), and (b)
Tobramycin free base heated to 208OC (Form 11).
SY6 ALEKHA K. DASH

Table 4

Powder X-Ray Diffraction Data for Tobramycin

Diffraction pattern of Diffraction pattern of

Tobramycin base (Form I) Tobramycin base (Form 11)

Peak d-Spacing Relative Peak d-Spacing Relative

No (4 Intensity No (A) Intensity
(%I
1 15.07 27 1 15.77 10
2 10.35 48 2 7.69 18
3 8.87 49 3 7.08 89
4 8.14 36 4 5.90 29
5 7.48 33 5 5.50 29
6 6.17 53 6 4.98 84
7 4.97 57 7 4.72 25
8 4.79 100 8 4.64 20
9 4.69 47 9 4.27 100
10 4.56 35 10 3.95 31
11 4.39 36 11 3.77 27
12 4.24 36 12 3.60 31
13 4.08 54
14 3.91 31
15 3.79 53
 TOBRAMYCIN 591

5.2 Spectrophotometric Methods

A colorimetric method based on the reaction between tobramycin and

copper sulfate has been developed for the quantification of this compound
in injectable formulations [17]. A colorimetric method based on the
reaction between tobramycin and 2,4-dinitrofluorobenzene(Sanger's
reagent) has been reported for the quantification of tobramycin in topical
formulations [181.

A spectrofluorimetric method for the determination of tobramycin in

biological fluids using a fluorescent dihydro-lutidine derivative has also
been reported [191. The fluorescent derivative is formed by condensation of
the primary amino groups of tobramycin with acetyl-acetone and
formaldehyde under acidic conditions (pH=2.4).

Sampath and Robinson have reported a spectrophotometricmethod for the

analysis of tobramycin and compared their method with the existing
methods [20].

5.3 Chromatographic Methods

5.3.1 Thin Layer Chromatography [13]

Tobramycin solution is prepared in distilled water (0.6% w/v). A 3 pL

portion of this solution is applied to a silica gel (0.25-mm layer) TLC plate.
The chromatogram is developed by equilibrating the plate for 5.5 hours in a
chromatographic chamber containing a mixture of methanol, ammonium
hydroxide and chloroform (60:30:25; v/v/v). The plate is removed from the
chamber and heated at 11O°C for 15 minutes. The spots are detected by
spraying with a 1 in 100 solution of ninhydrin in a mixture of butyl alcohol
and pyridine (100:1, v/v), and tobramycin is visualized as a pink spot.

5.3.2 High Performance Liquid Chromatography (HPLC)

The very low absorptivity of tobramycin in the UV and visible region does
not permit its direct quantification at low concentrations. This problem can
be solved by derivatizing this compound with a suitable absorbance-
598 ALEKHA K. DASH

enhancing or fluorescence-producing agent. This deficiency can be

overcome through the use of either pre-column or post-column
derivatization. Various HPLC methods using pre-column [21-261 and post-
column [27,28] derivatization to quantify tobramycin in pharmaceuticals
and biological fluids have been described and are summarized in Table 5 .

5.3.2 Gas Liquid Chromatography (GLC)

A CiLC method has been described by Mayhew et al. [29] for the assay of
tobramycin in biological fluids. A silanized Pyrex column (2 m by 3 mm
id) packed with 3% OV-101 coated onto 80-100 mesh Chromosorb WAW
was utilized in this method. Nitrogen gas was used as a carrier. The
injector and detector temperature were maintained at 272' and 287OC
respectively, and a electron captured detector was used in this study.

5.4 Biological Methods

5.4.1 Microbiological assay

Various microbiological assay methods for the analysis of tobramycin have

been described [24,30,3 11. The method developed by Maitra et al. [24] used
an agar diffusion method using Bacillus subtilis (ATCC 6633) as a test
organism. The organism was grown on seeded agar at 37°C for 16-18
hours. These microbiological assays are reliable and simple but they are
time consuming and less specific.

5.4.2 Radioimmunoassay (RIA)

Radioimmunoassays have been developed for measuring tobramycin in

biological fluids [32,33]. The RIA is based on the competition between
1251-tobramycinand unlabeled tobramycin in the sample to be analyzed for
the antibody binding sites. Unbound 251-tobramycin is separated by
centrifugation, and the radioactivity of the bound tobramycin is counted and
the levels calculated from a standard curve. This method is highly sensitive
and specific.
 Table 5

HPLC Methods for the Analysis of Tobramycin

Type of Derivatizing Mobile Phase Column Detection Sample Type Referenc

Derivatization Agent Mode e

Pre-column 1-fluoro-2,4- Water:acetone:acetic C18 uv Biological 21

dinitrobenzene acid (30:70:0.1; v/v/v) 30 cm x 3.9 mm (365 nm) fluids
Flow = 3 mL/min

Pre-column 2,4,6-trinitro- Acetonitri1e:phosphate CIS uv Biological 22

benzenesulfonic buffer (70:30 v/v) 30 c m x 4 mm (365 nm) fluids
acid Flow = 3 mL/min

Pre-column 1-fluoro-2,4- 75% V/V (NH4)3P04 c 18 uv Formulations 23

dinitrobenzene and 25% v/v 30cmx4mm (365 nm)
Acetonitrile
Flow = 2 mL/min

Pre-column o-phthalaldehyde, Methano1:water (72:26) C18 Fluorescence Biological 24

mercaptoethanol and 0.005 EDTA 30 cm x 4 mm (360 nm EX fluids
Flow = 1 mL/min 430 nm EM)
Pre-column o-phthalaldehyde. 250 mL of 0.5 M Tris p-Bondpak C18 Fluorescence Biological 25
mercaptoethanol buffer + 10 mL of (Waters} (360 nm EX fluids
triethylamine, and qs to 430 nm EM)
I L with methanol
Flow = 2 mL/min

Pre-column 2,4,6-trinitro- Acetonitrile5OmM CIS uv Formulations 26

benzenesulfonic Phosphate buffer 25 cm x 4.6 mm (340 nm)
acid (62:38 v/v)
Flow = 2.5 mL/min

Post-column o-phthalaldehyde, Sodium. sulfate (0.1 p-Bondpak C18 Fluorescence Biological 27

mercaptoethanol M), sodium 30 cm x 3.9 mm (340 nm EX fluids
pentasulfonate (0.02 (Waters) 4 18 nm EM)
M), and 17.4 mM
acetic acid in 1 L of
water
Flow = 2 mL/min

Post-column o-phthalaldehyde water:methanol:acetic Lichosorb 5 RP Fluorescence Biological 28

acid (99.7:0.2:0.1 mole C8 (15 cm) (340 nm EX fluids
%) containing 0.2 (Chrompack) 418 nm EM)
moles of sodium sulfate
and 0.02 moles of
sodium pentane sulfate,
Flow = 1 mL/min
 TOBRAMYCIN 60 1

5.4.3 Radioenzymatic assay

Radioenzymatic assays for the assay of tobramycin in biological fluids have

been reported [34-361. The method involves the specific enzymatic transfer
of a radioactive modifying group to the drug. These enzymes are present in
organisms that carry resistant (R) factors which are responsible for the
activation of the drug. The entire reaction process is stoichiometric, and the
amount of radioactivity incorporated is proportional to the concentration of
the antibiotic. These assays are simple, accurate and precise, but the need to
work with radioactive material may pose a disadvantage for some clinical
laboratories.

5.4.4 Fluorescence polarization immunoassay

Fluorescence polarization immunoassay (FPI) is a method that combines the

principles of competitive protein binding with the principles of fluorescence
polarization, and has also been utilized to determine the tobramycin
concentration in serum [37,38].

5.4.5 Fluorescence immunoassay (FIA)

FIA uses the principle of competitive protein binding, and has been used to
quantify tobramycin in biological samples [28, 39-41]. Competitive
binding reactions are set up with fluorogenic tobramycin reagent, a limiting
amount of antibody against the drug, and the serum sample to be analyzed.
Tobramycin in the serum sample competes with the fluorogenic tobramycin
reagent for the antibody binding sites. The unbound fluorogenic reagent is
then hydrolyzed by P-galactosidase to produce the fluorescence which is
detected as the observable parameter.

6. Stability, degradation and incompatibility

Tobramycin solution in water at pH 1 to 11 was reported to be stable for

several weeks at temperatures from 5 to 37OC, and could be autoclaved
without loss of potency [12]. When aqueous tobramycin was adjusted to pH
602 ALEKHA K. DASH

1.2 with HC1 and autoclaved for 30 minutes at 120°C in sealed glass
ampules, an extra peak was observed in the chromatogram. This was
attributed to a possible degradation product [26].

Tobramycin is compatible with most available IV fluids, but is not

compatible with heparin solution. In addition, it can interact chemically
with p-lactam compounds of the penicillin, cephalosporin, and cephamycin
family [ 3 ] . This interaction depends upon the concentration and pH of both
tobramycin and f3-lactam compounds. Solutions of tobramycin sulfate and
clindamycin phosphate have been reported to be unstable in dextrose
injection [42].

The stability of tobramycin in 30 and 50% dextrose peritoneal dialysate

concentrate (PDC) fluids have been reported [43]. This study indicated that
if tobramycin is to be added to PDC fluids containing 50% wlv of dextrose,
it should be used within 12 hours of admixture.

7. Pharmacokinetics

7.1 Absorption

Tobramycin is not appreciably absorbed when taken orally, but does exert
an antibacterial effect in the intestine. When applied to the skin, the drug is
not absorbed to a degree sufficient to produce any therapeutic effects. There
is no significant absorption of the drug from the bronchi and lungs after
administration as an aerosol [44]. Studies in rabbits suggest that tobramycin
is absorbed into the aqueous humor following topical installation of 3
mg/mL solution of the drug onto the eye and absorption is greatest when the
cornea is abraded [45].

Owing to these adsorption characteristics, tobramycin should be

administered either intramuscularly (IM) or intravenously (IV). Absorption
after IM injection is rapid, with the peak serum concentration being
achieved at 20-45 minutes. Senun concentrations of tobramycin following
a single IM injection are given in Table 6 [46-511. Mean peak serum
concentrations of tobramycin following various rates of IV injections are
given in Table 7 [49,50,52,53].
 TOBRAMYCIN 603

Table 6

Mean Peak Serum Concentration of Tobramycin Following

Single Intramuscular Injections

Dose Serum concentrations References

(I.ldmL)

50 mg/m2 4.6 46
100 mg 5.1 47
2.5 mgkg 7.1 48
100 mg 3.8 49
100 mg 5.2 50
40 mg 2.4 51
80 mg 3.7 51
604 ALEKHA K. DASH

Table 7

Mean Peak Serum Concentration of Tobramycin

Following Intravenous Injections

Rate of Injection Dose Peak serum References

levels (yg/mL)

1 hour infusion 1 mgkg 4.4 52

30-45 min 1 mg/kg 5.5 49

30-45 min 1.5 mgkg 6.0 49

1 hour infusion 100 mg 4.6 50

bolus injection 80 mg 11.2 53

(2.5-3 min)

bolus injection 1 mgkg 10.0 53

(2.5-3 min)
 TOBRAMYCIN 605

7.2 Distribution

The distribution of tobramycin in human tissue and body fluids is

summarized in Table 8 [54-601. The half-life of the drug in serum ranges
between 1.6 and 3.5 hours in normal individuals. Ragamey et al. have
reported the apparent volume of distribution (AVD) value for tobramycin to
be 24.5 liters [50]. However, Simon et al. have reported the AVD value to
be 16.9 liters [51].

7.3 Protein binding

Using equilibrium dialysis, Ramirez-Ronda et al. have reported that

approximately 70% tobramycin is bound to plasma proteins at a
concentration of 10 mg/mL or less [61]. However, Gorden et al. [62], and
Neber et a!. [63] have reported that under conditions of physiological pH
and temperature, no serum protein binding of tobramycin occurred at a
concentration of 5 mg/mL. A similar effect was also reported by Ullmann
et al. using steady state gel filtration and frontal analysis [64].

7.4 Excretion

Tobramycin is rapidly excreted unchanged in the urine after an IM or IV

injection [65]. However, Pechere and Dugal[66] have suggested that 10%
of the drug is eliminated by extrarenal mechanisms. The renal excretion of
tobramycin takes place entirely by glomerular filtration [46,50]. The total
plasma clearance of tobramycin from IV infusion studies have been reported
to be 113.7 mL/min/1.73 m2 [50] and 87.9 mL/min/1.73 m2 [51]. The rate
of recovery of tobramycin from urine over a 6 hour period is 60% and 80-
85% during the 24 hours after injection [67,68]. During the first 6 hours
after a dose of 1 mgkg (given by infusion over a period of 1 hour), urinary
concentrations between 70 and 300 mg/mL have been reported [52].
 Table 8

Distribution of Tobramycin in Various Tissues and Body Fluids

Tissue or other Dose and Routes Time Concentration References

body fluids of administration elapsed detected (pg/mL)

Breast milk 80 mg by IM 1 hour 0.60 54

8 hours 0.85 55

Amniotic fluid 80 mg by IM 3 hours 1.2 56

Cerebrospinal fluid 3-4.5 mgkg by IV <I 57

sputum 5 m g k g IM injection 1 to 3 hours 0.3 58

in three divided doses

Bile 80 mg im 1 hour 1.4 59

Palatine tonsil 80 or 160 mg im 1 hour 0.5 to 1.3 60

samples
 TOBRAMYCIN 607

Table 9

LD50 of Tobramycin in Rat and Mouse [69]

Animal species Route of LD50 (mg/kg)

Administration

Rat intraperitoned 1030

Rat subcutaneous 969
Rat intravenous 104
Rat intramuscular 913
Mouse intraperitoneal 445
Mouse subcutaneous 3 67
Mouse intravenous 72.5
Mouse intramuscular 440
Pig subcutaeous 676
 ALEKHA K.DASH

8. Toxicity

The LD50 values determined for tobramycin in different animal species are
given in Table 9 [69]. The LD50 for tobramycin is found to be 50-66% of
that of gentamicin in mice, and 70% of that of rats [70,71]. Lethal and
sublethal doses of tobramycin produce hyperactivity, decreased respiration
and prostration in mice, rats and guinea pigs. No noticeable changes in
behavior, appearance, or in hematological or biochemical values were
noticed in dogs when 3.75 and 7.5 mgkg of tobramycin is administered for
90 days. There was evidence of slight cloudy swelling of proximal portions
of nephrons after 30 mgkg dose of the drug for 90 days.

9. Acknowledgments

The useful suggestions of Professors. Raj Suryanarayanan (University of

Minnesota, College of Pharmacy) and Shanker L. Saha (Creighton
University) are gratefully acknowledged.

10. References

1. C. E Higgens and R. E. Kastner, Antimicrob. Agents Chemother.,

1967, 324-331 (1971).

2. R. Q. Thompson and E. A. Presti, Antimicrob. Agents Chemother.,

1967,332-340 (1971).

3. H. C. Neu, J. Infect. Diseuse., 134 (Suppl.), S3-Sl9 (1976).

4. The Merck Index, S. Budavari, ed., 1l* Edition, Merck and Co.,
Inc., Kahway, N.J., USA, 1989, p. 1499.

5. K. F. Koch and J. A. Rhoades, Antimicrob. Agents. Chemother.,

1970,309-313 (1971).

6. Y. Takegi,T. Miyake, T. Tsuchiya, S. Umezaya and H. Umezaya, J

Antibiotics., 26,403-406 (1973).
 TOBRAMYCIN 609

7. A. K. Dash, unpublished data.

8. K.F Koch, J. A. Rhoades, E. W. Hagaman and E. Wenkert, J. Am.

Chem. Soc., 96,3300-3305 (1974).

9. Clarke's Isolation and Identification of Drugs in Pharmaceutical,

Body Fluids and Post-mortem Material, A. C. Moffat, ed., 2nd
Edition., The Pharmaceutical Press, London, 1988, p. 1027.

10. T. Mills I11 and J. C. Roberson, Instrumental Data for Drug

Analysis, 2"d Edition, Vol3, Elsevier, NY, 1987, pp. 2256-2257.

11. A. K. Dash and R. Suryanarayanan, Pharm. Res., 8, 1159-1165

(1991).

12. Martindale, The Extra Pharmacopoeia, J. E. F. Reynolds, ed., 29*

Edition,The Pharmaceutical Press, London, England, 1989, pp. 326-
328.

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