A study of two anhydrous water-soluble ointment bases for ophthalmic use

MISSING IMAGE

Material Information

Title:
A study of two anhydrous water-soluble ointment bases for ophthalmic use sterilization, preservation, rheology and application
Physical Description:
xxv, 415 leaves : ill. ; 29 cm.
Language:
English
Creator:
Rogers, A. Garnell, Jr., 1943-
Publication Date:

Subjects

Subjects / Keywords:
Ophthalmic Solutions   ( mesh )
Pharmacy thesis Ph.D   ( mesh )
Dissertations, Academic -- Pharmacy -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Dissertation (Ph.D.) -- University of Florida.
Bibliography:
Bibliography: leaves 394-413.
Statement of Responsibility:
by A. Garnell Rogers, Jr.
General Note:
Photocopy of typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000342534
oclc - 08038594
notis - ABX8673
System ID:
AA00009118:00001


This item is only available as the following downloads:


Full Text











\ A STUDY OF TWO ANHYDROUS WATER-SOLUBLE
OINTMENT BASES FOR OPHTHALMIC USE1:
STERILIZATION, PRESERVATION, RHEOLOGY AND APPLICATION















BY

A. GARNELL ROGERS, JR.


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1981














DEDICATION



This dissertation is dedicated to my wife, Beth,

and my son, Christopher, whose unfailing support and

encouragement through the extended period necessary

for the completion of this project made its accomplishment

possible.
































Copyright 1981

by

A. Garnell Rogers,Jr.














ACKNOWLEDGEMENTS


I wish to express my sincere gratitude to Dr. Charles

H. Becker for serving as Chairman of my Supervisory Com-

mittee until his retirement, for his special contribution

to my educational development, and for his continued sup-

port, his donation of personal time after his retirement

from active teaching, and his friendship throughout the

time required for this achievement.

I wish to thank Dr. John H. Perrin for giving freely

of his time to serve on my Supervisory Committee and for

assuming the duties of Chairman upon the retirement of

Dr. Becker.

I wish to thank Dr. Arnold S. Bleweis for numerous

discussions which resulted in the completion of major

portions of this work, and for his unfailing encouragement

as a member of my Supervisory Committee.

I wish to express my sincere appreciation to Dr. Oscar

E. Araujo, Dr. Dinesh O. Shaw, and Dr. L. Gene Gramling

for their contributions to my doctoral research program,

their advice on specific research problems, and for

serving on my Supervisory Committee.

I wish to acknowledge the help given by the personnel

of several departments: Mrs. Esther B. Jones and the









reference staff of the Health Center Library, for guidance

during the initial literature search; Dr. Alvin F.

Moreland, his staff of veterinarians and the employees of

Animal Resources, whose constant care, advice and concern

for my animals allowed the completion of important segments

of this project; the staff of the Bioelectronics department,

for several pieces of special apparatus; and the members of

the College of Pharmacy stock room, for the rapid procure-

ment of many items critical to the completion of this

research project. My thanks are expressed also to Dr. Ronald

G. Marks and Dr. Jane F. Pendergast for their help in the

statistical analysis of several sections of this work.

I wish to express my gratitude to my parents and my

wife's parents for their continued caring and support, and

to my grandfather, the late A.D. Rogers, whose encouragement

many years ago sustained my interest in education and who

would have been delighted to see this achievement.

My heartfelt thanks are expressed to my typist,

Mrs. Susan Mahon, for her excellent work and for reading

my handwritten notes; and to Miss Marie Dence, for her

valued friendship and for providing the opportunity for me

to finish the writing of this dissertation.

I wish to acknowledge the financial assistance provided

by a graduate assistantship in the College of Pharmacy and

support by the U.S. Army Research and Development Command.
















TABLE OF CONTENTS


Chapter

ACKNOWLEDGEMENTS . .

LIST OF TABLES . .

LIST OF FIGURES . .

ABSTRACT . .

I. INTRODUCTION . .

II. REVIEW OF THE LITERATURE .

Definition of Terms .

Solution. . .

Microbial Bioburden .

Organogels . .

Ionic Deswelling ...

Ophthalmic Ointments .

CarbopolR and Polyethylene Glycol
Gel Bases . .

Ophthalmic Irritation Testing .

Test Animals for Ophthalmic Testing

Microbiology of the Anterior Segment
of the Eye . .

Sterilization Methods of Ophthalmic
Ointments . .

Sterility Assurance Tests for
Ophthalmic Ointments .


Page

. iv

. xiii

. xvi

. .xxiii


. 10

. 10
. 10



. 11

. 11

. 11

. 12


Mechanisms of Preservative Action .


rr





















Surface Tension: Applicability to
Ophthalmic Preparations. .. 55

Definition. . 55

Measurement of Surface Tension. 55

Applications to Ophthalmic
Preparations . ... 58

Rheology: Applicability to
Ophthalmic Preparations. ... 61

Definitions . ... 61

Measurement of Semi-Solids. ... 64

Applications to Ophthalmic
Preparations . 66

III. EXPERIMENTAL METHODS. . ... 69

Materials ... . 69

Equipment . .... 69

Ointment Base Composition and
Preparation. . 69

Microorganisms. . ... 91

Test Animals and Examination Facilities 91

Photomicrography. . 96

Preliminary Studies: Irritation
Potential. . 96

Test Preparations ... 97

Scale of Evaluation ... 99

Test Animals. . .. 99

Testing Procedure .. ... 103

Sterilization and Sterility Assurance
Tests. . . 105


vii


Chapter


Page









Chapter


Preparation and Standardization
of Heat-Resistant Spore Sus-
pension of Bacillus
stearothermophilus. ... 106

Culture characteristics of
B. stearothermophilus ..... 106

Sporulation medium .. .107

Method of spore production
and harvest ... .107

Standardization of heat-
resistant spore suspensions
of B. stearothermophilus 109

Methods of Sterilization and
Sterility Testing of Ointment
Bases E-3 and E-4 ... .112

Media. . ... 112

Growth promotion tests .. .113

Bacteriostatic effects of test
samples on growth of
B. stearothermophilus 114

Sterility test procedure for
direct transfer to test
media . ... 115

Sterility test procedure for
membrane filtration .. .116

Dry heat sterilization method. .117

Moist heat sterilization
method: oil bath simulation. 120

Methods of Sterilization and
Sterility Tests of Individual
Components. . ... 121

Polyethylene glycols 200, 400,
and 4000. . ... 121

1,2,6-Hexanetriol. .... 122

Ethomeen C/25. . 122

viii


Page









Chapter


Carbopol-940 resin: dry
heat sterilization. ... 122

Carbopol-940 resin: gaseous
sterilization ... .123

Aseptic combination of
sterilized components .. .123

Heat Transfer Characteristics of
Covered and Uncovered Samples
of Ointment Bases E-3 and E-4 124

Antimicrobial Preservative Effectiveness
in Bases E-3 and E-4. .. ... 125

Test Organisms . .. 125

Media. . ... 125

Microbial Characteristics. ... 126

Coagulase production ...... 126

Fluorescin and pyocyanin
production. . ... 126

Antimicrobial Preservative System. 127

Composition. . ... 127

Method of sample preparation 127

Preservative Effectiveness Testing:
Tube Dilution Method. .. .130

Specific preservative inactiva-
tion media. . ... 130

Preparation of inoculum. ... .131

Preparatory testing. .. .131

Tube dilution procedure. ... .133

Preservative Effectiveness Testing:
Zone of Inhibition Method 136

Preservative Effectiveness Testing:
Test for Fungal Growth
Inhibition by Surface Inocula-
tion Method . .. 138
ix


Page











Rheological Characteristics of
Nonsterile and Sterilized Bases E-3
and E-4 Upon the Addition of Water,
Atropine Sulfate, and Various
Preservatives. . 139

Purposes. . .. .139

Rotating Viscometer . .. .140

General Method. . .. .142

Effects of Water Addition 143

Effects of Sterilization by Moist
Heat and Dry Heat. ... 143

Effects of Atropine Sulfate Addition. 144

Effects of Preservative on the
Rheological Properties of
Sterilized Bases .. 146

Surface Tension Effects of Bases E-3 and
E-4 Dilutions. .. . 147

Relative Apparent Bioavailability of 1%
Atropine Sulfate Ophthalmic Ointment 153

Test Animals. . .. .153

Experimental Design ... 153

Supportive Studies. . 158

Determination of Apparent pH. ... 158

Tonicity Effects of Aqueous Dilutions
of Bases E-3 and E-4 on Suspended
Red Blood Cells. . .. .159

Approximate Melting Point of
Bases E-3 and E-4, With and
Without Atropine Sulfate and
Varying Water Content. .. .161

IV. RESULTS AND DISCUSSION . .. 163

Preliminary Studies . ... 163

Control .. . .163
x


Chapter


Page









Chapter Page

Standard. . 163

Ethylene glycol . .. .164

Base E-3 . .167

Base E-4. . ... 167

Base E-5. . .. 167

Base M-13N. . 168

Statistical Comparisons . 169

General Comments. . .. .169

Sterilization and Sterility Assurance
Tests. . . .179

Preparation and Standardization of
Heat-Resistant Spore Suspension
of B. stearothermophilus .. .180

Methods of Sterility Testing of
Ointment Bases E-3 and E-4 ... 188

Dry Heat Sterilization Method ..... 190

Moist Heat Sterilization Method:
Oil Bath Simulation. ... 191

Moist Heat Sterilization Method:
Steam Sterilization. .. .193

Sterilization of Individual
Components . .. 193

General Comments . .. 195

Heat Transfer Characteristics of Covered
and Uncovered Samples of Bases E-3
and E-4. . ... 198

Antimicrobial Preservative Effectiveness
in Bases E-3 and E-4 .. 209

Tube Dilution Method. . 209

Preparatory testing ... .209

Tube dilution procedure .. .213
xi









Chapter Page

Zone of Growth Inhibition Method. 218

Surface Inoculation Method for
Fungal Growth Potential .. 236

General Comments. . .. 237

Rheological Characteristics of Modified
Bases E-3 and E-4. . 241

Effects of Water Addition 241

Effects of Sterilization. .. .262

Effects of Atropine Sulfate Addition. 279

Effects of Preservative Addition. 297

General Comments. . ... 297

Effects of Bases E-3 and E-4 on Apparent
Surface Tension of Water ... 328

General Comments. . ... 337

Relative Bioavailability of Atropine
Sulfate from Bases E-3 and E-4 in
Rabbit Eyes. . ... 344

General Comments. . ... 360

Supportive Studies . 372

Determination of Apparent pH. ... .372

Tonicity Effects of Aqueous Dilutions
of Bases E-3 and E-4 on Suspended
Red Blood Cells. . ... 377

Approximate Melting Points of
Bases E-3 and E-4. ... 382

General Comments. . ... 385

V. SUMMARY AND CONCLUSIONS . .. 387

REFERENCES . . 394

BIOGRAPHICAL SKETCH .... 414


xii














LIST OF TABLES


Table Page

1. Materials. . ... 70

2. Materials for Preparation of Ointment Bases. 80

3. Equipment. . . 83

4. Per Cent (w/w) Composition and Method of
Preparation of Experimental Ophthalmic
Ointment Bases . .. 89

5. Microorganisms Used in Study and Related
Characteristics. . ... 92

6. Scale of Evaluation of Ophthalmic Irritation 100

7. Irritation Testing: Summary of Individual
Responses to a Sterile Commercial
Petrolatum-base Ointment . ... 165

8. Irritation Testing: Summary of Individual
Responses to Experimental Ophthalmic
Ointments With and Without Dimercaprol 170

9. Krushall-Wallis Statistical Analysis of
Ophthalmic Irritation Test Results .. .174

10. Results of Standardization Tests for
Spore Suspension, Lot 071676,
B. stearothermophilus, at 1210 ........ 185

11. Heat Transfer Characteristics of Uncovered
and Covered 50-g Samples of Ointment
Bases E-3 and E-4 in Oil Bath at 1210 for
Various Times. . ... 199

12. Specific Preservative Inactivation Media 210

13. Preparatory Tests of Specific Inhibitory
Media and Test Specimens . .. 211


xiii











14. Results of Tube Dilution Tests for
Antimicrobial Preservative Effectiveness. 214

15. Zones of Growth Inhibition of Plate
Cultures of S. aureus, and P. aeruginosa,
Produced by Samples of Base E-3 and
Base E-4, Plain or Containing Specific
Preservative Systems. . .. 221

16. Studentized Range Multiple Comparison
Procedure Analysis of Zones of Inhibition
for Ophthalmic Ointment Preservative
Against S. aureus and P. aeruginosa .. 226

17. Dunnett's Multiple Comparison Procedure
Analysis of Zones of Inhibition for
Base E-3 and E-4 Ophthalmic Ointments with
Preservatives Against S. aureus and
P. aeruginosa . . 234

18. Rotational Viscometer Conversion Factors
for Instrument U-Values to RPM. ... 242

19. Rotational Viscometer Values of Non-Sterile
Bases E-3 and E-4 Containing Various
Amounts of Water at 250 . 243

20. Rotational Viscometer Values of Bases E-3
and E-4 Sterilized by Moist Heat at 1210 or
Dry Heat at 1400 for 1 and 2 hours. ... 263

21. Rotational Viscometer Values of Sterilized
Bases E-3 and E-4 Containing Atropine
Sulfate, 1% (w/w), or Atropine Sulfate
(1% w/w) and Water, 4% (w/w). ... 280

22. Rotational Viscometer Values of Sterilized
Bases E-3 and E-4 Containing Various
Preservatives. ... . .. .305

23. Effects on the Apparent Surface Tension of
Deionized Water by Increasing
Concentrations of Dodecyl Sodium Sulfate,
Sterilized and Non-Sterilized Bases E-3
and E-4, and Various Contributory
Components. . . .. 329


xiv


Table


Page











24. Preliminary Bioavailability Results.
Per Cent Change in Rabbit Eye Pupil
Diameter following Single-Dose
Administration of either Commercial
Standard, Base E-3, or Base E-4 Sterile
Ophthalmic Ointments Containing 1% (w/w)
Atropine Sulfate. . ... 346

25. Average Per Cent Change in Right and
Left Rabbit Eye Pupil Diameters following
Single-Dose Administration of either
Commercial Standard, Base E-3, or
Base E-4 Sterile Ophthalmic Ointment
Containing 1% (w/w) Atropine Sulfate. ... .348

26. Summary of Averaged Bioavailability-
Related Parameters for Commercial Standard,
Base E-3, and Base E-4 Ophthalmic
Ointments Containing 1% (w/w) Atropine
Sulfate . . .. .. 352

27. Analysis of Variance for Maximum
Individual Per Cent Dilatation Response to
Single-Dose Administration of Commercial
Standard, Base E-3, and Base E-4
Ointments Containing 1% (w/w) Atropine
Sulfate. .. . .. 357

28. Analysis of Variance for Time Required
to Attain Maximum Individual Per Cent
Dilatation Response to Single-Dose
Administration of Commercial Standard,
Base E-3, and Base E-4 Ointments
Containing 1% (w/w) Atropine Sulfate. ... .359

29. Results of Apparent pH Determinations for
Various Aqueous Dilutions of Bases E-3
and E-4, With and Without 1% (w/w)
Atropine Sulfate at Zero Time and 200 373

30. Tonicity Effects of Various Dilutions of
Bases E-3 and E-4, With and Without 1%
(w/w) Atropine Sulfate, on Human Red
Blood Cell Suspensions. ... .. .378

31. Approximate Melting Points of Bases E-3
and E-4, With and Without 1% (w/w)
Atropine Sulfate, with Various Concentrations
of Water. . . .. 384


Table


Page















LIST OF FIGURES


Figure


Page


1. Growth characteristics for Bacillus
stearothermophilus in Tryptic Soy
Broth incubated at 60. Turbidimetric
determinations of 625 um. . .. 181


2. Survivor curve for aqueous suspension
of heat-resistant spores of Bacillus
stearothermophilus, Lot 071676, at
oil bath exposure temperature of 121.0 .

3. Heat transfer characteristics of 50-g
sample of base E-3 in uncovered glass
container immersed in oil bath at 121.00.

4. Heat transfer characteristics of 50-g
sample of base E-4 in uncovered glass
container immersed in oil bath at 121.00.

5. Difference in temperature (AT) between
edge and center temperatures vs time
in 50-g samples of bases E-3 and E-4
in uncovered glass containers immersed
in oil bath at 121.00 . .

6. Heat transfer characteristics of 50-g
sample of base E-3 in covered glass
container immersed in oil bath at 121.00.

7. Heat transfer characteristics of 50-g
sample of base E-4 in covered glass
container immersed in oil bath at 121.00.

8. Difference in temperature (AT) between
edge and center temperature vs time
in 50-g samples of bases E-3 and E-4
in covered glass containers immersed
in oil bath at 121.00 . .

9. Example of developed plate with zones
of inhibition surrounding wells of
samples . . .


. 187



. 202



. 203





. 204



. 206



. 207





. 208



. 220


xvi









Figure

10. Characteristic rheogram of triturated
anhydrous base E-3. . .

11. Characteristic rheogram of base E-3
containing 0.99% (w/w) water. .

12. Characteristic rheogram of base E-3
containing 2.91% (w/w) water. .

13. Characteristic rheogram of base E-3
containing 4.76% (w/w) water. .

14. Photomicrograph of nonsterilized
base E-3, brightfield illumination.

15. Characteristic rheogram of triturated
anhydrous base E-4. . .

16. Characteristic rheogram of base E-4
containing 0.99% (w/w) water. .

17. Characteristic rheogram of base E-4
containing 2.91% (w/w) water. .. ..

18. Characteristic rheogram of base E-4
containing 4.76% (w/w) water. .

19. Photomicrograph of nonsterilized base
brightfield illumination. .

20. Photomicrograph of nonsterilized
base E-4, polarized light .

21. Photomicrograph of nonsterilized
base E-4, brightfield illumination.

22. Photomicrograph of nonsterilized
base E-4, polarized light .


Page


. 246


S. 248


. 249


. 250


. 252


. 253


. 254


. 255


. 256

E-4,
. 259


. .. 259


S ... 261


. 261


23. Characteristic rheograms of nonsterilized
anhydrous bases E-3 and E-4, undisturbed
standard preparations for use in
comparisons . ... .267

24. Characteristic rheograms of anhydrous
base E-3 sterilized by moist heat
(autoclaving) for 1 hour or 2 hours
at 1210, fast exhaust . ... 268

25. Characteristic rheogram of anhydrous
base E-3 sterilized by dry heat for
1 hour or 2 hours at 1400 .... 269
xvii









Figure


26. Characteristic rheograms of anhydrous
base E-4 sterilized by moist heat
(autoclaving) for 1 hour or 2 hours
at 1210, fast exhaust. .

27. Characteristic rheogram of anhydrous
base E-4 sterilized by dry heat for
1 hour at 1400 . .

28. Characteristic rheogram of anhydrous
base E-4 sterilized by dry heat for
2 hours at 1400 . .

29. Photomicrograph of anhydrous base E-4
sterilized by moist heat (autoclaving)
for 1 hour at 1210, fast exhaust.
Brightfield illumination .

30. Photomicrograph of anhydrous base E-4
sterilized by moist heat (autoclaving)
for 2 hours at 1210, fast exhaust.
Brightfield illumination .

31. Photomicrograph of anhydrous base E-4
sterilized by moist heat (autoclaving)
for 2 hours at 1210, fast exhaust.
Brightfield illumination .

32. Photomicrograph of anhydrous base E-4
sterilized by dry heat for 1 hour
at 1400. Brightfield illumination .


. 272


273



274


. 276


. 276




.. 278



. .. 278


33. Characteristic rheograms of nonsterilized
anhydrous base E-3 containing 1% (w/w)
atropine sulfate incorporated as crystals,
and nonsterilized base E-3 containing
1% (w/w) atropine sulfate and 4% (w/w)
water. . .. 286

34. Characteristic rheograms of anhydrous
base E-3 containing 1% (w/w) atropine
sulfate incorporated as crystals,
sterilized by moist heat (autoclaving)
for 1 hour or 2 hours at 1210, fast
exhaust. . . ... 287

35. Characteristic rheogram of anhydrous
base E-3 containing 1% (w/w) atropine
sulfate incorporated as crystals,
sterilized by dry heat for 1 hour or
2 hours at 1400. . 288


xviii


Page















Figure


36. Characteristic rheograms for anhydrous
base E-3 containing 1% (w/w) atropine
sulfate and 4% (w/w) water, sterilized
by moist heat (autoclaving) for 1 hour
or 2 hours at 1210, fast exhaust. .. .289

37. Characteristic rheograms of anhydrous
base E-3 containing 1% (w/w) atropine
sulfate and 4% (w/w) water, sterilized
by dry heat for 1 hour or 2 hours
at 1400 . .... 290

38. Characteristic rheograms of nonsterilized
anhydrous base E-4 containing 1% (w/w)
atropine sulfate incorporated as crystals,
and nonsterilized base E-4 containing
1% (w/w) atropine sulfate and 4% (w/w)
water . . .. ... 293

39. Characteristic rheograms of anhydrous
base E-4 containing 1% (w/w) atropine
sulfate incorporated as crystals,
sterilized by moist heat (autoclaving) for
1 hour or 2 hours at 1210, fast exhaust 294

40. Characteristic rheograms of anhydrous
base E-4 containing 1% (w/w) atropine
sulfate incorporated as crystals,
sterilized by dry heat for 1 hour or
2 hours at 1400 . .. 295

41. Photomicrograph of nonsterilized
anhydrous base E-4 containing 1%
(w/w) atropine sulfate incorporated
as crystals. Brightfield illumuniation 299

42. Photomicrograph of anhydrous base E-4
containing 1% (w/w) atropine sulfate
incorporated as crystals, sterilized
by moist heat (autoclaving) for 1 hour
at 1210. Brightfield illumination. .. .299

43. Photomicrograph of anhydrous base E-4
containing 1% (w/w) atropine sulfate
incorporated as crystalline fine powder,
sterilized by moist heat (autoclaving)
for 2 hours at 1210, fast exhaust.
Brightfield illumination ... .301


xix


Page









Figure


44. Photomicrograph of same sample presented
in Figure 43. Polarized light. ... 301

45. Photomicrograph of different section of
same sample presented in Figure 43.
Polarized light . .. 303

46. Photomicrograph of nonsterilized base E-4
containing 1% (w/w) atropine sulfate
and 4% (w/w) water. Brightfield
illumination . . 303

47. Photomicrograph of nonsterilized base E-4
containing 1% (w/w) atropine sulfate and
4% (w/w) water. Polarized light. ... 304

48. Characteristic rheograms of sterilized
base E-3 containing 0.01% (w/w)
benzalkonium chloride, or 0.01% (w/w)
benzalkonium chloride and 0.01% (w/w)
disodium ethylenediaminetetraacetate ... 309

49. Characteristic rheograms of sterilized
base E-3 containing 1000 units of
polymyxin B sulfate per gram of base
and 0.01% (w/w) disodium ethylene-
diaminetetraacetate, or 0.1% (w/w)
methylparaben and 0.02% (w/w)
propylparaben . . 310

50. Characteristic rheograms of sterilized
base E-3 containing 0.03% (w/w)
p-chloro-m-xylenol, or 0.5% (w/w)
chlorobutanol . .. 311

51. Characteristic rheograms of sterilized
base E-4 containing 0.01% (w/w)
benzalkonium chloride, or 0.01% (w/w)
benzalkonium chloride and 0.01% (w/w)
disodium ethylenediaminetetraacetate ... 312

52. Characteristic rheograms of sterilized
base E-4 containing 1000 units of
polymyxin B sulfate per gram of base
and 0.01% (w/w) disodium ethylene-
diaminetetraacetate or 0.1% (w/w)
methylparaben and 0.02% (w/w)
propylparaben . .. 313


Page









Figure


53. Characteristic rheograms of sterilized
base E-4 containing 0.03% (w/w)
p-chloro-m-xylenol or 0.5% (w/w)
chlorobutanol. . ... 314

54. Effect of increasing concentrations
of dodecyl sodium sulfate on the
apparent surface tension of deionized
water. . . 334

55. Effects of increasing concentrations of
nonsterilized and sterilized base E-3
on the apparent surface tension of
deionized water. . ... 336

56. Effect of increasing concentrations of
polyethylene glycol 200/400 vehicle
on the apparent surface tension of
deionized water. . ... 338

57. Effects of increasing concentrations
of nonsterilized and sterilized
base E-4 on the apparent surface
tension of deionized water ... .339

58. Effect of increasing concentrations of
polyethylene glycol 400/4000 vehicle
on the apparent surface tension of
deionized water. . ... 340

59. Effect of increasing concentrations of
1,2,6-hexanetriol on the apparent
surface tension of deionized water .. .341

60. Average per cent change in rabbit eye
pupil diameter vs time after single-
dose administration of commercially
available standard ophthalmic ointment
containing 1% (w/w) atropine sulfate .. 354

61. Average per cent change in rabbit eye
pupil diameter vs time after single-
dose administration of base E-3
containing 1% (w/w) atropine sulfate
and 4% (w/w) water . ... 355

62. Average per cent change in rabbit eye
pupil diameter vs time after single-
dose administration of base E-4
containing 1% (w/w) atropine sulfate
and 4% (w/w) water . .... 356
xxi


Page











63. Apparent pH determinations of various
aqueous dilutions of bases E-3 and E-4,
with and without 1% (w/w) atropine
sulfate, immediately after solution and
at 200 . .. 375


xxii


Figure


Page














Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy



A STUDY OF TWO ANHYDROUS WATER-SOLUBLE
OINTMENT BASES FOR OPHTHALMIC USE:
STERILIZATION, PRESERVATION, RHEOLOGY, AND APPLICATION

By

A. GARNELL ROGERS, JR.

December, 1981



Chairman: John H. Perrin
Major Department: Pharmacy



Two anhydrous water-soluble ointment bases, E-3 and

E-4, were evaluated as ophthalmic vehicles. Both exhibited

low irritation potential in rabbits' eyes.

Base E-3 contained polyethylene glycols 200 and 400

thickened by an amine-neutralized CarbopolR 940 resin to

form a stable transparent gel. Base E-4 contained

polyethylene glycols 400 and 4000 plasticized by 1,2,6-

hexanetriol.

Each base was sterilized satisfactorily using both

moist and dry heat. A specific biological indicator,

Bacillus stearothermophilus, provided sterility assurance.


xxiii









Several common preservative systems were evaluated

against standardized test organisms of Staphylcoccus

aureus, Pseudomonas aeruginosa, and Aspergillus niger,

using specific inhibitory media, a zone of inhibition

procedure, and direct surface inoculation. Results

indicated inherent inhibitory activity in both unpreserved

bases. Base E-3 was most effectively preserved against

S. aureus using benzalkonium chloride with disodium

ethylenediamine tetraacetate (Na2EDTA) and against

P. aeruginosa by use of polymyxin B sulfate with Na2EDTA.

The most effective preservative against either organism

for base E-4 was polymyxin B sulfate with Na2EDTA.

Rotational viscometry data and physical and micro-

scopic observations provided information on theological

effects of sterilization methods, and the addition of

atropine sulfate, water, and preservatives to both bases.

Base E-3 proved to be stable except for a dramatic

decrease in viscosity resulting from ionic charge neutrali-

zation when adding atropine sulfate. Evidence of

crystalline structure growth resulted from base E-4

sterilization.

Base E-3 demonstrated a small amount of surface

activity; increasing aqueous concentrations resulted in

decreasing apparent surface tension values.

The relative bioavailability of atropine sulfate

from single doses of ointments containing the drug in each


xxiv









base compared with a commercial standard showed no

significant differences in rate, degree, or duration of

rabbit-eye pupil dilatation. A randomized three-way

crossover study was possibly complicated by some animals

having an atropine-destructive enzyme, tropinesterase.

Adjunctive studies addressed the apparent pH of each

base, their tonic effects on human red blood cell suspen-

sions, and melting point ranges.

Based on these results, base E-3 proved to be the

more favorable preparation for ophthalmic use.


xxV












CHAPTER I
INTRODUCTION



The development of an ointment for ophthalmic use is

a complex and exacting process requiring the involvement

of widely varying disciplines.

The ideal ophthalmic ointment should possess several

characteristic properties. Ophthalmic preparations are

sterile products, essentially free from foreign particles,

suitable compounded and packaged for either topical

application to the eyelids, or instillation into the space

(cul-de-sac) between the eyeball and eyelids (1). Incor-

porated ophthalmic drugs must be stable for a practical

and reasonable amount of time after preparation. The

hydrogen-ion concentration of a solubilized ointment may

affect the therapeutic action of the medication, the

irritation potential of the instilled preparation, and

the length of time the medication remains in contact with

the ocular surface; therefore, an optimum pH must be

achieved by use of buffers, adjunct solution instillation,

or by other means. An ophthalmic ointment should produce

little, if any, irritation response upon instillation; and

such irritation that occurs should be limited to a

transitory response, never resulting in permanent injury

to the eye. The tonicity of water-soluble ophthalmic








vehicles dissolved in the lacrimal fluid should be such

that instillation does not cause wide-spread tissue or

cell destruction or creation, with resultant irritation

and discomfort. Ophthalmic ointments must possess a

consistency which allows ease and comfort of instillation,

extended retention time in the eye, rapid spreading on

the ocular surface, and non-leakage from the packaging

container. The melting point of a water-insoluble

ophthalmic ointment should be at or slightly below body

temperature. The melted or dissolved preparation should

produce a film on the ocular surface which is optically

clear and free from particulate matter. Bioavailability

of the drug from the ointment must be demonstrated to

the extent that the preparation is effective for its

intended use. Multiple-dose products must contain preserva-

tives or antimicrobial agents which prevent contamination

and subsequent infection or reinfection by application of

the ointment. Finally, the completed preparation must be

compatible with the packaging container for the established

shelf-life of the product.

It is in the course of the developmental process for

an ophthalmic preparation that each formulation must be

tested to determine the extent to which it possesses the

characteristics of the ideal product. The initial formu-

lation phase may produce several hundred preparations.

Preliminary screening tests for physical and chemical

stability reduce this number to a few possessing suitable

characteristics for further study.









In 1956, Becker et al. published a report on the

formulation, preparation and packaging of BAL (dimercaprol)

ointment (2). This study was done because the dimercaprol

ointment in use at that time leaked from the packaging

tube, was brittle and hard at 00 yet liquid at body tempera-

ture, and showed indications of dimercaprol instability in

the vehicle employed. As a result of this investigation,

recommendations were made concerning appropriate packaging

containers, sealing methods and two types of vehicles for

increased stability of the dimercaprol, one of which was

a polyethylene glycol-type base, the other a gel-type base

using a copolymer of methyl vinyl ether and maleic anhydride

(PVM/MA). As additional factors to increase the stability

of the dimercaprol component, an antioxidant was recommended

for addition and water-free ingredients were to be used

in preparations. The compositions of these ointments

were listed as follows:

Percent
Base Materials Composition (w/w)

CarbowaxR base a Polyethylene glycol 4000 31.7
Polyethylene glycol 400 63.2
Tenox BHA 0.1
BAL 5.0

PVM PVM/MA (0.1-0.5) 5.0-6.0
Ethylene glycol 78.8-79.0
Carbowax 4000 10.9
Tenox BHA 0.1
BAL 5.0



aCarbowax is a registered trademark of the City Chemical
Corporation, New York, N.Y.









The PVM/MA gel-type base was recommended as the best

base of those tested, noting that the polyethylene glycol-

type base showed some syneresis of BAL and was very hard

and brittle at 00. The content of BAL for each formulation

was suggested to be increased to 6.0% (w/w) to allow for

the initial rapid degradation which levels off on longer

storage. The intended use of these ointments was as a

topical preparation.

Interest in the ophthalmic use of a dimercaprol

ointment in the detoxification of ocular exposure to heavy

metal compounds such as chlorovinylarsine dichloride

(Lewisite), and the availability of new materials for the

formulation of ointment bases and for new packages, resulted

in a later investigation at the University of Florida College

of Pharmacy in which the formulation, testing and packaging

of several dimercaprol ophthalmic ointments was studied

(3,4). Eighty-four anhydrous, water-soluble ointment bases

and ninety-two anhydrous, oleaginous ointment bases, semi-

solid and homogeneous at the time of preparation, were

evaluated for physical and theological stability at

temperatures between 00 and 500. Spreadability, flow

properties, and degrees of transparency and solidarity at

the various temperatures were studied. Single-point

apparent pH values for several diluted water-soluble bases

were determined.

Based on physical spreadability characteristics and

acidic apparent pH values, five of the water-soluble









exploratory bases were selected for viscosity studies at

various temperatures. Three of these bases showed

acceptable and promising characteristics. Their composi-

tion are as follows:


Base

E-3 (x-61)




E-4 (x-77)



E-5 (x-83)


Materials

Carboxy vinyl polymer 940
Polyethylene glycol 200
Polyethylene glycol 400
Poly-o-et (15) coc

Polyethylene glycol 4000
Polyethylene glycol 400
1,2,6-Hexametriol

Carboxyl vinyl polymer 940
Polyethylene glycol 200
Polyethylene glycol 400
Poly-o-et (15) coc
Hexadecyl alcohol


Percent
Composition (w/w)

1.36
29.24
68.83
0.57

16.0
24.0
60.0

1.50
38.40
58.12
0.53
1.44


A similar investigation on the oleaginous exploratory

bases showed only one base which exhibited acceptable

characteristics. The composition of this base is as

follows:


Materials

Micronized silica
Stearyl alcohol
Hexadecyl alcohol


Percent
Composition (w/w)

12.0
2.0
86.0


New instrumentation and advanced methods of analysis

enabled the investigators to study various chemical and

physical aspects of pure dimercaprol to support with

fundamental information the development of a stable BAL

ophthalmic ointment. Stability studies of dimercaprol in

solution under conditions of varying pH were done for


Base

M-13N









stability baseline data for use in later accelerated

stability work. In vitro release studies of BAL from the

various ointment bases reported that the rate of release

of dimercaprol from the water-soluble base E-4 was faster

and more complete than any of the other water-soluble bases

studied, and that the oleaginous base M-13 (using stearic

acid instead of the stearyl alcohol used in M-13N) showed

good release characteristics (5). Gas-liquid chromatography

provided a useful means to determine, quantitatively, the

purity of dimercaprol, revealing the existence of at

least two contaminants, 3-mercaptopropylene sulfide (MPS)

and 1,2,3-trimercaptopropone (TSH), which may be toxico-

logically important (6). A qualitative method of deter-

mining the composition of BAL was also reported (7).

Using the results of these basic studies, the stability

of dimercaprol was investigated in the selected water-

soluble (8) and oleaginous (9) exploratory bases at various

temperatures and in several packaging containers. In

addition, dimercaprol ointments using the water-soluble

bases E-3, E-4 and E-5, and the oleaginous base M-13N,

were biologically tested in rabbit eyes for irritation

potential and for their effectiveness in the treatment of

ophthalmic exposure to Lewisite and to Mustard Gas, a

highly toxic vesicatory irritant (10). As a result of

these investigations, two water-soluble, anhydrous ointment

bases--base E-3, a carboxyl vinyl polymer organogel, and

E-4, a plasticized polyethylene glycol preparation--were









shown to be effective vehicles for dimercaprol as an

ophthalmic ointment, producing minimal irritation on instil-

lation and retaining desirable physical and theological

characteristics over a broad temperature range.

Jurgens and Becker reported on the use of some of the

exploratory semi-solid oleaginous bases developed as

ophthalmic vehicles for pilocarpine hydrochloride, including

determinations of softness point, water absorption, vis-

cosity characteristics and in vitro release rates (11).

It was felt that an investigation of the general applications

of the water-soluble exploratory bases E-3 and E-4 regarding

methods of sterilization, preservation, the incorporation

and effectiveness of a drug other than dimercaprol, and

some associated supportive studies would be appropriate

and interesting.

The purposes of this investigation are as follows:


1. To review and summarize the study of irritation
potential in rabbit eyes of selected water-soluble
or oleaginous exploratory bases E-3, E-4, E-5 and
M-13N, plain or containing dimercaprol, as necessary
and appropriate to the later presentation of a
bioavailability study for atropine sulfate;

2. To investigate various methods of sterilization
of bases E-3 and E-4, including moist heat, dry heat
and bacterial filtration, chracterizing the heat
transfer characteristics of each base, and using
a standardized suspension of heat-resistant bacterial
spores as a biological indicator for sterility
assurance tests;

3. To determine the effectiveness in bases E-3 and E-4
of antimicrobial agents or preservatives commonly used
in ophthalmic preparations against standardized inocula
of Staphylococcus aureus, Pseudomonas aeruginosa,
and spores of Aspergillus niger, using various test
methods;









4. To determine the theological effects on bases E-3
and E-4 of the addition of various components to
the anhydrous preparation, such as water, atropine
sulfate, and various preservatives, and the effects
of sterilization by moist heat or dry heat for
varying exposure times, relating these changes to
physical and microscopic observations;

5. To investigate the effects on aqueous surface tension
resulting from increased concentrations of bases
E-3, E-4 or various base components, compared to a
known surface-active material, dodecyl sodium sulfate;

6. To study the relative bioavailability of atropine
sulfate from bases E-3 and E-4, compared with a
commercial standard atropine sulfate ophthalmic
ointment, in rabbit eyes; and,

7. To report the results of supportive studies on bases
E-3 and E-4 concerning pH profiles of increasingly
diluted bases, melting point data, and tonicity effects
of diluted bases on human red blood cells in vitro.


The results of this investigation have shown that the

water-soluble, anhydrous organogel ointment base E-3 shows

promise for use as an ophthalmic vehicle and warrants

possible further testing, while the modified polyethylene

glycol ointment base E-4 exhibited definite long-term

stability problems, separating on storage and becoming

increasingly less extrudable. Based on the data reported,

base E-3 proved to be stable on sterilization and extended

storage, extrudable over a wide temperature range, unaffected

by the addition of various preservatives or small quantities

of water, and low in irritation potential. The ophthal-

mological bioavailability of two different medications,

dimercaprol and atropine sulfate, from this carboxyl vinyl

polymer-thickened base has been shown to be effective in

animal tests using rabbit eyes. Although susceptible to





9


consistency changes upon the addition of monovalent and

divalent salts, base E-3 may prove to be an acceptable

vehicle for other commonly used ophthalmic medications.














CHAPTER II
REVIEW OF THE LITERATURE





Definition of Terms



Several terms are used repeatedly throughout this

study and are defined for clarification and continuity.

Solution

Takruri (12) defines a solution as a thermodynamically

stable, homogeneous system of two or more components.

Within this definition is included the macromolecular

solution, where the molecular size and weight of the

molecular size and weight of the macromolecules are of

such magnitude that the system acquires unique properties.

These solutions are recognized now as true monophasic,

thermodynamically stable systems and the old concept of

considering them as heterogeneous dispersions is believed

to be inaccurate. Solutions of polymers such as neutral-

ized CarbopolR 940a fall under this classification.



aCarbopol is a registered trademark of the B.F. Goodrich
Chemical Co., Cleveland, Ohio.









Microbial Bioburden

This term is used to indicate the probable types

and numbers of microorganisms present in a product

immediately prior to sterilization (13).

Organogels

A gel is a solid or semisolid system of at least

two constituents, consisting of a condensed mass enclosing,

and interpenetrated by, a liquid (14). Gels may be

classified either as two-phase or as single-phase systems.

The gel mass may consist of floccules of small particles

dispersed in a liquid, forming a two-phase system which

may be thixotropic but not always stable. Alternatively,

a gel may consist of macromolecules existing as twisted

matted strands, bound together by van der Waals forces,

forming a single-phase system. Gels which contain an

organic liquid are termed organogels.

Gels are characterized by a comparatively high degree

of elasticity (15), undergoing rather large elastic defor-

mations, sometimes as much as 10-30%, at shear stresses

below the yield value, from which they recover their

shape when the stresses are removed. True gels are one

of two classes of semisolid materials which exhibit yield

stresses, or yield values, the other being pastes. Typical

gels will exhibit plastic behavior rheologically.

Ionic Deswelling

CarbopolR resins are very mild acids which readily

react with neutralizing agents to form salts. When the









proper salt is prepared in the given solvent, the polymer

uncoils and extends into its form of highest solvent-

holding capacity with attendant solvent thickening. The

addition of soluble salts decreases the efficiency of

CarbopolR mucilages by neutralization of the ionic charges

in the Carbopol salt, causing the polymer to return to a

more tightly coiled configuration with resultant thinning

of the preparation. Monovalent salts of all types, such

as sodium chloride, affect the Carbopol resins, while

divalent salts, such as calcium chloride, produce a more

drastic loss of thickening efficiency. This loss of

thickening capability by the described mechanism is known

as ionic deswelling.





Ophthalmic Ointments



Ophthalmic ointments are semisolid preparations

intended for application to the eye, contacting the

outside and edges of the eyelids, the conjunctiva, the

cornea, and affecting the iris and ciliary processes of

the lens. Special precautions must be taken in their

preparation (16). Ophthalmic ointments are manufactured

from sterilized ingredients under rigidly controlled

conditions and meet the requirements of the official

sterility tests. They must contain a suitable antimicrobial

substance if the preparation is intended for multiple-use.









The active ingredient may be added as a micromized

powder or as a solution, resulting in a finished ointment

which is free of large particles, that is, dispersed

material must be impalpable. The official compendium

provides tests designed to limit to a level considered to

be unobjectionable the number and size of discrete metal

particles that may occur in the ophthalmic ointment (17),

usually as a result of manufacture. Particles no larger

than 50 um and limited in number are acceptable.

The ointment must be comfortable and convenient to

apply, contain a minimum number of ingredients, and retain

the activity of the medicament for a reasonable period of

time under proper storage (18). It must also permit the

diffusion of the drug throughout the secretions bathing

the eye (19).

In ophthalmology, the primary use for an ointment as

a vehicle is to increase the ocular contact time of a

drug. Studies have indicated that with the use of radio-

active technetium the ocular contact time is about two

times longer in the blinking patient and four times longer

in the non-blinking patient than with the use of a saline

vehicle (20).

Semisolid ophthalmic vehicles frequently contain

soft petrolatum, a bland absorption base, or a water-

soluble base (21). The water-soluble base may be prepared

from polyethylene glycols or with a water-soluble gum.

Some absorption bases, water-removable bases, and water-









soluble bases may be desirable for water-soluble drugs (22).

Such bases allow for better dispersion of water-soluble

medicaments, but they must be non-irritating to the eye.

Petrolatum is used as a principal base for ophthalmic

drugs. A survey of currently available ophthalmic ointments

(23) showed that white petrolatum combined with mineral

oil and/or non-ionic lanolin derivatives was used

frequently. White petrolatum and mineral oil gelled with

a polyethylene resin served as a vehicle also. Petrolatum

is often used when an anhydrous vehicle is desired for

reasons of stability. Non-aqueous organogels may offer

advantages over the hydrocarbons, and only sparsely have

been investigated.

Problems with the use of petrolatum as an ophthalmic

ointment base are numerous. Mineral oil is frequently

added to petrolatum to lower its fusion point, but its

addition introduces the problem of separation on storage

(24), especially in warmer climates. Although this

problem can be prevented by the addition of small amounts

of natural wax such as ozokerite, ceresin, or microcrystal-

line wax, the addition of extra ingredients is not preferred.

The release of a medication from a petrolatum-type

base has been compared with release from other types of

ophthalmic ointment bases. The influence of twenty-five

different ointment bases on the release of chloramphenicol

in vitro, in the conjunctival sac, and also on its

penetration into the eye by testing aqueous humor of healthy









rabbit eyes was investigated by Richter (25). His studies

indicate that the activity of chloramphenicol is dependent

on the type of ointment vehicle in which it is incorporated,

and showed that the pure hydrocarbon bases exhibited pro-

tracted and incomplete drug release, though they remained

within the inferior formix in contact with the cornea for

5 hours or more. Good release of the medication was found

to occur from an o/w emulsion vehicle composed of Lanette

WaxR N, Lanette WaxR O, white petrolatum and water.

The composition of petrolatum can vary, depending on

the source. Petrolatum is available in the form of a

long or short "fiber" (26). The type of "fiber" possessed

by petrolatum generally is determined by dipping the

index finger into the sample and then slowly withdrawing

it. The long "fiber" type tends to form a transparent

continuous film joining the finger and the sample, while

the short "fiber" ruptures easily. Different "fiber"

types result in variation in spreading properties. The

official compendia permits wide density and melting point

ranges, as well as variation in chemical composition for

petrolatum throughout the world.

Instillation of a petrolatum-type base in the eye

can cause interference with vision. One disadvantage to

ophthalmic ointment use is the blurred vision as the

ointment base melts and spreads across the ocular

surface (27).









It is possible and probable that the use of non-

aqueous water-soluble organogel vehicles for ophthalmic

use will solve some of the problems associated with the

use of petrolatum. Organogels are stable, can be made

transparent, can be water-soluble, and therefore may

release medications more quickly than petrolatum when

used in the eye.




CarbopolR and Polyethylene Glycol Gel Bases



A gel has been defined as a form of matter inter-

mediate between a solid and a liquid (28). It consists of

polymers, or long-chain molecules, cross-linked to create

a tangled network and immersed in a liquid medium. The

properties of a gel depend strongly on the interaction

of these two components. The liquid prevents the polymer

network from collapsing into a compact mass; the network

prevents the liquid from flowing away. Depending on the

chemical compositions and other factors, gels vary in

consistency from viscous fluids to fairly rigid solids,

but typically they are soft and resilient.

CarbopolR resins are carboxy vinyl polymers of

extremely high molecular weight supplied as dry, fluffy

powders in acid form, requiring neutralization to develop

optimum properties (29). Carbomer is a generic term for

the CarbopolR resins replacing carboxypoly methylene.








While these resins are very mild acids, weaker than acetic

acid, they readily react with neutralizing agents to form

salts (30). It is necessary to choose a neutralizing agent

that forms a CarbopolR salt which is soluble in the solvent

chosen. When the proper salt is prepared in a given

solvent, the polymer uncoils and extends into its form of

highest solvent-holding capacity with attendant solvent

thickening. The choice of neutralizing agentss, which

may be metallic hydroxides or amines, determines whether

or not the resulting salt is hydrophilic, lipophilic, or

amphipathic, a combination of hydrophilic and lipophilic

properties.

CarbopolR resins have been used to thicken many

liquids, chiefly aqueous, though many non-polar solvent

systems have also been used. 1,2,6-Hexanetriol has been

gelled using CarbopolR 934 neutralized by diisopropanola-

mine (31). Use of these gels, especially non-aqueous

organogels prepared with CarbopolR, as cosmetic vehicles

is common.

Schoenwald et al. investigated the use of carbomer

(CarbopolR 940) gels containing 2% pilocarpine, 0.01%

benzalkonium chloride, 0.01% edetate disodium, 2% mannitol,

and purified water in the rabbit eye to determine if high-

viscosity polymer systems could overcome the suspected

thinning that occurs because of eye movements and/or

blinking of the eyelids, thus prolonging the effect of

pilocarpine in the eye (32). Gels contained 0.9-5.0%









carbomer, neutralized with sodium hydroxide-pilocarpine

combination to pH 5.9-6.2. Their results indicate that

increased duration of the gel in the eye for gels con-

taining not less than 2.7-3.0% carbomer occurred, giving

correspondingly prolonged miotic effects.

Schoenwald and Boltralik conducted a study in rabbits

to determine whether a high viscosity gel would show

increased bioavailability for two steroids when compared

to commercially available, reference preparations (33).

A 1% aqueous gel suspension of CarbopolR 940 was used as

the vehicle for labeled prednisolone acetate, a water-

insoluble steroid, and a 1% aqueous gel of CarbopolR 934

was prepared for use with labeled prednisolone sodium

phosphate, a water-soluble steroid. At specific times

after instillation, the prescribed number of rabbits were

sacrificed, and aqueous humor and cornea were sampled.

Measurements indicated that the uptake of both steroids

was significantly increased as a result of gel formulation

use, with prednisolone acetate showing a 4.5-fold increase

and prednisolone sodium phosphate a 5.5-fold increase over

the respective reference preparations. It was also noted

that significant quantities of gel were observed in rabbit

conjunctival sacs through 8 hours, and that the quantity

of gel in the rabbit eye slowly diminished over time.

This indicated that the drug was probably made available for

absorption partly by diffusion through the bulk of the gel

and partly by uptake from.the surface of the gel as it con-

tinuously erodes.









No reports of the ophthalmic use of CarbopolR

preparations similar to base E-3, described in this investi-

gation, were found.

Plasticizers are added to plastic materials to improve

flow and to reduce brittleness of the product. The basic

requirements which must be met by a plasticizer are compat-

ibility and permanence (34). The plasticizer must be

miscible with the plastic system, exhibit similarity in the

intermolecular forces active in the system, have a low

vapor pressure and low diffusion rate within the polymer

network.

In 1956, Collins and Zopf reported on an improved

polyethylene glycol ointment base containing 1,2,6-

hexanetriol (35). Brittleness in polyethylene glycol

semi-solid preparation can be a problem when storage

temperatures are lowered. The new formulation was reported

to show improved appearance, have less tendency to be

granular and to be compatible with all major ointment

ingredients. The composition was as follows:


Base CHA

Polyethylene glycol 4000 34 g
Polyethylene glycol 400 50 g
1,2,6-Hexanetriol 16 g


The melting point for this base was listed as 51.7.

Softening did occur at 420, but no separation was noted.

This anhydrous base is similar to base E-4, described in

this investigation, except that base E-4 contains a









substantially higher proportion of 1,2,6-hexanetriol, the

plasticizer. The incorporation of more than 10% water to

Base CHA resulted in an unsatisfactory product, correctable

by the addition of greater amounts of polyethylene glycol

4000. Base CHA was formulated for topical use.





Ophthalmic Irritation Testing



According to Friedenwald et al. quantitative

evaluation of the severity of predominate disease manifesta-

tions by a numerical method was widely used in vitamin

studies as early as 1909, by Holst and Trohlich (36).

Rothschild, Friedenwald, and Bernstein described a method

for grading the severity of ocular reactions in allergies

in 1933, but gave no illustrations (37). Friedenwald,

Hughes, and Herrman, in 1944, were the first to give a

complete method for the evaluation of the severity of the

reaction produced so as to yield data which could be

studied statistically and graphically (38). The significant

symptoms were specified and varying maximal values were

assigned to them, the rating being dependent upon their

relative importance.

The development of the test procedure currently used

occurred in 1944 and was reported by Draize, Woodard, and

Calvery (39). The grading system is a slightly modified

interpretation of that used by Friedenwald, and the









principle was extended to other physiological effects.

A complete scheme was presented. McCowan and Husa (40)

later presented another method, known as the Modified

Whitehill Scale, for evaluating irritation in rabbit eyes

caused by various collyria.

Articles have been presented offering interpretation

(41), modifications (42), reviews (43), and criticism (44,45)

of the original Draize Test. Major objections to the

procedure were the inter- and intralaboratory variations

due to interpretation by the observers, and the difficulty

of producing a standard lesion from which to estimate the

extent of damage to the eye. Harley admits to the fact

that secondary infectious processes complicate the conduction

of reliable irritation and effectiveness studies, and mar

the ability of the observer to obtain clear-cut, decisive

results (46). New pieces of apparatus, such as the corneal

applicator (47) and the cup-aspirator (48), have been devel-

oped to improve the reproducibility of test results, but

no significantly new method of grading has evolved.

Commercial eye irritation studies have relied on some form

of the Draize testing method (49).

Recently, additional objections have been presented

from antivivisectionists and lay critics concerning the

Draize Eye-Irritancy Test (50). The cosmetic industry,

as a result, has allocated large quantities of money and

time to find an alternative method of conducting such

tests. Among those tests currently under development are









methods for culturing ocular material, either tissue

fragments or cellular elements, and a test using the

cultured peritoneal cells from a rat, to replace the in

vivo eye irritancy tests with in vitro tests of equal or

better sensitivity. Mathematical modeling and statistical

analysis can be used to reduce the number of animals needed

to yield significant data.




Test Animals for Ophthalmic Testing



The application of irritation test results from

animal studies to man is still unclear. Beckley (51)

has suggested that several test animals be used, including

the rabbit, dog, and monkey, in an attempt to more closely

duplicate the effects seen in the human eye. However,

since the rabbit is readily available as a strain-specific

test animal with a large corneal surface, and is economical

to raise in the large numbers necessary for this type of

testing, a large amount of information relating to this

animal has been compiled, making the prospects of its use

more desirable. The animal most frequently used is the

healthy, adult, New Zealand White albino rabbit, both male

and female sexes.

The National Formulary XIV describes an eye irritation

test procedure for the evaluation of extractables in

plastic containers for ophthalmics (52). Samples of the









plastic container are extracted with sodium chloride

injection and with cottonseed oil. Instillation of 200

ul of these extracts is made into the retracted lower

eyelid sac of a specified number of albino rabbits' eyes.

Suitable blank solutions are used, and each animal serves

as its own internal standard. Examination for irritation

is done at 24, 48, and 72-hour intervals.

Anatomically and physiologically, the rabbit eye is

reported to be different in many respects from the human

eye (53). There is uncertainty of the relative sensitivity

of human and rabbit eyes. Stained cross-sections of human

and rabbit eyes revealed that the human eye exhibited a

relatively thicker epithelium and a distinct Bowman's

membrane. The membrane's thickness in the rabbit eye was

much less. The thicker epithelium of the human eye was

reasoned to be a factor in protecting the Bowman's membrane,

disruption of which results in scar formation in the cornea.

Healing of denuded corneal epithelium required about three

times longer in the rabbit than in the human eye. Obser-

vations showed, however, that the rabbit eye has enormous

regenerative power following very severe damage and that

injury to Bowman's membrane does not always result in

scar-tissue formation.

Mann and Pullinger also noted differences between the

eyes of humans and rabbits (54). Lesions produced in

rabbit eyes by ocular contact with mustard gas were

almost identical with those in human eyes, although the









rabbit required a larger dose to do so. It was postulated,

also, that the pathologic processes of rabbits are speeded

up in proportion to their shorter life span, so that

results occur approximately 10 times as rapidly as in man.

Prince indicates several differences which must be

kept in mind when attempting to extrapolate results

obtained in rabbit eyes to man (55). While the normal

coats of the eye do not differ greatly, the circulatory

systems show differences. Accommodation, the ability to

change the shape of the lens to focus, is much less in the

rabbit than in man, in fact being almost useless. The

rabbit, in addition, has several anatomical structures

which man does not possess, such as an active retractorbulbi

muscle, a Harder's gland (essentially a lubricating function),

an active nictitating membrane, and a nictitous gland.

The lacrimal gland is relatively much larger in the rabbit,

and the lacrimal drainage system is somewhat different.

The rabbit is also devoid, according to Prince, of a

significant Bowman's membrane. However, despite these

differences, the similarities between the eyes of the

rabbit and man are numerous enough to justify its use in

the ophthalmic laboratory.

Eye irritation studies have been made on the sodium

salt of CarbopolR 940 in aqueous solutions of 0.4 and one

percent concentrations (56). One milliliter of each

solution was introduced into the conjunctival sac of

rabbits' eyes and examinations were performed after 1, 24,








48, and 72 hours. Results indicated only insignificant

irritation and no damage to the ocular tissues.

The mixed sodium-Ethomeen C-25 salt of CarbopolR 934

has also been tested for eye irritation effects (57). The

salt was made into a gel with water, the concentration of the

chemical being 0.5% by weight. One drop of this material was

instilled into the right eye of albino adult rabbits using

the left eye as a control. The eyelid was held closed for

one minute after instillation and examinations were made at

one-half hours and 24 hours after application. Fluorescein

was used to check for corneal surface injury. In no instance

was there any detectable evidence of eye injury or irritation.

Glycols, also commonly called diols, are characterized

by two hydroxyl groups, which contribute to water solubility

and hygroscopicity. As a class the glycols are of a low

order of ophthalmic toxicity. Ethylene glycol exhibited no

evidence of surface damage in the rabbit eye upon instilla-

tion of the liquid chemical (58).

As polymers of ethylene oxide, the polyethylene glycols

comprise a group of unique chemicals widely used in pharma-

ceutical preparations. Tests for evidence of eye injury,

which is defined as surface damage produced by the liquid

or solid chemical or appropriate concentrations thereof,

have shown trace injuries for polyethylene glycolsa 200 and

400, and none for polyethylene glycol 4000(59).



aCarbowax brand of polyethylene glycols, Union Carbide
Corp., New York, New York.









Draize Test results for cosmetic grade hexadecyl

alcohol in rabbit eyes indicated transient irritation

confined to the conjunctivae; there was no effect upon

cornea or iris (60). Dilution of the alcohol to approxi-

mately half-strength with mineral oil reduced its eye

irritancy to a negligible level. Human studies using

solutions of 1 and 5% Hexadecyl alcohol in mineral oil

applied directly to the eyes of subjects also produced

only transient effects. A 10% solution gave slight sub-

jective irritation accompanied by temporary redness, and a

30% solution resulted in local discomfort and conjunctival

irritation which completely cleared within a few hours.

1,2,6-Hexanetriol is described as virtually non-toxic

and caused no reaction when applied to the skin of rabbits

(61). The results of eye irritation tests were not listed

for this chemical.





Microbiology of the Anterior Segment of the Eye



The eye harbors bacteria from the time of birth

throughout life. The normal bacterial flora in human eyes

consists most commonly of staphylococci species and

diphtheroids; streptococci species and pneumococci were

present infrequently (62). Such flora was modified little

by age, sex, or season. Bacteria cultured from the eye

are similar to those found on the skin and in the upper









respiratory tract; bacteria commonly found in the air are

rarely recovered from the eye (63). Of those bacterial

infections most often seen in the anterior eye, infection

of the conjunctiva, both chronic and acute, infection of

the eyelid and of the lacrimal sac and canaliculi are

caused most frequently by Staphylococcus aureus, either

alone or in combination with other organisms. Corneal

ulcers are caused by the same organism, though, in cases

of trauma, Pseudomonas aeruginosa is a major cause of

ulceration and is extremely difficult to treat (64).

In healthy adult eyes, Aspergillus species was the

fungus most commonly found upon culture (65).

The normal eye presents certain barriers to the intro-

duction and proliferation of microbial organisms. Among

these are the mechanical washing action of the normal

blink reflex, the flushing of the eye surface by tears, and

the lysozyme content of the tear fluid (66). The normal

bacterial flora of the eye may play a role itself in con-

trolling the invasion of new organisms, possibly by the

secretion of antibiotic substances (67). The mucin

component of tears plays a part in the lubricating and

thickening properties of the tears and may be subject to

certain regulatory factors, the absence of which may

promote disease (68). An intact epithelial surface on

the cornea prevents the invasion of many microbes, notably

Ps. aeruginosa and various fungal species.









It is not surprising, then, since a very large number

of common ophthalmic diseases are caused at least in part

by microbes most usually found in or around the eye, that

preservatives for ophthalmic ointments should be effective

against them.





Sterilization Methods of Ophthalmic Ointments



For many years, it was assumed that the anhydrous

nature of petrolatum-based ophthalmic ointments provided

an environment which was not conducive to the proliferation

of microorganisms. Improved sterility testing methods,

however, resulted in proof that a large number of com-

mercial ointments were indeed contaminated. Vander Wyk

and Granston (69) tested 83 commercial ointments for

contamination by bacteria and found 71 to contain micro-

organisms. The method used for testing consisted of

aseptically melting the ointment sample, dispersing it in

water, and plating aliquots of the dispersion.

The USP XX recognizes 5 methods of sterilization (70):


1) steam sterilization, denoting heating
in an autoclave employing saturated
steam under pressure at a minimum
temperature of 1210 for not less than
15 minutes;

2) dry heat sterilization, denoting exposure
to heated air in a specially designed and
regulated oven, generally at temperatures
of 1600 to 1700 for a period of not less
than 2 hours;









3) gas sterilization, denoting exposure to
sterilizing gases such as ethylene oxide
in specialized equipment;

4) sterilization by ionizing radiation, denoting
the exposure of the article to radiation
of varying dose, dependent upon the
microbial bioburden and the nature of
the article being sterilized; and,

5) sterilization by filtration, denoting the
physical removal of organisms by adsorption
on the filter medium or by a sieving
mechanism.


Additional methods of sterilization exist (71), but,

for various reasons, are not applicable to ointment

vehicles. Antimicrobial agents are regarded as preserva-

tives and discussed elsewhere in this review.

Sterilization is usually regarded as the use of any

physical or chemical method to eliminate all viable microbes

from a material (72). An organism is considered to be

non-viable when it loses the ability to propogate

indefinitely when placed in a suitable environment, or it

may be physically removed from the material, for example,

by filtration. The process of sterilization has as its

objective, then, the production of products that contain

zero microorganisms capable of growing and reproducing.

Absolute sterility of a product is, in a practical

sense, impossible to achieve. In the health industries,

the objective is to have the nonsterile or contamination

level less than 10-6, that is, that the probability of a

single unit being contaminated in a batch processed under









the same conditions is less than 1 in 1 million. An

excellent discussion of the concept of statistics and

sterilization processes is presented by Pflug (73).

The majority of available ophthalmic ointments are

petrolatum-based or mixtures of petrolatum and mineral

oil; these usually can be sterilized by dry heat sterili-

zation. Those bases using polyethylene-gelled mineral

oil cannot be sterilized by dry heat without separation of

their components (74).

Perkins has reported that in dry heat sterilization

an exposure time of 60 minutes at 320F (160C) is

approximately the equivalent of 15 minutes at 2500F (121C)

in moist heat (75). Additionally, the addition of 1% water

to fats, oils, or paraffin made sterilization possible in

the autoclave after 30 minutes at 1200 (76). Most oils

do contain a small amount of moisture.

Glycols, particularly polyethylene and propylene,

pose a problem with respect to sterilization. If these

compounds are to be sterilized in an autoclave, a minimum

water content of 10 to 20 percent is essential. When

there is little water present, sterilization by dry heat

is recommended at 1400 for 2 hours for polyethylene glycol

400 and at 1490 for 2 hours for polyethylene glycol 4000 (77).

The sterilization of dry powders similar to CarbopolR

resin has been done using dry heat. An exposure of 2 hours

at 1600 is adequate if the powder is restricted to a layer

inch thick in a Petri dish (78).









An ephedrine gel was formulated by neutralizing

CarbopolR 934 with ephedrine base (79). Sterilization

of this gel was effected by autoclaving at a temperature

of 121.20 and 15.3 lbs for 30 minutes; no change was

observed in the viscosity or pH reading upon repeatedly

subjecting the ephedrine gel to autoclaving.

Company literature states that heat aging of the dry

CarbopolR resins has little effect on subsequent water

swelling capacity or physical appearance at temperatures

up to 3000F (148.90C) for as long as 2 hours (80).

Decomposition proceeds rapidly above 3000F, producing hard,

insoluble granules which display no water-holding capacity.

Improved filtration equipment and materials have

allowed changes in industrial manufacturing of sterile

ophthalmic ointments. An example of such changes was

reported by Smith, Fonner, and Griffin (81), describing

the filtration of an ointment composed of anhydrous

lanolin, white petrolatum, and mineral oil at about 90-95.

Chlorobutanol was used as a preservative and was filtered

at room temperature because of stability problems.

Filtration was accomplished under pressure using a 0.45

um prefilter and a sterile 0.20 um sterilizing filter.

Smith reported another example of industrial

sterilization of ophthalmic ointments (82). Two base

formulations, minus active ingredients, were sterilized

by filtration under pressure through sterilizing porosity









membrane (0.22 um) filters. Rheological comparison of

the bases before and after filtration indicated no change.

No corresponding literature information was found

concerning the attempted filtration of nonaqueous organogels

such as the ones described in this present investigation.

Additionally, no information was found on another poten-

tially useful method of sterilizing this class of

preparation, that of ionizing radiation. Investigation of

this area would undoubtedly be worthwhile at some future

time.





Sterility Assurance Tests for Ophthalmic Ointments



USP XX provides several test methods designed to

detect the presence of viable forms of bacteria, fungi, and

yeasts in ointment vehicles (83). For water-soluble

materials, either a direct transfer procedure to the test

media or a membrane filtration method may be used. In

the direct transfer method, the ointment sample is

aseptically transferred from the test container to a

vessel of the appropriate culture medium, mixed, and

incubated at the correct temperature for not less than 14

days. Testing by membrane filtration requires dissolution

of the sample in either specific media or sterile water,

filtration through a 0.45 um, or less, sterile membrane

filter, and culturing the removed filter in the appropriate









media for not less than 7 days. Other methods or

modifications of these methods are allowable as long as

the results are comparable to those obtained by the

official methods.

Oils, oleaginous ointments or gels not soluble in

water may be dissolved in sterile isopropyl myristate

before testing by similar methods.

In a review article in 1956, Sykes (84) discussed

the state of the art in sterility testing techniques,

describing methods for testing many types of materials,

including oils, and types of media. Bowman (85) described

a membrane filtration method application to the sterility

testing of petrolatum-based antibiotic ophthalmic oint-

ments. Bowman (86) also reviewed the sterility testing

of pharmaceuticals in 1969, including sampling procedures,

culture media, incubation specifications, biological

indicators, and methods of testing. Additional reports on

membrane filtration sterility testing appeared, such as

that by Tsuji et al. (87) and application information for

membrane use (88).

Information concerning the application of membrane

filtration methods to water-soluble non-aqueous gels was

not included in any of the above references.

Until recently, the effectiveness of a sterilization

process had relied on either direct sampling of the

sterilized product, using a broad-range culture medium

in an effort to detect any one of many microorganisms which









might have been present, or a variety of chemical or

physical indicators, which would melt or turn color when

the conditions of temperature and pressure were applied

for the correct time. Both of these approaches exhibited

significant failures, and an improved, more reliable

method was developed in the form of a biological indicator.

Biological indicators are standardized preparations

of specific microorganisms relatively resistant to the

particular sterilization process, used to demonstrate in

a positive manner the adequacy of the sterilization

process (89). They are of two forms, each of which in-

corporates a viable culture of a known species of micro-

organism. One form is the addition of the culture directly

to representative units of the lot to be sterilized. If

this is not practicable, the culture may be added to a

carrier which shows no less resistance to sterilization

than the product to be sterilized.

Biological indicators, where applicable, are the

most effective means of demonstrating the adequacy of a

sterilization process (90).

One of the most outstanding characteristics of

bacterial spores is their resistance to heat activation.

For this reason, standardized preparations of bacterial

spores are used to monitor various sterilization processes.

Bruch discussed the insufficiencies of finished-product

sterility tests and encouraged the use of knowledge of

microbial death rate kinetics (D values) and biological









indicators to obtain low probabilities (10-6) of survivors

in sterilized materials (91). He stated that the best

proof to certify that a lot of sterilized materials has

a high probability of being sterile is the destruction

of calibrated doses of microorganisms of defined resistance

carried by a few samples from the lot.

Several spore-forming bacteria have been used as

biological indicators, but the most widely accepted for

use in steam autoclaving is Bacillus stearothermophilus.

Gillis (92) evaluated six commercially-available biological

indicators containing spores of B. stearothermophilus for

resistance parameters to 121.10 and 132.20 saturated steam

in a specially designed experimental steam autoclave.

He found large variations among the samples and that

labeled claims on some of the indicators were not sub-

stantiated by the study. Standards were recommended which

included a parameter that at 121.10, 100% of the spores

in a standard preparation would survive for 5 minutes and

100% would be killed after 15 minutes exposure to

saturated steam.

The carrier of spore preparations of B. stearothermo-

philus has received considerable attention. Brewer and

McLaughlin (93) reported on a study evaluating a steriliza-

tion control of paper impregnated with spores of this

organism and specific culture media containing an acid-base

indicator in dehydrated state. Spore survival after

exposure to sterilizing process is indicated by a color









change and turbidity. The type of spore carrier, among

other things, was evaluated concerning its influence on

germ count and heat resistance by Buhlmann, Gay and

Schiller (94). Their results showed that commercially-

available spore-impregnated filter paper strips and

ampuls of spore suspensions had a very low heat resistance.

The authors' own test strips showed that elevated

temperature and desiccation, associated with unsuitable

storage conditions or packing, led to fairly rapid decrease

in the number of living spores. Test ampoules containing

spore suspensions constituted a more reliable test object.

Mayernik (95) studied five types of biological

indicators containing either mixed cultures of B. stearo-

thermophilus and B. subtilis, or cultures of B. stearother-

mophilus alone, on various strip or ampul suspension

carriers. He found wide variation among the samples, and

also wide variation among the heat-up times and final

temperatures of supposedly equivalent autoclaves, leading

to an additional conclusion that the normal steam autoclave

cannot be used as a research tool for the determination of

spore resistance.

Many factors affect the heat-resistance of spores

of B. stearothermophilus, among them being the incubation

temperature and the nutritional content of the sporulation

medium. Several authors have discussed at length the

nutritional needs of thermophilic bacteria (96-98). All

seem to agree that a complex medium was necessary,









containing various amino acids and minerals, and that

temperature had an effect on the nutritional requirements

of a particular strain of organism, raising or lowering

its need for various components as the temperature of

incubation varied.

Heintz et al. (99) described a very comprehensive

method for the production of spores of B. stearothermo-

philus, their standardization with regard to heat-resistance

and their use as a biological indicator for injectable

solutions. A sporulation medium composed of Nutrient

Broth, Yeast Extract, MnC12 4H20, and Agar was used,

having a pH of 7.2 before sterilization. The isolated

spores were stored at 2-4.

Brown (100) presented a study concerning the heat

resistance of bacterial spores, using a sporulation medium

composed of a nutrient agar with manganese to produce

spores of B. stearothermophilus.

Thompson and Thames (101) used a sporulation medium

composed of tryptone, potassium phosphates and manganese

sulfate at a pH of 6.8 after autoclaving. Growth and

sporulation of five strains of B. stearothermophilus

were studied, with increased manganese content resulting

in increased spore production.

A chemically-defined synthetic medium for sporulation

of B. stearothermophilus was reported by Anderson and

Friesen (102). Its composition was listed as including

glucose, 1-glutamic acid, ammonium phosphate (dibasic),









potassium phosphate (monobasic), ammonium sulfate, magnesium

sulfate, ferrous sulfate, manganese sulfate, and calcium

chloride. The final unadjusted pH of the medium was 6.5.

These references suggest the following conclusions:


1) that commercially-available biological
indicators show wide variation in their
heat resistance and must be standardized
for research use;

2) that a good choice for an organism for use
as a biological indicator is B. stearothermo-
philus;

3) that use of a biological indicator provides
the best available assurance of the
adequacy of a sterilization process; and,

4) that the normal autoclave is unsatisfactory
for exacting research regarding heat-
resistant organisms specialized equipment
is necessary.


Further support for the use of B. stearothermophilus

results from the fact that the organism is considered to

be non-pathogenic and will not grow at normal body

temperature.





Relevant Pharmacological Effects of Dimercaprol



Dimercaprol

Because of the inclusion of BAL in the formulations

tested for irritation potential, a brief description of

its principal pharmacological effects is presented.








Dimercaprol (BAL, 2,3-dimercaptopropanol) is a dithiol

compound which forms a very stable and relatively non-toxic

chelate ring with heavy metal compounds, especially those

containing arsenic, allowing them to be eliminated from

the body and preventing the harmful inhibition of essential

sulfhydryl enzymes by metals. It is readily absorbed in

the eye, and a 5% solution in ethylene glycol results in

little irritation when instilled ophthalmically (103,104).





Preservation of Ophthalmic Ointments



Ophthalmic ointments must contain a suitable substance

or mixture of substances to prevent growth of, or to

destroy, microorganisms accidentally introduced when the

container is opened for use (105). A survey of com-

mercially-available ophthalmic preparations indicated that

the antimicrobial agents currently used most often are

methyl- and propylparabens, chlorobutanol, benzalkonium

chloride (alone and in combination with disodium edetate),

and phenylmercuric nitrate.

The ideal preservative should exhibit a number of

characteristics: 1) possess a broad spectrum; 2) have

continuing activity; 3) possess rapid action; 4) be

nonallergenic and nonsensitizing; 5) be nontoxic and

nonirritating; 6) possess chemical and pharmacological

compatibility with the other ingredients; 7) be chemically









and physically stable; 8) be readily inactivated for

testing purposes; and, 9) exhibit ready solubility in

the appropriate vehicles (106). The ideal preservative,

of course, does not exist. Compromises among these

characteristics are a necessity to achieve an acceptable

preservative which is effective in use.

A large quantity of literature has been written

concerning the effects of preservatives in ophthalmic

preparations, largely solutions. For example, Cooper (107)

presents an overview of preservatives used in ophthalmic

solutions, listing concentrations and probable mechanisms

of action. Russell, Jenkins, and Harrison (108) review

preservative concentrations and characteristics for eye

solutions, indicating those which are irritant. Kohn,

Gershenfeld, and Barr (109,110) reported on the effective-

ness of a number of antibacterial agents against several

strains of Ps. aeruginosa, recommending the use of

benzalkonium chloride.

It has been reported that CarbopolR resins do not

support mold and fungus growth. They do not, however,

prevent mold growth, and certain bacteria grow well in

CarbopolR mucilage. Such growth can be prevented by

autoclaving the CarbopolR mucilage and including an

antimicrobial substance such as 0.1% methyl paraben,

propylparaben, or p-chloro-m-cresol. These do not affect

the theological properties of the gel (111,112).









USP XX (113) describes procedures for the estimation

of the number of viable aerobic microorganisms present and

for freedom from designated microbial species in

pharmaceutical articles by either conducting tests for a

total aerobic microbial count, or tests for the presence

of Staphylococcus and Pseudomonas species, or for

Salmonella species and E. coli.

Microbial growth requires the presence of moisture.

In petrolatum-based ointments, this moisture usually exists

as surface condensation; therefore, inoculation of the

surface of such an ointment with a specific microorganism,

followed by incubation, should provide a measure of the

ability of the product to support growth of the organism.

As an example, Jurgens (114) inoculated non-aqueous

oleaginous bases, some of which contained antimicrobial

substances, with a standard inoculum of Ps. aeruginosa.

Antimicrobial substances used were benzalkonium chloride,

0.025%, benzyl alcohol, 2.0%, or chlorobutanol, 0.5% and

1.0%. His results (115) stated that all substances except

for chlorobutanol 0.5% were found to be effective.

Gross (116) used a similar technique for micro-

biological testing of some CarbowaxR and polyoxyethylene

stearate suppository bases in combination with water.

The organisms used were E. coli, B. subtilis, A. niger,

Mucor, and S. cerevisiae, representing Gram-positive and

Gram-negative bacteria, molds and yeasts. Anaerobic and

aerobic tests, in addition to air-exposure of plated









samples, were done, and the plates and tubes examined

macroscopically and microscopically for growth over a

period of 2 weeks. No growth was seen in any of the

samples.

Hugo and Russell (117) state that (microbiological)

tests performed on completed formulations should present

some similarity to the actual conditions of stress which

the product could encounter in real life, and that the

use of accelerated testing procedures with extremes in

temperature, pH, etc., is not generally applicable to

microbiological testing. Temperature cycling (i.e.,

intermittent storage at two or more temperatures) may be

used to detect localized weak spots in the microbial

resistance of the formulation, such as the formation of

dilute moisture films on the surface of the product.

In a discussion of some of the shortcomings of the

U.S.P. XVIII Microbiological Test, Leitz (118) states that

the method described which has not been changed

substantially in the current U.S.P. is inefficient

in its requirements for testing individual samples, each

challenged by a different single organism; she recommends

a mixed inoculum and use of differential recovery media.

In addition, she feels the panel of organisms recommended

is unrealistic in two respects: first, that the high

initial cell count is far in excess of that allowed in

a product made under good manufacturing practices and

is probably unlikely in anything other than a grossly









user-contaminated multiple-use product; secondly, the

cells used are too young and too uniform in age to be

representative of chance contamination, and no spore-

forming bacteria are included in the panel. Time require-

ments are the same regardless of the type of product tested

by the U.S.P. procedure; Leitz feels a differentiation

should be made for products of different use, for example,

parenterals and ophthalmic products vs. oral or topical

preparations.

Kellogg (119) reported on the use of preservative

testing of parenterals and ophthalmics from the standpoint

of Quality Control, and discusses streamlining the

biological test so that the procedure can be used for

routine control analysis on a batch-to-batch basis.

Yablonski (120) argues against using the composite

or mixed culture inoculum approach in preservative testing,

and notes that organisms carefully grown on rich media

may or may not be able to adapt to a particular product

environment and so may not be as reliable an indicator

of preservative effectiveness as inoculi of microbial

contaminants isolated from the spoiled product itself.

A significant problem in antimicrobial testing of

formulations is the inactivation of the preservative,

allowing any viable microbes present to proliferate to

a detectable level. The earliest approach, and still

the primary one, was dilution of the sample to the point

where the preservative is no longer active. Many times,









this method is adequate and its simplicity makes it

desirable; in some cases, however, simple dilution is

ineffective, especially when dilution volumes are

abnormally high and the formulation being tested contains

low microbial contamination levels. The use of specific

inactivation media is then the preferred method.

Specific inactivation media have been developed for

many of the commonly-used preservatives. USP XX (121)

states that soy lecithin, 0.5%, and polysorbate 20, 4.0%,

are examples of ingredients which may be added to the

culture medium to neutralize inhibitory substances present

in the sample. Kohn, Gershenfeld, and Barr (122) list

several neutralizing media for inactivation of quaternary

ammonium compounds, amphoteric surfactants, iodophors,

a partially polymerized silver mannuride, chlorhexidine,

and colistin. Lachman, Lieberman, and Kanig (123) give

several selected antibacterial substances and their

inactivation methods for sterility testing, most employing

the use of either glycerin, lecithin, polysorbate 20

or polysorbate 80 as added ingredients. The principal

methods of inactivation use either neutralization of

ionic charge, dilution to non-effective concentration,

complexation, absorption, adsorption or combinations of

these processes.

Mechanisms which serve so well in the selective

inactivation of preservatives in culture media can cause,

in turn, problems in formulations to which preservatives









are added. Lachman (124) indicates that reduced preserva-

tive concentration can occur through chemical complexation

with a surfactant or gum, for example, polyethylene

glycol (CarbowaxR) 4000 will bind approximately 16% of

methylparaben and 19% of propylparaben present in a

preparation of these materials, while 5% polysorbate 80

binds 80% of total methylparaben present in the aqueous

phase of a preparation.

In a study of the compatibility of certain preserva-

tives with CarbopolR 934, Schwarz and Levy (125) state that

a marked viscosity decrease and precipitation were observed

in solutions containing 0.5% neutralized Carbopol and

0.1% benzalkonium chloride, but neither a viscosity decrease

nor precipitation was observed in solutions which contained

0.01% of the preservative. The reason for this result

is stated to be the reaction between the high molecular

weight anionic CarbopolR and the cationic preservative,

causing precipitation of the resin and resultant decrease

in viscosity. The mechanism is termed "ionic deswelling,"

and is the cause of viscosity decreases in CarbopolR

solutions on addition of sodium benzoate, benzoic acid,

and high concentrations of thimerosal, as well.

Crooks and Brown (126), in a study of mixtures of

polyethylene glycol and sodium lauryl sulfate, state that

methylparaben is bound to polyethylene glycol by an

interaction governed by a very low association constant.









At any particular polyethylene glycol concentration, the

fraction of preservative bound is independent of the total

preservative concentration.

Reports of preservative binding to polymer containers

or rubber closures are numerous. For example, Blackburn,

Polack, and Roberts (127) assayed a selection of com-

mercially available ophthalmic solutions obtained at

random from shelves of community pharmacies for chloro-

butanol content and compared the results with the labeled

0.5% concentration. The assay revealed a range of

0.02-0.29% actual chlorobutanol content, and losses were

attributed to disappearance of the chemical during the

sterilization process or the sorbtion by plastic containers

during subsequent storage. Richardson et al. (128)

state that the preservative content of thirty-four com-

mercially available contact lens solutions were assayed

and over half of the solutions contained less than 90%

of the labeled content. Experimental tests indicate that

thiomersal and chlorobutanol appear to be sorbed by the

container (and volatized on the outside, in the case of

chlorobutanol), while benzalkonium chloride and chlor-

hexidine gluconate interacted mainly by a surface adsorption

process. The extent of the interaction was dependent on

the type of plastic polymer used.

The processing or manufacturing methods of ophthalmic

product preparation can affect the preservative content.

Reference has been made to the degradation of chlorobutanol









by heat sterilization. Naido, Price, and McCarthy (129)

report that losses of preservative may occur during

sterilization of ophthalmic solutions by bacterialogical

filtration, losses being considerable with fibrous abestos

pads, significant with porcelain candles and sintered

glass, and slight with membrane filters. Separate buffered

solutions of chlorhexidine acetate, 0.01%; phenylmercuric

nitrate, 0.002%; benzalkonium chloride, 0.02%; and phenyl-

ethyl alcohol, 0.5%, were tested.

The effects of pH on the efficiency of the alkyl

esters of p-hydroxybenzoic acid were investigated by

Bandelin (130). He states that, of the compounds tested,

the esters of p-hydroxybenzoic acid were least affected

by pH changes, with only a slight reduction in activity

at the higher pH levels. He also notes that the activity

of these esters increases with increasing molecular weight.

The concentration of preservatives which can be used

in ophthalmic preparations is limited by the potential of

each for producing eye irritation or injury on prolonged

use. Jaconia (131) reviewed the use of several preserva-

tives and reports that "safe" levels for some have been

determined, while other require additional investigation.

Phenylmercuric nitrate, 1:10,000 (0.0%), is recommended,

as are levels of 1:5000 (0.02%) for benzalkonium chloride,

and 0.8% for chlorobutanol. A total ester concentration

of 0.08% for the parabens is considered to be bacterio-

static, and the combined 0.2% level to be bactericidal,









though the latter concentration is irritating to the eye.

Interesting results have been reported from combinations

of preservatives, such as benzalkonium chloride, 0.01% and

tetracemin, 0.05%, or ethylenediaminetetraacetate.

USP XVIII (132) recommends levels of 1:10,000 (0.01%) for

benzalkonium chloride, 1:50,000 (0.002%) for phenylmercuric

nitrate or phenylmercuric acetate, and 0.5% for chloro-

butanol, noting that chlorobutanol hydrolyzes to hydro-

chloric acid with a resultant decrease in solution pH.

Similar concentrations are recommended by Russell, Jenkins,

and Harrison (133).

Several authors have presented mathematical models

for the prediction of preservative action, mainly from

true solutions and solubilized and emulsified systems.

Allawala and Riegelman (134) discuss the concept of

availability in terms of bacterial agents of the kinetic

type, that is, their toxicity is dependent upon time,

among other factors. Availability may be equated with

permeability, the grams per unit time required for an

antibacterial agent to traverse a bacterial membrane; with

diffusion; with transfer across an interface, as a function

of the partition coefficient; or, with the rate of

adsorption, when the action of the drug is on the surface

of the bacterium. The experimental data are explained in

terms of the fundamental driving forces behind this

availability concept, in that the driving force measures

how far short of equilibrium the system is; the availability









measures the rate at which this equilibrium is approached.

Further, the effects of solubilization of preservatives

by the formation of micelles is discussed in terms of

concentration of both preservatives and surface-active

agent, saturation of aqueous phase with preservative, and

killing time change corresponding to increases in surface-

active agent concentration.

Garrett (135) presented a basic model for the evalua-

tion and prediction of preservative action in oil/water

emulsion systems in which he develops several concepts

such as macromolecular binding, effect of pH, and

preservative activity in the external aqueous phase of

o/w emulsions. The result is an integrated mathematical

model for the estimation of preservative action which

yields a total concentration of preservative, P and the

principles related to use of combinations of preservatives

are discussed.

Kazmi and Mitchell, in a series of articles (136-138),

present mathematical models for the calculation of free

preservative necessary for inhibitory or bactericidal

action of chlorocresol in solubilized systems and o/w

emulsions stablized by a nonionic macromolecular surfactant,

cetomacrogol. They develop the concept of capacity for

these systems, allowing first calculations in an effort

to avoid much "trial-and-error" formulation when purely

microbiological techniques are employed.









Wedderburn (139) presented a comprehensive review of

the chemical and physical aspects of preservative action

in general, and specifically in emulsions. Rigler and

Schimmel (140) reviewed the use of preservatives in

various cosmetics and suggest that in preparations having

a low moisture content (as low as 12%), little nutrient

resources, and relatively low pH (pH 4 to 6), fungi are

the most likely organism to be found, probably as a

surface growth, when storage of the product is at room

temperature (20-240). Support for the single organism

challenge test can be seen in the reasoning that cosmetic

preparations contaminated with multiple organisms usually

provide an environment favorable to the growth of one

organism over another; thus, a given preparation may

eventually harbor what is essentially a pure culture of

a single species.

Little quantitative work has been reported on the

preservation of anhydrous semi-solid preparations, possibly

because of the basic assumption that the vehicle for

growth of the microorganism is aqueous and the biological

activity of preservative action must be exercised in the

aqueous phase (141). Dried bacterial cells, spores, and

certain fungi can survive, however, for extended periods

in anhydrous vehicles, notably vegetable oils (142).

Furthermore, in spite of apparent successes in calculating

preservative levels necessary for effective activity, the








most reliable method remains a combination of the microbial

challenge approach interpreted by an appreciation of the

factors which affect preservative performance.

Ansel (143) lists several considerations in his

discussion on the selection of preservatives: 1) that the

preservative should be effective against types of microbes

most likely to contaminate the particular type of

preparation; 2) that the preservative be soluble enough

in water to achieve adequate antimicrobial concentrations;

and, 3) that the proportion of preservative remaining

undissociated at the pH of the preparation be sufficient

to penetrate the organism and destroy it.

Preservative "capacity," as defined by Hugo and

Russell (144), is a measure of the total quantity of

microorganisms a formulation can inactivate before signifi-

cant deterioration of efficiency becomes evident, and

varies with preservative type.

Within these restrictions, a limited number of

chemicals have been found useful for preservation of

ophthalmic preparations; a brief description of possible

mechanisms of action follows for a selected number of them.



Mechanisms of Preservative Action

Ansel (143) separates the mode of preservative action

into four categories, indicating that one or more may

be applicable at the same time:









1) Modification of membrane permeabiility;

2) Denaturazation of enzymes or other
cellular proteins;

3) Oxidation of cellular constituents; and,

4) Hydrolysis.


Several examples are given. The esters of p-hydroxybenzoic

acid exert their action by denaturazation of proteins;

phenolic compounds act by lytic and denaturing mechanisms

on membranes, as do alcohols such as chlorobutanol;

quaternary ammonium compounds operate by lytic action alone

on membranes.

Hedgecock (145) reports that the action of benzal-

konium chloride at low concentration appears to be that of

protein precipitation and dissociation of conjugated pro-

teins. He states that phenolic materials combine with the

bacterial cell by an adsorption process (146) which may

involve hydrogen bonding, with the hydroxyl group as the

active component which reacts with the cell; the mechanism

of action at low concentrations is reversible protein

precipitation, which at higher concentrations becomes

irreversible and involves permanent enzyme damage.

The polymyxins, a family of polypeptides produced by

Bacillus polymyxa, exert their action by binding to the

outer surface of sensitive cells, and producing a leakage

of cell constituents including purine and pyrimidine

derivatives (147). The antibiotic is stated to be bound

to particles of lipoprotein, and the completing of









polymyxin with ionized phosphate groups of phospholipids

in the protoplast membrane presumably leads to disruption

of the lipoprotein and the integrity of the membrane.

Studies on organisms other than Gram-negative bacteria

suggest action on the cell walls.

Davis and Dulbecco (148) discuss specific chemical

agents used as antimicrobial substances. Some surface-active

agents, especially cationic detergents such as benzalkonium

chloride, are active against all kinds of bacteria, acting

by disrupting the cell membrane, causing a release of

metabolites. Phenol is stated to be both an effective

denaturant of proteins and a detergent; its bactericidal

action involves cell lysis. The compound p-chloro-m-xylenol

is an example of a chlorinated phenolic derivative.

Numerous authors mention the combination of disodium

edetate (ethylenediaminetetraacetic acid, disodium salt;

Na2 EDTA) with polymyxin B sulfate or benzalkonium chloride

to increase their antipseudomonal activity. Jaconia (149)

reviews this aspect of preservation. MacGregor and Elliker

(150) adapted a strain of Ps. aeruginosa to grow on

Tryptone-Glucose-Yeast Extract Agar containing 2000 ppm

alkyldimethylethylbenzyl ammonium chloride (QAC-A);

treatment of these resistant cells with 100 ppm EDTA in the

presence of 100 ppm QAC-A resulted in a return of QAC-A

sensitivity and a kill rate approaching 100% in 300 sec.

It was postulated that the QAC-A resistant strains of Ps.

aeruginosa owe their resistance to their impermeability to

QAC-A.









Brown and Richards (151) used a spectrophotometric

method to investigate the effects of EDTA on the action of

several antimicrobial agents against cultures of Ps.

aeruginosa, NCTC 8203, in nutrient broth. It was found

that the separate activities of polymyxin B sulfate (1

unit/ml), benzalkonium chloride (35 ug/ml), and chlorhexidine

diacetate (0.7 ug/ml) were increased substantially in the

presence of EDTA (6.5 ug/ml). Magnesium and calcium ions

were found to block this action. The postulated mechanism

indicates that inhancement by EDTA may be related to its

chelating properties and it is noted that sodium salts of

EDTA are effective, magnesium salts are not. A hypothesis

presented states that EDTA exerts a lytic action and is

synergistic with antibacterial agents by a mechanism

involving removal of calcium or magnesium ions or both from

cell membranes.

A study utilizing electromicroscopy (152) concluded

that part of the mechanism of action of benzalkonium chloride

in subinhibitory concentrations is to damage severely the

external membrane of Ps. aeruginosa. Edetate disodium also

damages the outer layers of the cell envelope and, when

used in combination with benzalkonium chloride, appears to

have a potent effect on sites internal to the external

membrane of the cell. Levels of benzalkonium chloride

used range from 1:1450 (0.07%) to 1:20,000 (0.005%) and of

disodium edetate from 1:20,000 (0.005%) to 1:40,000 (0.0025%).









Surface Tension: Applicability To Ophthalmic Preparations



Definition

The cohesive forces between adjacent molecules in

liquids are well developed. Molecules in the bulk of the

liquid are surrounded in all directions by other molecules

for which they have an equal attraction. Molecules at the

surface, or liquid/air interface, experience attractive

cohesive forces from molecules in the bulk of the liquid

and adjacent to them at the surface; very little attractive

force is exerted by molecules in the vapor phase above the

surface. The net effect is that molecules at the surface

experience an inward force toward the bulk of the liquid;

this force effectively contracts the surface and pulls

the molecules at the interface together, for example, as

in the concentration of soap films and the formation of

spherical liquid droplets. The force which must be applied

parallel to the surface to counterbalance exactly this

inward pull is known as the surface tension and is expressed

in the units of dynes/cm or ergs/cm2

A more complete discussion of the theoretical aspects

of surface tension can be found in texts by Martin (153)

from which this definition was condensed, and by Adamson

(154) and Davies and Rideal (155).



Measurement of Surface Tension

Several methods have been described (156) for the

measurement of surface tension. They include the









Drop-weight Method, where the weight (or volume) of a

liquid drop which detaches itself from the tip of a

vertical tube is measured and related to the liquid surface

tension force determining its size; the Wilhemy Plate

Method, where the force pulling on a thin plate attached

to a balance mechanism is determined and is equal to the

product of the plate perimeter and the surface tension of

the liquid; and, the Pendant Drop Method, where a pendant

drop of the liquid is photographed or projected on to

squared paper and the shape of the drop used to compute the

surface tension.

A method used for many years is the ring method, where

the surface tension determines the force required to detach

a metal ring from the surface of the liquid. The simplest

theoretical interpretation of the results equates the pull

required to detach the ring from the surface to the total

perimeter of the ring multiplied by the surface tension.

The total perimeter of the ring is twice the length, since

the meniscus pulls on each side of the wire. The combina-

tion of a readily-cleaned platinum ring attached to a

torsion wire which can be calibrated has been incorporated

into an instrument known as the du Nouy tensiometer.

Surface-active agents generally possess polar and

non-polar characteristics, with at least one of the two

regions of the molecule being very asymmetrical and of

considerable size. Solutions of such agents in water

depend on the interaction of the polar portion of the agent









molecule with water molecules, and solubility is limited

by the existence of the non-polar portion of the agent

molecule interpositioned among the molecules of water.

Solubilized systems of higher surface-active agent concen-

tration occur when the molecules form aggregates known as

micelles, in which the non-polar portions of the agent

molecules orient themselves together, leaving the polar

portions to point outward toward the water molecules.

Formation of these micelles is associated with specific

changes in physical measurements of the solution properties,

such as surface tension, and is known as the critical con-

centration for micelle formation (C.M.C.). A more

complete discussion of this phenomenon is provided by

Mulley (157).

At concentrations below the C.M.C., surface-active

agents concentrate at the air/liquid interface of a solution,

orientating themselves molecularly so that the polar

portion is associated with the water molecules and the

non-polar portion is repelled, altering the surface

characteristics of the liquid, sometimes dramatically.

When these changes occur in tear fluid due to the instilla-

tion of ophthalmic preparations containing surface-active

materials, alterations in irritation potential, bioavaila-

bility of the active ingredient, tear volume and thickness,

spreadability and the preparation, and wettability of the

corneal surface may be seen.









Application To Ophthalmic Preparations

The importance of an appreciation for the surface

chemistry aspects of ophthalmic preparations containing

polymers has been reported by Benedetto, Shah, and Kaufman

(158,159). Using in vitro and in vivo methods, they

studied the effects on the thickness of the preocular tear

film (PTF) of alterations in viscosity and surface-active

properties using fluorescein solutions of polymers such

as hydroxypropylmethylcellulose and polyvinyl alcohol,

comparing these results with those from the use of several

commercially-available ophthalmic wetting solutions. Their

results indicate that increases in PTF thickness can not

be attributed to increased viscosity alone, but may be

explained by at least three mechanisms: 1) by increasing

the volume of fluid available in the marginal tear strip;

2) by absorption of polymers at either the corneal/aqueous

or mucin/aqueous interface; or, 3) by a film of polymer

solution at the air/tear interface supporting a layer of

water beneath it or by dragging a layer of water with it

as it spreads over the ocular surface with each blink.

These effects are directly related to the surface-active

properties of the polymers studied, noting that polymer

molecules which are capable of rapid adsorption at the

air/tear interface will move from the bulk solution to the

surface and continue to provide a surface-pressure gradient

which induces spreading until equilibrium is reached. The

duration of polymer-induced tear film thickening would be










a function of the retention time of the polymer in the

eye, and highly water-soluble polymers which are less

surface active would have a short retention time in the

PTF since they would be removed by the normal tear flow.

The relative importance of viscosity-mediated mechanisms

and those due to degree of polymer surface activity are

stated to depend on use of the ophthalmic product, that

is, whether the concern is for drug bioavailability,

spreading properties as a wetting preparation, or

"cushioning" characteristics in the case of contact lens

use.

Brauninger, Shah, and Kaufman (160) demonstrated by

simple physical means the existence of an oily layer on

the tear film surface, further enhancing interest in

polymers which possess surface-active properties.

Wolff and Meyer (161) indicate that CarbopolR

polymers provide repeating carboxyl groups which can be

modified to produce a molecule with surface-active

character. Neutralization of a portion of the carboxyl

groups with long chain alkyl amines yields a molecule with

a lipophilic portion which can orient itself effectively

at oil/water interfaces. Neutralization of some or all

of the remainder of the carboxyl groups with a common

base such as sodium hydroxide provides control over pH,

viscosity, and yield value of a preparation. One of the

more effective amines used for neutralization of CarbopolR









resins is EthomeenR C/25a, a polyethylene oxide modified

dodecylamine which has some advantage over the use of

unmodified amines because of increased water solubility

and lower interfacial tension at equivalent usage levels.

Thus, some effects on the anticipated ophthalmic charac-

teristics of a vehicle gelled with long chain amine-

neutralized Carbopol resin may be attributed to surface

activity.

The effects of surface-active agents can extend also

into the area of sterility assurance and preservative

testing. Wedderburn (162) notes that variations in surface

tension of culture media are well known to microbiologists

as a means of influencing growth, and thus the surface

tension of an aqueous phase of an emulsion is certain

to be a factor in determining what organisms grow in it.

Gram-positive organisms do not grow well at surface tension

levels much below 50 dyne/cm, while many Gram-negative

bacteria, and the coliforms in particular, flourish in

environments abounding with surfactants, even though

cationic surfactants are toxic to many organisms.

Therefore, results of direct dilution tests of water-

soluble semi-solid vehicles containing surface-active

polymers could be altered by dramatic decreases in media

surface tension. It apparently has not been determined



aEthomeen is a trademark of the Armour Industrial Chemical
Co., Chicago, Ill.









to what extent CarbopolR polymers influence such tests

and whether these polymers are toxic to microbial growth

under these conditions.





Rheology: Applicability To Ophthalmic Preparations



Definitions

The term rheology is used to describe the deformation

and flow properties of matter. Viscosity is an expression

of the resistance of a fluid to flow, and is usually

applied to simple liquids, although the term has been used

to describe more complex systems.

The consistency of non-Newtonian materials is attributed

to the summation of many complex properties such as

elasticity, yield value, texture, and viscosity, and cannot

be expressed adequately by any single index. However,

several fundamental theological properties which are

indicative of consistency can be determined, and an

inspection of the complete consistency curve of rate of

shear vs. shearing stress gives an indication of the

behavior of the material when it is subjected to manipu-

lation (163).

Materials classified by types of flow and deformation

are placed in one of two categories: Newtonian or non-

Newtonian systems. Liquids such as water, glycerin and

chloroform; true solutions such as syrups; and very dilute








colloidal systems exhibit flow properties where the rate

of shear is directly proportional to the shearing stress.

The isothermal flow of such liquids can be described by

a single value, the absolute viscosity.

Semi-solids, and many other pharmaceutical preparations,

fail to follow this simple relationship and are classified

as non-Newtonian systems. Such systems may exhibit charac-

teristic flow properties which can change with changes in

pH, temperature, time and speed of deformation, additives,

and the previous treatment which the material has undergone,

among other factors.

Non-Newtonian materials can be analyzed in a suitable

viscometer to yield characteristic consistency curves or

rheograms for various types of flow, such as plastic,

pseudoplastic, and dilatant. Plastic flow is characterized

by a rheogram in which the curve is linear over most of its

length, but does not pass through the origin; instead,

the linear portion intersects the horizontal shearing stress

axis at a point known as the yield value. This type of

flow is associated with the presence of flocculated particles

in concentrated suspensions, resulting in a continuous

structure throughout the system, and only a shearing stress

above the yield value provides sufficient force to break

this structure and cause movement or flow.

Pseudoplastic flow is characterized by a rheogram in

which no yield value is apparent, and the flow curve is

non-linear, approaching the origin of the graph. Viscosity








decreases with increasing shear rate, also known as

"shear rate thinning". This type of flow is exhibited

by polymers, such as the natural gums, in solution, and

results from the shearing action on the long-chain molecules

of linear polymers which are disarranged and matted at low

shear rates, but tend to become aligned at higher rates

of shear.

Dilatant flow is characterized by an increase in

resistance to flow with increasing shear rates, that is,

the material returns to a fluid state when agitation is

stopped. This type of flow is termed "shear rate

thickening" and is essentially the reverse of pseudoplastic

flow. It is characteristic of suspensions containing high

concentrations of very fine particles which are deflocculated

at lower rates of shear and flocculated at higher rates.

Some non-Newtonian materials can show flow properties

characterized by an initial breakdown in structure as

rates of shear are increased followed by a resetting of the

material to a semi-solid consistency when allowed to stand

undisturbed. This phenomenon is termed thixotropy and is

a reversible isothermal sol-gel transformation produced

by a structural breakdown and reaggregation of floccules,

crystallites, linear polymers or finely-suspended particles

superimposed on the basic flow types. The rheogram will

exhibit a hysteresis loop.

A more complete theoretical treatment of theological

characteristics can be obtained from one of the texts from








which these definitions were condensed, such as those by

Martin and Banker (164), Martin et al. (165), Wilkinson

(166), and Van Wazer et al. (167).



Measurement of Semi-Solids

Manufacturers of therapeutic ointments have recognized

for a long time the need to provide preparations which

have uniform consistency from batch to batch, and

theological measurement is included as a part of the pro-

cedures for quality control. Martin and Banker (168)

discuss the use of multi-point vs. one-point measurements

and indicate that the most accurate method for non-

Newtonian materials is the construction of multi-point

rheograms for comparison of one batch to another.

A rotational viscometer can be used to supply data for

the construction of specific rheograms. It is desirable to

have the instrument designed so that the speeds of the

rotating elements can be changed without interruption,

particularly important in the measurement of thixotropic

materials.

Kostenbauder and Martin (169) used a Stormer rotational

viscometer with a modified cup and bob to measure the

theological properties of several semisolid preparations.

Meyer and Cohen (170) used the Brookfield Viscometer to

determine flow plots for several natural and synthetic

polymer solutions, including 0.2% solutions of CarbopolR 934.

These carboxy vinyl polymer solutions were determined to








exhibit plastic flow properties, while the others studied,

all natural gums, showed pseudoplastic flow characteris-

tics. Wolff and Meyer (161), also using the Brookfield

instrument, confirmed these findings for other CarbopolR

polymers, stating that they are found to be unique in the

field of natural and synthetic polymers in their ability

to develop plastic theological behavior and yield value in

water solutions.

Marriott (171) discusses the use of rotational

viscometers, specifically the Brookfield instrument, in

determining the theological properties of various cosmetic

preparations, and indicates that a water dispersion of a

substance such as CarbopolR becomes liquid on the applica-

tion of a very small stress, then returning to a rigid

state when the stress is removed, that is, displays

thixotropic characteristics.

Araujo (172) used a Stormer viscometer, in which the

cup was replaced by a 100-ml glass beaker already containing

the sample and the bob was replaced by a star-shaped rotor,

to study the thixotrophy of bentonite magmas of different

concentrations.

Barry and Meyer (173), using a Ferranti-Shirley cone

and plate rotational viscometer, studied fifteen gels

prepared by neutralizing aqueous solutions of carboxypoly-

methylene 940 or 941 (CarbopolR) with triethanolamine.

Such gels did not exhibit time-dependent behavior

(thixotropic character) on shearing.









Applications to Ophthalmic Preparations

Eriksen (174) discusses the role of viscosity in

ophthalmic preparations, stating that if all other facts

are equal, thicker products, in general, stay in the eye

longer and produce greater penetration. There are limits

to this concept, however; for example, the continued

increase of viscosity of methylcellulose solutions produces

diminishing returns. He indicates that the increase of

viscosity beyond 10 cps produces little significant improve-

ment in penetration, but decreased mixing and irregular

optical surfaces produce visual disturbances.

Kostenbauder and Martin (169) studied the theological

changes in petrolatum-based ointments due to various

additives and proposed a classification method according

to relative hardness and stiffness. Three classes were

designated. Class I consists of ophthalmic ointments,

which are the softest type, generally petrolatum containing

20-30% mineral oil. Class II consists of the common

medicated ointments, and Class III of the protective

ointments, which are hard and stiff enough to remain in

place even when applied to moist, ulcerated areas.

The closeness of the eyeball to the eyelids exposes

a non-Newtonian material to considerable shearing stress

during the normal process of blinking, and can result

in shear-thinning with resultant changes in drug availa-

bility from the preparation. Sieg and Robinson (175)

studied the effects of shear on the release of pilocarpine








from ophthalmic ointments. They found that good correla-

tion between in vivo and in vitro test results could be

obtained only if the contribution of shear to the release

rate of the drug was considered. Controlled mechanical

shearing was used in the in vitro tests and produced

results which showed acceptable drug release for prepara-

tions which would have been rejected on the basis of simple

standard partition-diffusion methods.

Recent interest in aqueous CarbopolR gels for

ophthalmic use has resulted in the preparation and testing

of several medicated gels. Schoenwald et al. (176), studied

a series of carbomer gels containing pilocarpine, indicating

that these gels exhibited plastic flow characteristics

and definite yield values. Results of in vivo tests using

rabbits showed that increased miosis duration was obtained

by gels containing 2.7-3.0% carbomer, and that some degree

of controlled release of drug from these gels was evident.

The control is attributed to either diffusion within the

gel structure or slow erosion of the gel surface with time

in the eye. Shear-facilitated release is also a factor, in

that the increased duration was a consequence of the gel's

increased yield value such that appreciable in vivo thinning

of the gel does not take place with eyelid and eyeball

movement.

Schoenwald and Boltralik (177) compared the relative

bioavailability of prednisolone sodium phosphate, 1%, and

prednisolone acetate, 1%, from aqueous suspension or








solution, and from aqueous CarbopolR 940 gel or gel

suspension. It has been mentioned previously that increased

viscosity, within limits, prolongs contact of drug with

the eye and a lowering of surface tension improves mixing

with the precorneal tear film. The results of the experi-

ments showed that significant quantities of gel were observed

in the rabbit conjunctival sacs through 8 hrs., and that

the gel quantity slowly diminished over time, probably partly

as a result of diffusion and partly by surface erosion.

A significant increase in the bioavailability of both the

soluble and the practically insoluble steroids was observed

with the gel dosage form, making it possible to reduce the

concentration of the drug in the gel and still maintain

a maximal therapeutic effect.

Giroux and Schrenzel (178) studied ocular and

percutaneous tolerances of jellies made with Carbopol 934

and Carbopol 940 to which various drugs were added,

including atropine. Results indicated that gels made with

Carbopol 940 were preferred and that the neutralizing agent

played a part in the absorption of the drugs. Correlations

of absorbance and viscosity variations were not always

possible.













CHAPTER III
EXPERIMENTAL METHODS





Materials



Chemicals used in the preparation of ointment bases

for these studies are listed in Table 1 and were selected

as a result of previous work. Additional materials and

chemicals are listed in Table 2. All chemicals conform

to U.S.P. or N.F. standards unless otherwise specified.





Equipment



The specialized equipment and apparatus employed are

listed in Table 3. Other equipment not indicated and

glassware used were of good quality and conventional

design congruent with accepted research standards.





Ointment Base Composition and Methods of Preparation



The percent (w/w) composition of the ointment bases

and methods of preparation used are listed in Table 4.

69




















4
( 4


O)
0




4J


























04
-
















4















()
C






































Cd
r4-














4-)
Cd
























-r


U

H


0 -H

HO

r-4 0





.,I r-



0to
0) (v

.U C















Ln
Cd













0
-4





VI)
iar
H Q0
H *O


* 0 *(



-H 0
un


CdZ
c .

H
H*
U


O )
O d
rHl 50


O
0
u
0 *
*.r >
4 -i-l H

4-J
C Z
C)4-I



-H 3 -l
0C (*
02 n

5. 4 0

44 .U r
U) C) -
*H. Cd-


.4
O
0)









C)
oH
S4-)

































0) (a U
JCd
(0 4- .


























r4 CN
0U















--Hq 0
m4 Z
-H 0
QU
C c
-, 0 *



Od r
O QO


OO
0
U

-H-
AI
C) *



c- r-
en 4 oo


) --H 4.
-rH d 0O


U



.4
C.


U 0
SdC


-4
c -
m












N
,--IC


















S4-1
U1


0





0
r-a
m




0

Ho
*0


O0







M C)
S











>



C)
rE


0
U

U
-rH
r.f


1 .
. z


,)


























a0
,4 -



















O
1 C
U -H
,-H



















Q).
t0 .
4 0














Cr0
H .













CS
0C)












0)


0)
-H


rd

E






71



0U 0

S* H i- .
S0 a) 0 0 >
-4 U 3 0H I u U -H
0,) I44 H "-- H H4

O)0 S O= O U U u
3 r -~ -r t O *.4 .H
S-4 0- H 44 -- 0 -I 4 Z
44 0 -A I Z Z fu Z) %1 4J : r Z-:q
:j 4 "4 r- a) 0o r" a a) ..- A ..

S0 0 --U *H -4H H a cn
r U n) m U m u r-l4 .Zo
U -XE r-4 r-AC 3 -- U3:1 U 0NOM
OdQ r,-H-.I U,-I ,- crr-

0 a) r*l UO) U) 41 O -*
3 H *. c P.. r5 *H (U )
-P 4 4 H O Ua) r-l 4 C-H4) -r- 4J r :I P 4
M 0 0 *H 04-l4Z 0 (a U40(0 dJ 0 -Hl w O O
SZ ) Zr -i4 U 4 r4I44 5 Pr i U S








o o C rD


-. (
0 0 0 H
S0 $4 0- N 5- O


"- r 00
-, H o
En 4J r ra mN r4:


4 >1 0 >1 CN O rd 0 .
E-1 0o r O L' n 4 12 p 0 tO .
cou 0 ( 3 0 Cl)
O 0 -. H H .U) H .




o 0A 00
4)U 0 3 U ( C 4 H 4 (n
-H C,4 -I D C,4 r = ri u -i = a)
CU S -C >N 2 0s .




-- U +070 r f.- C 50 U (U




rd
C1) vo 4-4
:H a o i
0 U a-I ( ) u) 4 U

>1 U A U 4H (d 44
4-z 0 0 0C d Hi

u ^a : u (a rq o

4 0 rrl ::I >i U) r-l 4J O tn
-. + 0-) U-H ^
U )X ) U 4.D')
a 0
> U. U *

















s-4
s) 6

4 -


S4-J


























0
Ec






















CD
O
Sri


4-
04


























,-I
a)
(0d
-d
0)
&

r


0*
0m
r-A
4-)



IN N



CN *N

S















H
0 5
c4

























0 (
OIm












C
P4-


0
U

*H

*H 1
4 *
r


0) 0


0 14-
d(U


-H 0d 0
,C ** o
*(0 h -


-H U

0

4*
M 0
U) -4


--4



.-H


O
0

O 0
QA i


0

2 H
-H

O -
0
.-i
* J
-P 0
cn 1-4


(4
r-A
CN

cn
H C
O-4
O -H Cn
+rO r
4- -tH r

A, o
fd -l
1-



H 0








ir
0 0


0




'-4 En l
0O
10 0





C.
>i (U
O a









,r a
a r.


*0 0 -z
Hr U0 -r
r-I n rT


-H
r0
I 0)
a5
a)


>iU
o
> o
r-4 *n









O

4. -r- ,-i
a0 t




-HO 4,i
., QaC(
^ U *
r-4M


CO

0 ,
O &>
4-14


OOh
0 2 **





p4 4J 4J




0-H






>4J.




& M
Cd U2





I-H

-,4 -I








o


4o *:1 (-

n 0 tn .
4*)-1 0)









pi


o
0 a







r-H



-,-
tn


4 N



f:A
Hd 3
f-I t~
0~ tr








-H-

-H C


-HI
a





*
0)





0-4





Q,
o1
r4
*a0

r* T
(U4U4


rel
a)
-H




(fU
0



U)
U 0


0
-
Od
*r4


0
O




m c

ca
coz
0) 0

2=




















0p ,
a )





00
.H (0
n e





O p

HO-
0 Q


0


N )
4-4
S4J

























-r
0
4-


04

























a)
*^


0 ri


CO
4-) .


.-H I


00
U -
H p
S- )
H


I I
0 0 .
4 N4
tn 0 04 U)
>144 0

a a-H
>1i r
(r U3 Nu
> ) 0)o

0 N

0 EN


S-H9l 4

CU 0 :z
4-i
4U -r1 Z t0|4l
S4-I -10 -H r-4
4 Q J -I



t0 0 4 z
-H0 0)0 -H


-4 o aU) rd-r
4-O ca
-004-0 0


O)
- c



N- (H
0 i


O-H



00
O )



Q Q 4


4-i
*l


0 Ci



N> Q)
60 CU

N-H




O0 0 Ln
a- 4-
4d- 0
00 0
SCO 0-H
Cr(U C 4
4- *CUQ a














4-)-





O0



0t
O(1
3PZ


p)
0- r
4-) ie
N -H
0 to





00
U -4
rZ4I- 4-
-H)
0OO

QQA


UCO >
(1U ) 44
m> --
( -H 4-J



r -4q N
)r Cl E4-4
> 4 .J
-H4 ty
r3 .,- C-







0 0-I) C



0 0 CU .
O ( 4-i p
rd. CU 4






.-H 0 (1) t
CO U 4-) d U)1
a N FO 0 -H-







-In
C(


r-


(i2
(0000























,-4


0
U
0


I 0
0


4 .4 -
-N N
b-'0 0

S00 (
Q)- 4Q U
> U) o
0 4 Cq


oU 20




-.H 0 0
4) -H >P Q 4



0 04-4 ()I|

3 0 0 z0

-n 0 4-) 0 4
-4l U t->r


0


)0
*>-


I 44
0 0
N U
p (a) o

4-


>
)0
*0
0in
S4-I






4,j
04



4 U
0
+
0 *











0
4 0







CO













0
4-i







r04











) 0

3 o 3n .- i o .o
4 > H 4 4
SI, 00 4. H *H )
0 ) c 0 4 0
3 0 n HH ( 4
0 C N 0 0 -O 0 >
(Z OHCN O Q Q o 0
441 U %D U 4-)j :5 A --)
3$ 4- 0 I rI 0
C O O H cn O o N
o 1 I % O 0m o,- oE <- 3
E + o Q) 4 X. 4J o0 o o 4-M; 0 1 o-0
Mtd om U Ht *tiN U 3cq O ul
S--r C CC C O O 0
U El *r- D rA H 4*HJ
0 ** d ( ** ... .. r4p 4
W 04-" rHl Ur- +J Hr-4 4- r- .- ) 3 4J
t OO 4- C-,r- 0 U -r 0 (d 4 O Q) O0




I
g o
0) Q)
>1 4-) +J
-4 ZO(,
4 ( D M 0

U 4 000 04H-


SC U 0) o I H 0$ o-
O 0 -*- 'ZH N$ O 0 4O-I *Zi C
U *H e *d -(0)Z c -X1Z 0C! N rl
w 0 l 0 C) l 0 0 04-
*H U -.4 00 0 C 0M
z ai 4) C3 IC -0) c C) -,4o 4-4
U I I:- r 0>i 0 H a) z r.m z rH 04 N- < 0
40 ) 4 9 d (a- d r. 0U ].
04 (U }i + 3(H -p m a g 0)


H4 0) l i d >i3 rl 40 O 4-

S10 a r0i OHO3 4r4 r3 3- S (C : pH
H1 >i 0 3Hr >iO v I (. -e 0 :
dPH H(mH H>C1)H U a) 0) 4 l MJ$4

in a) 0 0 -O* 0 0- 0 CU H (0)
Cr >1 04E > U) 104 > U -) S u u tm





rd
0


4-)H
00 4-

*--I6
43 41) U) -H
4C Q Q)* 0 *
a) -H P4P4 $4 xfa 04
4l 0 0 r-* *
E >4* C* N *
O H:D HD 0:D
04 a4 4











r-l





0 a
H
T)







OH

zz H
WOa


.4
4 j



CO
rdr
4-
0












O











0
-.l
.4



0r












4-











-4
Cdl


*


a
4-)4

0)

00
P4 4


r-A
>1
04
0
p4
tr

4-H
rn

OO
N




0 0
g a\

>i0)
(1) 4-) 0







i a) e
M 4) 0
1 M .


0
o
U
U
.-I

4-) .
-H .




* i-l

- ,- 0










I
Hr
Q) C











-I -- o
>1 r-i






0 0)
OQ*
0 -r *




-lH* u
H 0-
I C


P4






0
*





O


0 0
4 .


C0
4 M
U


0
H


o -H


Om
(0d


U -n




or- 0I0
0 U



LO
l

ur-












,o
(U r-I







U 3












o
H n
H




























I
H *

P 0

o
I*
Hin
r-H i

I 0
I*
OH
HO)

il *


X (
















0



I d


U
Z
*M
0 ,-
Ok
P
au




Go
O(
0r
OQ








mo
H C
E I
rn r-l
















4
O
o r
0 (













On Cd
p4 EQ


-l1 -*-
mu m
:s 0) 0 -
-P















H -
0
4-1 0D






04-4
Sa0 0

ro Np
0 in r0















0 0









*H
>
)
4-













1-
0,


0






.- ;
-O


u
-Hi
4)



(0
0i 0







0 .

O1
4-Hrl








co
-rl




00)



CU





















0)
E G '





















C,


>1
4-,




0 a




0 .


00
dro




H 0)
Q -
0


l ^

a40




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EWCCDA2YK_VAXBYX INGEST_TIME 2012-03-02T21:46:18Z PACKAGE AA00009118_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES