Colored 5-aminolevulinic acid crystals useful for photodynamic therapy are disclosed. Preferably the colored 5-aminolevulinic acid crystals have the color imparted by irradiation of the crystals, such as gamma radiation. The colored ALA crystals are preferably pharmaceutically pure and sterile and can be contained in a sealed sterile container. Also disclosed is sterile aqueous ALA solution which includes the colored ALA crystals contained in water. Also disclosed is a method for preparing colored ALA crystals which includes exposing non-irradiated ALA crystals to a radiation source at a dose sufficient impart a color which is different than any color present in the non-irradiated crystals. Preferably the irradiation is sufficient to sterilize the ALA crystals. The sterile colored ALA crystals can be used in a kit for internal or external treatment and/or detection of a condition in a mammal, which includes the sterile, colored ALA crystals and sterile diluent, and in the case of internal treatment and/or detection optionally a catheter for administration of the ALA.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates generally to the field of medicine, particularly 
pharmacotherapeutics and pharmacodetection, using photosensitizing agents 
and precursors thereof, especially 5-aminolevulinic acid, also known by 
the acronym "ALA." More specifically, this invention relates to colored 
ALA, especially sterilized ALA, which is stable under commercial 
conditions. 
2. Description of Related Art 
Photodynamic therapy involves the administration of a photosensitizing 
agent to a subject, including administration of a precursor of a 
photosensitizing agent such as ALA, and subsequent irradiation with light 
of the target cells or tissue of the subject. The photosensitizing agent 
preferentially accumulates in the target cells, namely cells or tissues 
that are more rapidly proliferating or growing than other cells or tissues 
in the target environment. The target cells may be more rapidly 
proliferating because they are malignant or non-malignant, of infective 
agent origin, e.g. viral, bacteria, parasite or fungal origin or not of 
infective agent origin; are normally hyperproliferative, such as the 
endometrium of pre-menopausal women, or are abnormally hyperproliferative, 
such as cells infected with an infective agent. 
Although not intending to be bound by any particularly theory, it has been 
proposed that following administration of ALA, as a result of their more 
rapid proliferation, the target cells or tissue contain relatively greater 
concentrations of light sensitive porphyrins and thus are more sensitive 
to light. 
The target cells or tissue containing sufficiently high concentrations of 
the photosensitizing agent, including the metabolites of ALA, selectively 
absorb greater amounts of light and can be selectively localized and 
distinguished from the adjacent cells or tissues via fluorescence, or 
damaged or destroyed. The effect of the light, as is well known in the 
art, depends on the photosensitizer selected; the wavelength, intensity 
and duration of administration of the light; and the timing of irradiation 
vis-a-vis the administration of the photosensitizing agent, and results in 
fluorescence or impairment or destruction of the target cells or tissue. 
A variety of photosensitizing agents have been used for photodynamic 
therapy. The only commercially available photosensitizing agent is 
Photofrin.TM., a hemato porphyrin or HPD, sold by QLT Phototherapeutics, 
Inc. Vancouver, British Columbia. Synthetic porphyrins often have the 
disadvantage of having longer half-lives and lowered sensitivity to the 
rapidly growing cells as contrasted with normal cells than do naturally 
occurring porphyrins. The half-life of the photosensitizing agent is 
significant, since the buildup of excess porphyrins in the skin can result 
in reddening or burning of the skin. 
An alternative to synthetic porphyrins are natural porphyrins. Natural 
porphyrins appear to have shorter half-lives than their synthetic 
counterparts, but are difficult to synthesize and more importantly, are 
unstable ex vivo under environmental conditions to which drugs are 
subjected in normal commercial distribution and storage channels. 
A revolutionary discovery made in the late 1980's was that the naturally 
occurring amino acid ALA, a precursor in the metabolic pathway to heme, 
could be used in photodynamic therapy instead of synthetic porphyrins. ALA 
appears to act in the body as a precursor to naturally occurring, light 
sensitive porphyrins, which avoids the ex vivo problems associated with 
natural porphyrins noted above. This discovery has brought photodynamic 
therapy to a world wide interest level never before achieved with 
synthetic porphyrins. 
ALA has a very short half-life, depending on the route of administration, 
is highly tissue specific and, as a naturally occurring amino acid, 
minimizes complications and side effects which arise when foreign 
substances are administered to the body. Unlike synthetic porphyrins, ALA 
also makes it possible to distinguish small or flat tumors, e.g. in the 
bladder, from normal tissues, visually by means of fluorescence 
excitation. 
It is believed that ALA is converted by the cells and tissues in vivo or ex 
vivo into protoporphyrin IX and related endogenous biochemicals, which 
fluoresce or are degraded by light of the appropriate wavelength. The 
preferential accumulation of such naturally occurring porphyrins in 
rapidly growing cells permits the targeting of such cells. 
One of the roadblocks to the commercial use of ALA has been its extreme 
liability to destruction under ambient conditions. Aqueous solutions of 
ALA maintained under ambient conditions are progressively, degraded quite 
rapidly, resulting in degradation products, primarily 2,5-pyrazine 
dipropionic acid and intermediate degradation products which have not been 
able to be identified due to their transient nature. However, the 
intermediate degradation products are believed to include 2,5 
(beta-carboxymethyl)-dihydropyrazine. FIG. 1 depicts the degradation of 
ALA to 2,5 (beta-carboxymethyl)-dihydropyrazine and then to 2,5-pyrazine 
dipropionic acid. Formulating ALA in nonaqueous creams and gels did not 
prevent this degradation. Even ALA pressure-sensitive adhesive mixtures 
did not totally stop oxidative reactions. Likewise, the preparation of 
pharmacological equivalents of ALA such as functional derivatives of the 
carboxylic acid group, substitution of the amino group, blocking of the 
oxo group or the use of simple or more complex acid addition, acid, base 
or neutral salts has not completely overcome this problem because the more 
stable the product, the greater effect there may be on the metabolism of 
the product in the body. 
SUMMARY OF THE INVENTION 
One object of the invention is to provide ALA which does not suffer from 
the problems of the known art, particularly the extreme degradation of 
ALA. Another object of the invention is to provide sterile stable ALA 
which is pharmacologically active and can be used in photodynamic therapy 
and detection. Still another object of the invention is to provide a 
method for making sterile, stable ALA which does not suffer from the 
problems of the known art. Yet another object of the invention is to 
provide a combination of ALA and an endoscope for internal use in a 
mammal. Still another object of the invention is to provide a method for 
using ALA in the detection and/or treatment of a condition in a mammal. 
The foregoing objects and other objects are achieved according to one 
aspect of the present invention by colored 5-aminolevulinic acid, 
preferably 5-aminolevulinic acid HCl. In a preferred embodiment, the color 
is imparted by irradiation of crystals, preferably gamma radiation. 
According to another aspect of the invention, there has been provided 
colored 5-aminolevulinic acid crystals having an F-center point defect in 
the crystal lattice, where said F-center point defect imparts said color 
to the crystals. 
According to still another aspect of the invention, there has been provided 
a sterile aqueous ALA solution comprising the colored ALA crystals 
according to the invention, contained in water. According to yet another 
aspect of the invention, there has been provided a sterile package 
comprising colored ALA crystals according to the invention in a sealed 
sterile container. According to yet another aspect of the invention, there 
has been provided a method for preparing colored ALA crystals according to 
the invention, which includes exposing non-irradiated ALA crystals to a 
radiation source at a dose sufficient to impart a color which is different 
than any color present in the non-irradiated crystals. According to still 
another aspect of the invention, there has been provided a kit for 
internal and/or external treatment and/or detection of a condition in a 
mammal, which includes the sterile colored ALA crystals according to the 
invention and a sterile diluent, preferably water. In one mode of internal 
treatment and/or detection, the kit optionally includes a catheter and 
optionally an endoscope. 
According to still another aspect of the invention, there has been provided 
a method of administering 5-aminolevulinic acid in a stable form for 
internal and/or external mammal administration which comprises the 
administration of a solution of ALA derived from the colored ALA according 
to invention. 
Further objects, features and advantages of the present invention will 
become apparent from consideration of the detailed preferred embodiments 
which follow.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
5-Aminolevulinic acid is also known as 5-aminolaevulinic acid, 
.delta.-aminolevulinic acid, .delta.-aminolaevulinic acid and 
5-amino-4-oxopentanoic acid. 5-Aminolevulinic acid can be used as the 
salt, particularly a simple salt and especially the hydrochloride salt. 
5-Aminolevulinic acid can also be used in the form of a precursor or 
product of 5-aminolevulinic acid. 5-Aminolevulinic acid can also be used 
in its pharmacologically equivalent form, such as an amide or ester. 
Examples of precursors and products of 5-aminolevulinic acid and 
pharmacologically equivalent forms of 5-aminolevulinic acid that can be 
used in the present invention are described in J. Kloek et al., Prodrugs 
of 5-Aminolevulinic Acid for Photodynamic Therapy, Photochemistry and 
Photobiology, Vol. 64 No. 6, December 1996, pages 994-1000; WO 95/07077; 
Q. Peng et al., Build-Up of Esterified 
Aminolevulinic-Acid-Derivative-Induced Porphyrin Fluorescence in Normal 
Mouse Skin, Journal of Photochemistry and Photobiology B: Biology, Vol. 
34, No. 1, June 1996; and WO 94/06424, which are all incorporated by 
reference herein in their entirety. As used herein, all of these 
compounds, unless other wise noted, are referred to jointly and severally 
as "ALA." 
As used herein, the term "sterilized" refers to a product which has been 
processed to make it suitable for internal pharmaceutical use. 
As used herein, the term "colored" is defined as color that has been 
induced by irradiation. This is to be distinguished from non-irradiated 
ALA which is generally white, but in some instances may be off-white, 
probably due to the presence of varying amounts of degradation products. 
As used herein, a "pharmaceutically pure" substance is defined as a 
substance which is suitable for therapeutic and detection use in humans 
and other mammals. Preferably, the pharmaceutically pure substance is 
labeled for therapeutic and detection use in humans and other mammals. 
The present invention is based on the finding that sterile ALA can be made 
and prepared well in advance of its final use, despite the extreme 
degradation generally found with ALA as noted above. It was further and 
surprisingly found that despite the yellow color of irradiated crystalline 
ALA, the substance had not been degraded, despite the damaging effects 
that radiation, particularly gamma radiation, can have on 
thermodynamically unstable substances. In addition, it has been 
surprisingly found that the gamma irradiated material is stable for 
extended periods of time, at least one year, when stored in the sealed 
bottle in which it was irradiated. 
The sterilized colored ALA crystals are preferably substantially free of 
impurities, such as degradation products and pyrogens, especially when 
intended for systemic administration. The amount of impurities is 
generally .ltoreq.2.0 wt %, preferably .ltoreq.1.0 wt %, and more 
preferably .ltoreq.0.5 wt %. 
The crystalline ALA is colored to the naked eye by irradiation from a 
source which emits electromagnetic radiation, preferably gamma radiation 
or other ionizing radiation. For example when the hydrochloride salt of 
ALA is employed, the irradiation alters the appearance of the crystals to 
the naked eye from white or off-white to yellow. The intensity of the 
color, such as the yellow color for ALA HCl, is dependent on the 
crystalline form that is irradiated, such as the form as commercially 
supplied or a crystal that is further micronized. For example, when the 
ALA crystals are micronized, the irradiated ALA micronized crystals are 
less intensely colored than intact (i.e., non-micronized) ALA crystals. 
This is consistent with the F-center theory for colorization described 
below. The intensity is also dependent on the dosage of radiation that the 
crystalline ALA has been supplied with. 
This color disappears after dissolution of the irradiated crystals and does 
not appear again upon recrystallization from an aqueous solution under 
ambient (i.e., room temperature and pressure) conditions. The colored 
crystalline irradiated ALA has not been distinguished in any chemical 
property from non-irradiated ALA, either by spectroscopic, 
chromatographic, solution pH or solubility profile, except for the color 
of the irradiated material. This is explained more fully below with 
respect to FIGS. 2 to 14. 
Gamma irradiated ALA, as can be seen from FIGS. 2 through 14 is 
substantially physically and chemically the same, except for the color, as 
the non-irradiated ALA with respect to spectroscopic, chromatographic, 
solution pH and solubility profile. Although not intending to be bound by 
any theory, it appears that the color imparted by irradiation is due to 
F-centers. 
The calorimetric measurements of FIGS. 2 and 3 indicate that any major 
structural differences are present in quantities of less than 1% of the 
total ALA amount, or that the type of any structural modification is such 
that the sensitivity of the calorimetric assay to the change is below the 
capacity of the instrumentation. 
Spectral reflectance as shown in FIGS. 4 and 8-10 is very sensitive to 
spectral differences at or near the surface, while photoacoustic 
spectroscopy ("PAS") as shown in FIGS. 5-7 is sensitive to changes both at 
the surface and the interior of the crystal. Both of these techniques 
revealed significant spectral differences between the irradiated and 
nonirradiated reference samples of ALA in the solid crystalline phase. 
Since no differences were observed in the solution phase spectra, the 
yellow color of the irradiated material is likely to be a property of the 
crystalline solid only. Calorimetry failed to show gross crystal 
modifications, supporting the view that the crystal modifications are such 
that the overall crystal symmetry is not significantly affected. 
Although not wishing to be bound by any theory, gamma radiation is known to 
cause point defects in substances such as alkali halides. Such alterations 
do not change the overall symmetry of the crystal, since they involve only 
the removal or relocation of small numbers of specific ions, while 
essentially leaving the major crystalline structure intact. The most 
common type of point defect caused by the ionizing radiation of the 
F-(farben)-center, which is a negative ion vacancy with one excess 
electron bound at the vacancy. The time required to fill the vacancy 
formed by the electrons is on the order of minutes to days, and is 
dependent on the diffusion rate of electrons in the crystal lattice. 
F-centers have been extensively studied and are characterized by one or 
more absorbance band(s) at higher wavelengths (lower energy) than that of 
the normal absorbance of the molecules surrounding the excess electron. 
These allowed, red-shifted bands are believed to stem from the excess 
electron gaining state function properties from the surrounding molecules 
in a symmetrical manner. The location of the UV-visible absorbance bands 
seen in FIGS. 4, 5 and 6, detected by both PAS and reflectance 
spectroscopy, is consistent with the F-centers. 
The spectra shown in the photobleaching studies of FIGS. 7 and 8 is 
consistent with this F-center theory. The spectra depicted in FIGS. 9 
through 12, suggest that the color is due to an F-center defect rather 
than preformed chemical degradation. FIGS. 13 and 14 indicate that no 
microscopic changes take place upon irradiation of the ALA. 
Moreover, when ALA is irradiated, there is generally some delay before 
coloring of the crystal sets in. As noted above, the coloring is due to an 
ion being "kicked out" during irradiation, leaving a charged hole. The 
temporal delay in coloring is believed due to the basis of ion migration 
through the crystal lattice. This delay further supports the F-center 
theory. 
Although the above measurements and analysis were carried out on ALA HCl, 
which produces a yellow color, it is fully expected that other crystalline 
forms of ALA would also experience non-degradation related coloring upon 
irradiation. 
Another aspect of the invention provides methods for the preparation of 
sterile ALA suitable for internal use in human subjects and other mammals, 
namely by irradiating, particularly .gamma.-irradiation, of ALA. 
Preferably, the irradiation is carried out in a sealed container, such 
that both the ALA and the container are sterilized during irradiation. The 
irradiation procedure sterilizes the ALA in the conventional sense. 
Sterilization by radiation, particularly gamma radiation is well known in 
the art and will not be discussed at length. More detailed information can 
be found in Gamma Processing Technology: An Alternative Technology for 
Terminal Sterilization of Parenterals, by B. D. Reid, PDA Journal of 
Pharmaceutical Science & Technology, vol. 49, no. 2, March-April 1995, 
pages 83-89, which is incorporated herein in its entirety. 
The ALA is irradiated with a dose of radiation sufficient for 
sterilization. A sufficient dose of radiation can be determined by methods 
known to those skilled in the art. For example, after irradiating ALA with 
a selected dose, the ALA can be transferred to the appropriate media for 
encouraging the growth of viable microorganisms to determine if 
sterilization is sufficiently complete. 
For most applications using gamma irradiation, a dose of 5 kilograys or 
greater has been found to provide sufficient sterilization. However, as 
noted above, the crystalline ALA has been surprisingly found to resist 
degradation even at high doses of gamma radiation. Thus, it is possible to 
provide a dose of 25 kilograys or greater, without detectable degradation 
of the ALA. This is significant, in that the United States Food and Drug 
Administration's ("FDA") level of gamma irradiation for "overkill" is 25 
kilograys. This is a level at which the FDA presumes, without evidence, 
that virtually all microorganisms have been killed. 
The present invention also provides for methods of using the sterilized ALA 
in photodynamic therapy internally or externally on the tissues or cells 
of a mammal. For external use, the sterilized ALA may be applied by an 
applicator. For internal use, application may be orally, intravenously or 
administration by a catheter. For example, the administration of the ALA 
can be on internal surfaces of the body of the subject, typically in 
conjunction with an endoscope coupled to a light source. Numerous 
publications discuss photodynamic therapy, see e.g. Kriegmair et al., 
"Photodynamic Diagnosis (PDD) for Early Recognition of Carcinomata of the 
Bladder", ENDO World Uro No. 17-E, 1955, a publication of Karl Storz Gmbh 
& Co. and the equipment referred to therein. See also: 
1. Bahnson R. R. Editorial: Urothelial malignancy - Much promise but little 
progress. J. Urology 1996; 155:122. 
2. Baumgartner R., Kriegmair M., Jocham D., Hofstetter A., Huber R,, Karg 
O., Haussinger K. Photodynamic diagnosis (PDD). of early stage 
malignancies - Preliminary results in urology and pneumology. SPIE 1992; 
1641:107-112. 
3. Baumgartner R., Kriegmair M., Lumper W., Riesenberg R., Stocker S., 
Sassy T., Hofstetter A. ALA-assisted fluorescence detection of cancer in 
the urinary bladder. SPIE 1993:2081 (International Symposium on Biomedical 
Optics, September 1993, Budapest, Hungary) 
4. Chang S-C, MacRobert A. J., Bown S. G. The biodistribution and 
photodynamic effect of protoporphyrin IX in rat urinary bladders after 
intravesical instillation of 5-aminolaevulinic acid. SPIE 1995; 
2371:289296. 
5. Chang S-C, MacRobert A. J., Bown S. G. Biodistribution of protoporphyrin 
IX in rat urinary bladder after intravesical instillation of 
5-aminolaevulinic acid. J. Urology 1996;155:1744-1748. 
6. Chang S-C, MacRobert A. J., Bown S. G. Photodynamic therapy on rat 
urinary bladder with intravesical instillation of 5-aminolaevulinic acid: 
light diffusion and histological changes. J. Urology 1996;1 55:1749-1753. 
7. Forrer M., Glanzmann T., Mizeret J., et al. Fluorescence excitiation and 
emission spectra of ALA induced protoporphyrin IX in normal and tumoral 
tissue of the human bladder. SPIE 1995; 2324:84-88. 
8. Iinuma S., Farshi, S. S., Ortel B., Hasan T. A mechanistic study of 
cellular photodestruction with 5-aminolevulinic acid-induced porphyrin. 
Br. J. Cancer 1994; 70:21-28. 
9. Iinuma S., Bachor R., Flotte T., Hasan T. Biodistribution and 
photoxicitiy of 5-aminolevulinic acid induced PpIX in an orthotopic rat 
bladder tumor model. J. Urology 1995; 1 53:802-806. 
10. Jichlinski P. P., Mizeret J., Forrer M., Wagniere G., Van den Bergh H., 
Schmidlin F., Graber P., Leisinger H-J. Les tumeurs superficielles de la 
vessie. Rappel pathologique et clinique, et presentation d'une nouvelle 
methode diagnostique: la photodetection par fluorescence des carcinomas a 
epithelium de transition basee sur I'induction de protoporphyrine IX par 
I'acide delta-aminolevulinique (5-ALA). 
11. Jichlinski P., Forrer M., Mizeret J., Braichotte D., Wagnieres G., 
Zimmer G., Guillou L., Schmidlin F,, Graber P., Van den Bergh H., 
Leisinger H-J. Usefulness of fluorescence photodetection of neoplastic 
urothelial foci in bladder cancer following intravesical instillation of 
delta-aminolevulinic acid (5-ALA). SPIE 1996; 2671:340-347. 
12. Jichlinski P., Forrer M., Mizeret J., Glanzmann T., Ddraichofte D., 
Wagnieres G., Zimmer G., Guillou L., Schmidlin F., Graber P., Van den 
Bergh H., Leisinger H-J. Clinical evaluation of a method for detecting 
superficial transitional cell carcinoma of the bladder by light induced 
fluorescence of protoporphyrin IX following topical application of 
5-aminolevulinic acid. Preliminary results. Lasers in Surgery and Medicine 
1996;ln review. 
13. Jocham D., Baumgartner R., Fuchs N., Lenz H., Stepp H., Unsold E. Die 
fluoreszenzdiagnose porphyrin-markierter utothelialer tumoren. Urologe (A) 
1989; 28:59-64. 
14. Jocham D. Photodynamische verfahren in der urologie. Urologe 1994; 
3:547-552. 
15. Kriegmair M., Baumgartner R., Hofstetter A. Intravesikale instillation 
von delta-aminolavulinsaure (ALA) - eine neue methode zur photodynamischen 
diagnostik und therapies Lasermedizin 1992; 8:83. 
16. Kriegmair M., Baumgartner R., Knuchel R., Ehsan A., Steinbach P., 
Lumper W., Hofstadter F., Hofstetter A. Photodynamische diagnose 
urothelialer neoplasien nach intravesikaler instillation von 
5-aminolavulinsaure. Urologe 1994; 33:270-275. 
17. Kriegmair M., Baumgartner R.,, Knuechel R., Steinbach P., Ehsan A., 
Lumper W., Hofstadter F., Hofstetter A. Fluorescence photodetection of 
neoplastic urothelial lesions following intravesical instillation of 
5-aminolevulinic acid. Urology 1994; 44:836-841. 
18. Kriegmair M., Baumgartner R., Ehsan A., Lumper W., Hofstetter A., 
Knuechel R., Steinbach P., Hofstadter F. Detection of early bladder cancer 
and dysplasia by fluorescence cystoscopy. J. Urology 1995; 153:457 A. 
19. Kriegmair M., Stepp H., Steinbach P., Lumper W., Ehsan A., Stepp H. G., 
Rick K., Knuchel R., Baumgartner R., Hofstetter A. Fluorescence cystoscopy 
following intravesical instillation of 5-aminolevulinic acid: a new 
procedure with high sensitivity for detection of hardly visible urothelial 
neoplasias. Urol lnt 1995;55: 190-196. 
20. Kriegmair M., Baumgartner R., Knuchel R., Stepp H., Hofstadter F., 
Hofstadter A. Detection of early bladder cancer by 5-aminolevulinic acid 
induced porphyrin fluorescence. J. Urology 1996; 155:105-110. 
21. Kriegmair M., Baumgartner R., Lumper W., Waidelich R., Hofstetter A. 
Early clinical experience with 5-aminolevulinic acid for photodynamic 
therapy of superficial bladder cancer. British Journal Urology 1996; 
Accepted for publication. 
22. Kriegmair M., Baumgartner R., Lumper W., Riesenberg R., Stocker S., 
Hofstetter A. Fluorescence cystoscopy following intravesical instillation 
of aminolevulinic acid (ALA). 204 A. 
23. Kriegmair M., Baumgartner R., Susanne S., Riesenberg R., Hofstetter A., 
Knuchel, R., Steinbach P. Photodynamic treatment of urothelial cancer 
following intravesical application of 5-aminolaevulinic acid in a rat 
bladder tumor model. J. Urology 1994, 151: 518A, abstract 1163. 
24. Kriegmair M., Lumper W., Hofstetter A., Stenzl A., Holtl L., Bartsch G. 
Photodynamic therapy of superficial bladder cancer based on intravesical 
application of 5-aminolevulinic acid. Proceedings of the American 
Urological Association 1996; 155: 566A. 
25. Kriegmair M., Stepp H., Baumgartner R., Hofstetter A., Knuchel R., 
Steinbach P., Hofstadter F. Fluorescence controlled transurethral 
resection of bladder cancer following intravesical application of 
5-aminolevulinic acid. Proceedings of the American Urological Association 
1996;155: 655A. 
26. Leveckis J., Burn J. L., Brown N. J., Reed M. W. R. Kinetics of 
endogenous protoporphyrin IX induction by aminolevulinic acid: preliminary 
studies in the bladder. J. Urology 1994; 152:550-553. 
27. Moore R. B., Miller G. G., Brown K., Bhatnagar R., Tulip J., McPhee M. 
S. Urothelial conversion of 5-aminolevulinic acid to protoporphyrin-IX 
following oral or intravesical administration. SPIE 1995; 2371:284-288. 
28. Novo M., Huttmann G., Diddens H. Chemical instability of 
5-aminolevulinic acid used in the fluorescence diagnosis of bladder 
tumours. J. Photochem. Photobiol. B: Biol. 1996; Accepted for publication. 
29. Rodriguez M., Huttmann G., Diddens H. Chemical instability of 5 
aminolevulinic acid (ALA) in aqueous solution. SPIE 1995; 2371:204-209. 
30. Thomas S., Kaspers I., Schmitt-Conrad M., Svanberg K., Diddens H., 
Huttman G., Jocham D. Photodynamic imaging of urothelial bladder cancer 
after topical instillation of 5-aminolevulinic acid (5-ALA), 5th Biennial 
Meeting of the International Photodynamic Association, September 1994, 
Amelia Island, Fla. (U.S.A.). 
31. Steinbach P., Kriegmair M., Baumgartner R. Hofstadter F. Z. Knuchel R. 
Intravesical instillation of 5-aminolevulinic acid: the fluorescent 
metabolite is limited to urothelial cells. Urology 1994: 44:676-681 
32. Steinbach P., Weingandt H., Baumgartner R., Kriegmair M., Hofstadter 
F., Knuichel R. Cellular fluorescence of the endogenous photosensitizer 
protoporphyrin IX following exposure to 5-aminolevulinic acid. Photochem. 
Photobiol. 1995;62:887-895. 
All of the above references are incorporated herein in their entireties. 
Other photodynamic therapy or photodetection uses include treatment of 
actinic keratoses, hair removal, treatment of acne and endometrial 
ablation. 
Endoscopes coupled to a light source for use in photodynamic therapy are 
sold commercially but can be especially designed for use with ALA and its 
precursors. Suitable endoscopes for use with a light source are 
commercially available, e.g. from Karl Storz Gmbh & Co., Tuttligen, 
Germany; Circon ACMI; Olympus; and Richard Wolf. Another suitable 
endoscope is described in U.S. Pat. No. 5,441,531, incorporated herein by 
reference in its entirety. 
This invention also provides for commercial kits containing sterilized ALA, 
a sterile diluent such as an aqueous buffer solution, and, optionally a 
catheter for administering the ALA solution. An endoscope coupled to a 
light source such as those described above, for use in detection or 
treatment of the cells or tissues in which the ALA preferentially 
accumulates can also be included in the kit. Preferably instructions on 
the use of the ALA are packaged with the kits. 
Reference will now be made to the following non-limiting examples. 
EXAMPLES 1-4 and COMATIVE EX. 1 
ALA hydrochloride salt was obtained from Sochinaz, S. A., Vionnez, 
Switzerland. 1.65 grams of ALA hydrochloride salt was placed into 60 ml 
glass vials. 360 vials of crystalline non-irradiated ALA were prepared. Of 
these, 225 vials were sealed under ambient conditions, and were irradiated 
with gamma radiation as shown in Table 1. 
TABLE 1 
______________________________________ 
target dose 
Example no. of vials 
conditions (KGys) color 
______________________________________ 
1 45 crystalline, 
15 yellow 
atmospheric, 
room 
2 45 same as Ex. 1 
30 yellow 
3 45 crystalline, 
15 yellow 
atmospheric, 
packed in dry ice 
4 45 same as Ex. 3 
30 yellow 
Comp. Ex. 1 
45 same as Ex. 1 
0 off white 
______________________________________ 
EXAMPLES 5-6 AND COMATIVE EX. 2 
The other 135 vials as prepared above were sealed under an argon atmosphere 
and were irradiated as shown in Table 2. 
TABLE 2 
______________________________________ 
target dose 
Example no. of vials 
conditions (KGys) color 
______________________________________ 
5 45 crystalline, argon 
15 yellow 
atmosphere, 
room temperature 
6 45 same as Ex. 5 
30 yellow 
Comp. Ex. 2 
45 same as Ex. 5 
0 off white 
______________________________________ 
EXAMPLES 7-10 AND COMATIVE EXS. 3 and 4 
Crystalline ALA was first micronized by pulverizing into fine particles a 
few .mu.m in diameter by a jet mill by Micro-Macinazione, S.A. to 
determine if further reducing the crystalline size would have any effect 
on the color of the irradiated ALA. After micronization, 1.65 grams of ALA 
hydrochloride salt was placed into 60 ml glass vials. A total of 270 vials 
of crystalline non-irradiated ALA were prepared. Of these, 135 vials were 
sealed under ambient conditions and irradiated with gamma radiation as 
shown in Table 3. The other 135 vials were sealed under an argon 
atmosphere and irradiated with gamma radiation as shown in Table 3. 
TABLE 3 
______________________________________ 
target dose 
Example no. of vials 
conditions (KGys) color 
______________________________________ 
7 45 micronized 15 yellow 
crystalline, 
atmospheric, 
room temperature 
8 45 micronized 30 yellow 
crystalline, 
atmospheric, 
room temperature 
Comp. Ex. 3 
45 micronized 0 off white 
crystalline, 
atmospheric, 
room temperature 
9 45 micronized 15 yellow 
crystalline, 
argon, room 
temperature 
10 micronized 30 yellow 
crystalline, 
argon, room 
temperature 
Comp. Ex. 4 micronized 0 off white 
crystalline, 
argon, room 
temperature 
______________________________________ 
Subsequent analysis of the type described above with respect to FIGS. 2-14, 
confirmed that there was no discernible difference in structure or 
pharmacological activity between the irradiated examples and the 
non-irradiated control comparative examples. 
The gamma irradiation also results in a darkening of the glass bottle used 
to contain the ALA. Presumably the darkening is due to impurities in the 
glass that do not affect its pharmaceutical acceptability. In fact, this 
darkening is an advantage since it shades the bottle contents from direct 
light. 
Other embodiments of the invention will be apparent to those skilled in the 
art from consideration of the specification and practice of the invention 
disclosed herein. It is intended that the specification be considered as 
exemplary only, with the true scope and spirit of the invention being 
indicated by the following claims.