Method of controlling cryptosporidium infectons using protease inhibitors

A method for controlling infections caused by Cryptosporidium parvum. The method comprises using protease inhibiting compounds, preferably serine protease inhibitors, to inhibit excystation, invasion, and parasite maturation and development. The method is directed to therapeutic treatment of mammals, such as humans, exposed to C. parvum, and additionally as a prophylactic treatment in immunocompromised subjects at high risk for contracting cryptosporidiosis.

TECHNICAL FIELD 
The present invention relates to anti-protozoal therapy generally, and more 
specifically to a method of controlling infections caused by 
Cryptosporidium parvum (C. parvum) using protease inhibitors. 
BACKGROUND ART 
C. parvum is a coccidian protozoan that infects the epithelial cells lining 
the digestive and respiratory tracts of mammals. The protozoan 
preferentially invades the epithelial cells lining the microvilli lining 
the small intestine, but all sites of the gastrointestinal tract 
(including the esophagus, stomach, colon, common bile duct, gall bladder, 
liver, rectum, and pancreatic duct) can be involved. The most common 
clinical manifestations of the resulting infection, commonly called 
cryptosporidiosis, are characterized by voluminous watery diarrhea, 
cramping abdominal pain, and weight loss. Nausea, vomiting, fatigue, 
headache, and myalgia may also be present. In immunocompetent individuals, 
the infection causes a generally self-limited diarrhea and results in 
spontaneous eradication of the parasite from the intestinal mucosa and a 
protective acquired immunity. Severe consequences of cryptosporidiosis 
however can occur in hosts with immature or deficient immune systems. 
These include young children (usually under five years old), patients 
undergoing immunosuppressive drug therapy, geriatric hosts having 
decreased immune responsiveness, and patients with congenital or acquired 
immunodeficiencies (e.g. Human Immunodeficiency Virus ("HIV") infected and 
Acquired Immunodeficiency Syndrome ("AIDS") patients). These individuals 
frequently develop a profuse, protracted diarrheal illness which 
progresses to a chronic infection of the intestinal epithelium and, 
potentially, cryptosporidial dissemination into the alveolar and tracheal 
epithelium. 
At present, notwithstanding the fact that more than 120 different compounds 
have been tested, no effective treatment for cryptosporidiosis in man or 
animals has been disclosed. Most attempts at conventional chemotherapy 
with antiparasitic, antifungal, antibiotic or antiviral agents have been 
unsuccessful. Several colostrum preparations have also been used to treat 
infections, the best results being obtained with hyperimmune bovine 
colostrum harvested from dairy cows vaccinated with C. parvum antigens. 
Treatment of protozoal gastrointestinal disorders of parasitic protozoan 
origin by administration of hyperimmune milk products is disclosed in U.S. 
Pat. No. 5,106,618 (Apr. 21, 1992) to Beck et al. Despite promising 
indications, considerable variation has been observed in the therapeutic 
efficacies of different colostrum preparations. As a result, current 
treatments center around palliative remedies in addition to treatment with 
antidiarrheal compounds and fluid and electrolyte replacement to alleviate 
the dehydration accompanying diarrheal illness. 
Maintenance of homeostasis in tissues requires regulation of proteases 
(proteolytic enzymes) by endogenous protease inhibitors, which form 
complexes with and achieve selective inactivation of the proteases. 
Several classes of protease inhibitors, found primarily in blood plasma, 
exist. One family of such endogenous protease inhibitors, the "serine 
protease inhibitors" ("serpins"), is comprised of a number of structurally 
similar proteins. The reactive center of a serpin molecule consists of a 
reactive site loop structure that binds to the active site of a cognate 
target enzyme. The resulting serpin:enzyme complex is covalently 
stabilized as a 1:1 molecular complex under physiological conditions. 
Synthetic, non-protein based inhibitors are also known and are thought to 
interact with their target proteases to achieve the same effect. 
Protease activity has been described in the invasion, infectivity and 
pathogenicity of several parasites having structural and invasive 
characteristics similar to cryptosporidiosis. Protease-mediated events 
reportedly facilitate the process of host cell attachment in Eimeria 
vermiformis and Trichomonas vaginalis infections. Adams, J. H. & G. R. 
Bushell, International Journal for Parasitology, 18:683-685 (1988); 
Arroyo, R. & Aldrete J., Infection and Immunity, 57:2991-2997 (1989). 
Biochemical properties of sporozoans, such as Eimeria tenella and 
Plasmodium knowlesi, which appear to include one or more 
protease-dependent mediated steps in the host cell adhesion and invasion 
process have likewise been described. Fuller, A. & McDougald, L., Journal 
of Parasitology, 76:464-467 (1990); Hadley et al., Experimental 
Parasitology, 55:306-311 (1983). Proteinases are also known to affect the 
maturation and development stages of Leshmania, Trypanosoma, and Giardia. 
McKerrow et al., Annual Reviews in Microbiology, 47:821-853 (1993). 
Additionally, protease-mediated events that facilitate tissue migration, 
that degrade host proteins for subsequent use as a source of nutrition, 
and that provide a potential mechanism for parasites to elude host immune 
responses have been described in previous publications. 
More specifically, several investigators have reported a putative role for 
surface expressed glycoproteins or lectin-associated membrane affinity to 
aid in the initial attachment of C. parvum sporozoites to host cells. 
Bonnin et al., Parasitology 103:171-177 (1991); Mitschler et al., Journal 
of Eukaryotic Microbiology 41:8-12 (1994); Petersen, C., Clinical 
Infectious Diseases 15:903-909 (1992); Tilley, M. & Upton, S., FEMS 
Microbiology Letters 120:275-278 (1994). It is believed that the initial 
adherence of sporozoites to host cell membranes may facilitate the spatial 
orientation of parasite proteases that further enhance cell attachment 
and, potentially, contribute to the parasite's unique intramembranous 
location within infected cells. 
None of these aforementioned references and publications, however, is 
believed to disclose or suggest the use of protease inhibitors as a method 
of/for controlling or preventing infections caused C. parvum. 
DISCLOSURE OF THE INVENTION 
The present invention relates to a method of controlling or preventing 
infections caused by C. parvum using protease inhibitors. Preferably, a 
method of treating or preventing infections caused by C. parvum using 
serine protease inhibitors. Even more preferably, the present invention 
relates to a method of controlling C. parvum infections using 
pharmaceutically effective amounts of alpha-1-antitrypsin, antipain, 
aprotinin, leupeptin, soybean trypsin inhibitor, phenylmethylsulfonyl 
flouride, or combinations thereof. 
BEST MODE OF THE INVENTION 
The invention comprises administration of a protease-inhibiting compound 
(or compounds) to control infections caused by C. parvum. Administration 
of protease-inhibiting compounds are believed to inhibit various 
protease-dependent mechanisms, such as, parasite attachment, parasite 
nutrition, excystation, parasite invasion, and parasite maturation and 
development. As will be apparent from the hereinafter described Examples, 
protease inhibitors administered as described herein cause a significant 
reduction in the infectivity of C. parvum. 
The term "animal" is intended to mean, for the purpose of this invention, 
any living creature including mammals (e.g. humans, domestic animals, farm 
animals, and wild animals). The term "gastrointestinal disorder" as used 
herein means infections relating to the stomach, intestine, gall bladder, 
and/or biliary tract of a mammal that result in a disturbance of the same 
in terms of function, structure, or both. 
The terms "treating" and "treatment" are intended to mean the amelioration 
or complete elimination of the symptoms of the disorder and/or the 
pathogenic origin of the disorder, as part of therapeutic or prophylactic 
therapy. The term "administer" is intended to mean that any method of 
treating a subject with a substance, such as orally, intravenously, 
intramuscularly, subcutaneously, topically, rectally, or via inhalation 
therapy. 
The protease inhibitors of the present invention can be used in the 
therapeutic and prophylactic treatment of cryptosporidiosis in 
immunocompromised animals. The most severe consequence of C. parvum 
infection occurs in patients with human immunodeficiency virus where 
current medical intervention is commonly limited to the palliative 
treatment of symptoms such as diarrhea, cramping abdominal pain, weight 
loss, nausea, vomiting, fatigue, headache, and myalgia. Thus, prophylactic 
treatment of these immunocompromised patients can be used to avoid 
substantial morbidity. Patients undergoing immunosuppressive therapy and 
young children with insufficiently developed immune systems may likewise 
benefit from treatment with protease inhibitors to prevent, control, or 
eradicate infections caused by C. parvum. Further, protease inhibitors may 
be used to treat immunocompetent subjects who are exposed to C. parvum for 
the purpose of therapeutically treating or preventing the infection. 
The protease inhibitors of this invention may be used in conjunction with 
conventional anti-infective agents, antimicrobial agents, 
immunomodulators, and pharmaceutical preparations indicated for the 
treatment of symptoms associated with cryptosporidiosis. It is believed 
that the aforementioned medicinal agents may interact synergistically with 
the inhibitors of this invention, particularly in the treatment of 
immunocompromised patients that may otherwise succumb to this 
opportunistic infection and its morbid consequences. 
The protease inhibitors to be administered according to the method of the 
present invention include, but are not limited to, bases and 
pharmaceutically acceptable salts (e.g. acid addition salts) thereof. 
Examples of pharmaceutically acceptable acid addition salts include salts 
of inorganic acids such as sulfuric, nitric, phosphoric, and hydrochloric 
acid, as well as organic acids such as acetic, propionic, succinic, 
fumaric, maleic, citric, tartaric, cinnamic, lactic, mandelic, and 
ethanedisulfonic acid. The salts may be made by reacting the free base of 
the particular protease inhibitor with the chosen acid in a stoichiometric 
ratio in an appropriate solvent. The salts may be used, for example, in 
the preparation of oral and injectable formulations containing protease 
inhibitors as an active ingredient. 
Oral and injectable formulations to be used according to the method of the 
present invention may also be microencapsulated or otherwise incorporated 
into a pharmaceutical dosage form in order to protect the protease 
inhibitors from unwanted biodegradation processes or to create a 
sustained-release effect. Oral preparations may also be enteric coated to 
protect the protease inhibitors from acids and enzymes created in portions 
of the gastrointestinal tract. 
In another form of this invention there is provided a method for the 
treatment of infections with C. parvum in a mammal which comprises 
administering to the mammal a therapeutically effective amount of a serine 
protease inhibitor. The serine protease inhibitors target a specific 
biochemical component of the parasite in one or more of its developmental 
stages. Thus, the anticryptosporidial potential can be tailored to 
recognize proteolytic enzymes that are specific for the invading pathogen 
or that affect specific functional and developmental stages in the 
invading parasite's cycle. This course of action has proven clinically 
efficacious with regard to treatment of infection with HIV as well as the 
protozoan parasite Plasmodium species (i.e., etiologic agent of malaria). 
A preferred serine protease inhibitor to be used in accordance with the 
present invention is alpha-1-antitrypsin ("AAT"). AAT used is a human 
alpha-1-antitrypsin purified from plasma (available in research-grade 
purity from Chemicon International, Inc., Temecula, Calif.). A 
pharmaceutical preparation of human AAT proteinase inhibitor, manufactured 
by Miles, Inc. under the trademark PROLASTIN, is commercially available 
and has been approved by the Food and Drug Administration for use as a 
supplement for human patients with congenital AAT deficiencies. Other 
preferred serine protease inhibitors to be used include aprotinin 
(available from ICN Biochemicals, Inc., Aurora, Ohio), soybean trypsin 
inhibitor ("SBTI") (available from Calbiochem-Novabiochem Corp., La Jolla, 
Calif.), and phenylmethylsulfonyl flouride ("PMSF") (available from Sigma 
Chemical Co., St. Louis, Mo.). Other serine protease inhibitors, such as 
methoxysuccinyl-ala-ala-pro-valine chloromethylketone ("MAAPVCK"), can 
also be used, but may require higher concentrations for adequate protease 
inhibition. Pharmaceutically or therapeutically effective amounts of these 
compounds are already know or can be determined by one of skill in the 
art, but are generally amounts sufficient to interfere with the 
reproduction and/or infectivity of C. parvum). 
In yet another form of the invention, a method is provided for the 
treatment of infections with C. parvum in animals which comprises 
administering to an animal a therapeutically effective amount of an agent 
that inhibits both cysteine protease and serine protease. The 
cysteine/serine protease inhibitors which may be used in accordance with 
the invention include leupeptin (available from Calbiochem-Novabiochem 
Corp., La Jolla, Calif.), 
L-trans-epoxysuccinyl-leucylamindo-(4-guanidino)-butane ("E-64"), and/or 
antipain (available from ICN Biochemicals, Inc., Aurora, Ohio). 
Preferred protease inhibitors to be orally administered include those that 
are not protein-based such as antipain, MAAPVCK, and PMSF. These 
non-protein protease inhibitors are less susceptible to proteolysis by 
non-target proteinases which are found primarily in the stomach. 
Administration of pH-sensitive protease inhibitors can be accomplished by 
employing several methods to reduce acid hydrolysis in the stomach. These 
methods include the use of enteric coating, microencapsulation, lipid 
encapsulation, or administration of a buffer agent (e.g., with antacids) 
prior to, or concomitantly with, the administration of a formulation 
containing the protease inhibitor. 
The invention also includes a method of making pharmaceutical dosage forms 
containing a protease inhibitor for use in treating or preventing 
cryptosporidiosis.

Having now generally described the invention, the same will be further 
described by reference to certain specific examples which are provided 
herein for purposes of illustration only and which are not intended as 
limiting. 
EXAMPLES 
Example I 
Preparation of C. parvum in Cell Culture 
C. parvum (bovine isolate) oocysts used in this study were originally 
donated by the U.S. Department of Agriculture, Ames, Iowa. The oocysts 
were isolated from calf manure, preserved in 2.5% potassium dichromate, 
and used within 3 months of purification. Oocysts were decontaminated by 
suspension in 20% (vol/vol) 1.05% sodium hypochlorite on ice for 20 
minutes. Oocysts were then washed three times in Hanks' balanced salt 
solution ("HIBSS") and once in RPMI 1640 (available from Sigma, St. Louis, 
Mo.). Release of sporozoites was achieved by incubating oocysts in an 
excystation solution consisting of 0.25% (wt/vol) trypsin (available from 
Sigma, St. Louis, Mo.) and 0.75% (wt/vol) taurodeoxycholic acid (available 
from Sigma) in HBSS. The resulting suspension was incubated at 37.degree. 
C. for 45 minutes and microscopically observed to confirm sporozoite 
release. Sporozoites were completely separated from intact oocysts and 
oocyst walls by passage through sterile polycarbonate filters (3 micron 
pore size, available from Millipore Corp., Bedford, Mass.) twice prior to 
inoculation of bovine fallopian tube epithelial ("BFTE") cell monolayers. 
Primary BFTE cell cultures were prepared from bovine fallopian tubes (FT). 
Fat was trimmed from the serosal surfaces, mucus was gently squeezed from 
the lumens, and FT were decontaminated by being submerged in 70% ethanol 
for 30 seconds. The FT were then transferred to sterile culture petri 
dishes containing HBSS and washed twice. The BFTE cells were harvested 
either by flushing the FT with HBSS, using a 10 ml syringe equipped with a 
mouse feeding needle, or by opening the FT lengthwise with scissors and 
scraping the mucosal surfaces. The BFTE cells were subsequently washed in 
HBSS by centrifugation at 200.times.g (force of gravity) for 10 minutes, 
planted in 75-cm.sup.2 flasks containing RPMI 1640 and cultured in a 5% 
CO.sub.2 incubator at 37.degree. C. for 72 to 120 hours. When the cell 
lines reached confluency, they were trypsinized, split, and planted onto 
cover slips positioned on the bottoms of individual wells in 24-well 
tissue culture plates. When cells reached 80% confluency, they were 
inoculated with either 10.sup.5 oocysts or 4.times.10.sup.5 sporozoites 
per well. Plates were then maintained at 37.degree. C. in a candle jar 
environment (17% O.sub.2, 3% CO.sub.2, 80% N.sub.2). Growth medium was 
changed in each well every 72 hours. In wells inoculated with oocysts, the 
medium was first changed at 24 hours to remove any unexcysted oocysts. 
Coverslips were removed at 5, 24, 48, 72, 96, and 120 hours from the 
inoculated wells containing monolayers of BFTE cells. The coverslips were 
washed in RPMI 1640, fixed in 100% methanol for 10 minutes, stained with 
Giemsa stain for 1 hour, and washed three times with double-distilled 
water (ddH.sub.2 O). Coverslips were then mounted on glass slides and 
examined under oil immersion (1000X), using bright-field microscopy. 
Parasites were enumerated by counting all developmental stages of C. 
parvum present in a single scan (67 fields) through the center of each 
coverslip. The data were statistically compared for significance, using 
analysis of variance (Fischer's protected least-significant-difference 
test using a "STATVIEW" statistical analysis application developed by 
Abacus Concepts, Inc., Berkeley, Calif.). 
Successful infections were observed in BFTE cells inoculated with both 
oocysts and purified sporozoites. Parasites developed at the microvillous 
surface of BFTE cells in an intracellular but extracytoplasmic location. 
Significantly, multiple infections were common in individual cells, 
similar to those frequently observed in vivo. 
To confirm the production of infective oocysts in cell culture, an 
immunosuppressed mouse model for cryptosporidiosis was used. Three groups 
of adult female C57BL/6N mice (6 weeks of age, each weighing 14 to 16 
grams, purchased from Taconic, Germantown, N.Y.) were immunosuppressed 
with dexamethasone phosphate (available from Sigma, St. Louis, Mo.) 
provided in drinking water at a dosage level of 12 .mu.g/ml. At 120 hours 
post-inoculation, coverslips from individual 24-well plates were 
collected, and the surfaces were scraped and pooled for each plate. All 
mice in a group were gavaged on day 7 immunosuppression with an equal 
volume of the resulting cells, cell products, and parasites (plate 
product) harvested from a single plate. Group 1 (four mice) and group 2 
(five mice) received the plate product from plates inoculated with oocysts 
and sporozoites, respectively. Group 3 (five mice) was treated the same as 
group 1 except that the plate product was first suspended in 70% ethanol 
for 10 minutes to kill all stages of C. parvum except the oocysts. Fecal 
samples were collected from recta each day from mice in all groups and 
monitored for oocyst shedding, using oocyst-specific monoclonal 
antibody-based indirect immunofluorescence assay. Oocysts produced in BFTE 
cell culture were infective for immunosuppressed adult female mice. Also 
tested were 2 additional groups infected with non-cell culture derived 
oocysts. For further details on the complete experiment, see Yang et al., 
Infection and Immunity 64:349-354 (January 1996), the contents of which 
are incorporated by this reference. 
Example II 
Anticryptosporidial Effect of Alpha-1-Antitrypsin 
The anticryptosporidial potential of the protease inhibitor AAT was 
evaluated in a BFTE cell culture system inoculated with C. parvum oocysts 
in accordance with the methods detailed in Example I. AAT concentrations 
of 5, 10, 50 100, and 500 micrograms per milliliter (".mu.g/ml") in RPMI 
medium were mixed with C. parvum oocysts and used to inoculate BFTE cell 
monolayers. At 24 hours post-inoculation, the BFTE cells were rinsed with 
RPMI medium, fixed in methanol, and stained with Giemsa. Parasites were 
enumerated in cell monolayers by brightfield microscopy. Results, as set 
out in Table 1, represent the mean number of parasites counted per 
treatment group expressed as a percent, plus or minus the standard 
deviation, of the mean number of parasites counted in the infection 
control group. 
TABLE 1 
______________________________________ 
AAT Concentration (.mu.g/ml) 
Control Percent Survival 
______________________________________ 
0 (Control Group) 100% 
5 49.1 +/- 3.8% 
10 43.9 +/- 4.2% 
50 42.7 +/- 1.6% 
100 30.4 +/- 1.9% 
500 14.7 +/- 6.3% 
1000 1.8 +/- 0.4% 
______________________________________ 
These results show a concentration-dependent reduction in the number of 
surviving parasites, compared to infection control mean populations, 
following treatment with AAT. Significant reduction (P&lt;0.001) in parasite 
numbers were shown at concentrations as low as 5-10 .mu.g/ml. Not 
surprisingly, the most dramatic reduction in surviving parasite numbers 
were obtained with an AAT concentration of 1000 .mu.g/ml. During 
microscopic enumeration of parasites in BFTE cell monolayers treated with 
AAT, intact (i.e. unexcysted) oocysts were frequently observed. This 
observation was predominantly associated with AAT concentrations exceeding 
100 .mu.g/ml. There was no evidence of cytotoxicity for BFTE cells at the 
AAT concentrations evaluated in this study. 
Example III 
Anticryptosporidial Effect of ANTIPAIN 
The anticryptosporidial potential of the protease inhibitor ANTIPAIN was 
evaluated in a BFTE cell culture system inoculated with C. parvum oocysts 
utilizing the same methods and protease inhibitor concentrations as in 
Example II. Trial and data results are summarized in Table 2. 
TABLE 2 
______________________________________ 
ANTIPAIN Concentration (.mu.g/ml) 
Control Percent Survival 
______________________________________ 
0 (Control Group) 100 
5 67.7 +/- 7.2 
10 53.1 +/- 5.8 
50 42.1 +/- 6.1 
100 20.9 +/- 2.2 
500 15.6 +/- 1.4 
1000 6.6 +/- 1.4 
______________________________________ 
These results show a concentration-dependent reduction in the number of 
surviving parasites, compared to infection control mean populations, 
following treatment with ANTIPAIN. Significant reduction (P&lt;0.001) in 
parasite numbers were seen at concentrations as low as 5-10 .mu.g/ml, with 
the most dramatic reduction in surviving parasite numbers being obtained 
with an ANTIPAIN concentration of 1000 .mu.g/ml. During microscopic 
enumeration of parasites in BFTE cell monolayers treated with ANTIPAIN, 
intact (i.e. unexcysted) oocysts were frequently observed. This 
observation was predominantly associated with ANTIPAIN concentrations 
exceeding 100 .mu.g/ml. There was no evidence of cytotoxicity for BFTE 
cells at the ANTIPAIN concentrations evaluated in this study. 
Example IV 
Anticryptosporidial Effect of APROTININ 
The anticryptosporidial potential of the protease inhibitor APROTININ was 
evaluated in a BFTE cell culture system inoculated with C. parvum oocysts 
utilizing the same methods and protease inhbitor concentrations as in 
Example II. Trial and data results are summarized in Table 3. 
TABLE 3 
______________________________________ 
APROTININ Concentration (.mu.g/ml) 
Control Percent 
______________________________________ 
Survival 
0 (Control Group) 100 
5 83.9 +/- 6.6 
10 54.2 +/- 6.0 
50 36.1 +/- 4.7 
100 16.8 +/- 5.1 
500 10.9 +/- 4.7 
1000 9.4 +/- 1.8 
______________________________________ 
These results show a concentration-dependent reduction in the number of 
surviving parasites, compared to infection control mean populations, 
following treatment with APROTININ. As seen in previous examples, 
significant reduction (P&lt;0.001) in parasite numbers were shown at 
concentrations as low as 5-10 .mu.g/ml, with the greatest reduction in 
surviving parasite numbers were obtained with an APROTININ concentration 
of 1000 .mu.g/ml. During microscopic enumeration of parasites in BFTE cell 
monolayers treated with APROTININ, intact oocysts were frequently 
observed. This observation was predominantly associated with APROTININ 
concentrations exceeding 100 .mu.g/ml. There was no evidence of 
cytotoxicity for BFTE cells at the APROTININ concentrations evaluated in 
this study. 
Example V 
Anticryptosporidial Effect of Leupeptin 
The anticryptosporidial potential of the protease inhibitor Leupeptin was 
evaluated in a BFTE cell culture system inoculated with C. parvum oocysts 
utilizing the same methods and protease inhibitor concentrations as in 
Example II, except that a 1000 .mu.g/ml concentration was not included. 
Trial and data results are summarized in Table 4. 
TABLE 4 
______________________________________ 
Leupeptin Concentration (.mu.g/ml) 
Control Percent Survival 
______________________________________ 
0 (Control Group) 100 
5 89.1 +/- 5.4 
10 64.8 +/- 12.0 
50 58.7 +/- 11.2 
100 51.9 +/- 4.6 
500 43.4 +/- 6.6 
______________________________________ 
These results show a concentration-dependent reduction in the number of 
surviving parasites, compared to infection control mean populations, 
following treatment with Leupeptin. The number of parasites were 
significantly reduced (P&lt;0.01) following treatment with Leupeptin at 
concentrations of 10 .mu.g/ml and 50 .mu.g/ml. Greater reductions of 
surviving parasites were observed at Leupeptin concentrations of 100 
.mu.g/ml and 500 .mu.g/ml. 
Example VI 
Anticryptosporidial Effect of Soybean Trypsin Inhibitor 
The anticryptosporidial potential of the protease inhibitor SBTI was 
evaluated in a BFTE cell culture system inoculated with C. parvum oocysts 
utilizing the same methods and protease inhibitor concentrations as in 
Example II, except that a 1000 .mu.g/ml concentration was not included. 
Trial and data results are summarized in Table 5. 
TABLE 5 
______________________________________ 
SBTI Concentration (.mu.g/ml) 
Control Percent Survival 
______________________________________ 
0 (Control Group) 100 
5 89.1 +/- 5.4 
10 64.8 +/- 12.0 
50 58.7 +/- 11.2 
100 51.9 +/- 4.6 
500 43.4 +/- 6.6 
______________________________________ 
These results show a concentration-dependent reduction in the number of 
surviving parasites, compared to infection control mean populations, 
following treatment with SBTI. The number of parasites were significantly 
reduced (P&lt;0.01) following treatment with SBTI at concentrations of 50 
.mu.g/ml and 100 .mu.g/ml. A greater reduction of surviving parasites was 
observed at an SBTI concentration of 500 .mu.g/ml. 
Example VII 
Anticryptosporidial Effect of Phenylmethylsulfonyl Flouride 
The anticryptosporidial potential of the protease inhibitor PMSF was 
evaluated in a BFTE cell culture system inoculated with C. parvum oocysts 
in accordance with the methods detailed in Example I. PMSF concentrations 
of 1, 2, and 3 mM in RPMI medium were mixed with C. parvum oocysts and 
used to inoculate BFTE cell monolayers. The same methods of preparation 
and enumeration were used as in Example II. Trial and data results are 
summarized in Table 6. 
TABLE 6 
______________________________________ 
PMSF Concentration (mM) 
Control Percent Survival 
______________________________________ 
0 (Control Group) 100 
1 68.9 +/- 5.2 
2 55.9 +/- 4.7 
3 40.0 +/- 8.3 
______________________________________ 
These results show a concentration-dependent reduction in the number of 
surviving parasites, compared to infection control mean populations, 
following treatment with PMSF. The number of parasites were significantly 
reduced (P&lt;0.001) following treatment with PMSF at a concentration of 3 
mM. Significantly greater reduction in surviving parasites was observed at 
PMSF concentrations of 2 mM and 3 mM. 
Example VIII 
Anticryptosporidial Effect of MAAPVCK 
The anticryptosporidial potential of the protease inhibitor MAAPVCK was 
evaluated in a BFTE cell culture system inoculated with C. parvum oocysts 
utilizing the same methods and protease inhibitor concentrations as in 
Example II, except that a 1000 .mu.g/ml concentration was not included. 
Trial and data results are summarized in Table 7. 
TABLE 7 
______________________________________ 
MAAPVCK Concentration (.mu.g/ml) 
Control Percent Survival 
______________________________________ 
0 (Control Group) 100 
5 89.1 +/- 5.4 
10 64.8 +/- 12.0 
50 58.7 +/- 11.2 
100 51.9 +/- 4.6 
500 43.4 +/- 6.6 
______________________________________ 
These results show a concentration-dependent reduction in the number of 
surviving parasites, compared to infection control mean populations, 
following treatment with MAAPVCK. The number of parasites were 
significantly reduced (P&lt;0.01) following treatment with MAAPVCK at a 
concentration of 500 .mu.g/ml. 
Example IX 
Combined in vitro anticryptosporidial activities of AAT and paromomycin 
The anticryptosporidial potential of the protease inhibitor AAT in 
combination with the aminoglycoside paromomycin was evaluated in BFTE cell 
cultures. Each compound was tested at two different concentrations; AAT 
was evaluated at 250 .mu.g/ml and at 500 .mu.g/ml in combination with 
either 400 .mu.g/ml or 1200 .mu.g/ml of paromomycin. Purified oocysts 
(10.sup.5) were mixed with various combinations of the two compounds and 
1.0 ml of the resulting mixtures were used to inoculate confluent 
monolayers of BFTE cells on glass coverslips. Inoculated BFTE cells were 
maintained in a candle-jar environment at 37.degree. C. for 24 hours. 
After 24 hours incubation, inoculated cell monolayers were rinsed with 
sterile RPMI 1640 medium to remove residual inoculum and either collected 
(24 hour treatment group) or incubated at 37.degree. C. for an additional 
24 hours (48 hour treatment group) or 48 hours (72 hour treatment group). 
The remaining steps of the inoculation procedure were conducted in 
accordance with the methods detailed in Example I. Trial and data results 
are summarized in Table 8. 
TABLE 8 
______________________________________ 
Paromomycin 
AAT Conc. (.mu.g/ml) 
Conc. (.mu.g/ml) 
Control Percent Survival 
______________________________________ 
0 (Control Group) 
0 (Control Group) 
100 
250 400 19.6 (@ 24 hrs) 
34.8 (@ 48 hrs) 
29.5 (@ 72 hrs) 
500 400 6.3 (@ 24 hrs) 
10.9 (@ 48 hrs) 
8.2 (@ 72 hrs) 
250 1200 8.0 (@ 24 hrs) 
4.3 (@ 48 hrs) 
1.6 (@ 72 hrs) 
500 1200 3.6 (@ 24 hrs) 
4.3 (@ 48 hrs) 
3.3 (@ 72 hrs) 
______________________________________ 
The number of parasites counted in a single scan across the diameter of a 
coverslip was significantly reduced (P&lt;0.01) by each combination of AAT 
and paromomycin evaluated. The treatment group consisting of the highest 
concentrations of the two compounds (500 .mu.g/ml AAT and 1200 .mu.g/ml 
paromomycin) had the greatest quantitative effect on C. parvum infection 
for the 24 and 48 hour treatment groups evaluated. 
Although the invention has been described in detail with respect to use of 
specific protease inhibitors, methods of treating infections caused by C. 
parvum, it should be realized that certain modifications can be made 
within the scope and spirit of the invention by those skilled in the art.