UNEXPECTED ACTIVITY OF β-CYCLODEXTRIN AGAINST EXPERIMENTAL INFECTION BY ...
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1118 J. Parasitol., 86(5), 2000, p. 1118–1120 q American Society of Parasitologists 2000 UNEXPECTED ACTIVITY OF b-CYCLODEXTRIN AGAINST EXPERIMENTAL INFECTION BY CRYPTOSPORIDIUM PARVUM Jose ´ A. Castro-Hermida, Francisco Freire-Santos, A ´ ngel M. Oteiza-Lo ´ pez, and Elvira Ares-Maza ´s Laboratorio de Parasitologı ´a, Departamento de Microbiologı ´a y Parasitologı ´a, Universidad de Santiago de Compostela, 15706 Santiago de Compostela, La Corun ˜a, Espan ˜a ABSTRACT: An unexpected activity of b-cyclodextrin, an excipient used in pharmaceutical technology, was observed against Cryptosporidium parvum. The viability and infectivity of purified oocysts, exposed for 24 hr to b-cyclodextrin (2.5% suspension), were evaluated by inclusion/exclusion of 2 fluorogenic vital dyes and a suckling murine model, respectively. Results of the viability assay showed a high proportion of nonviable oocysts (81.5%). The intensity of experimental infection, determined 7 days postinoculation by examination of intestinal homogenates, was significantly lower (P , 0.05) than in the control litters. The preventive and curative efficacies of b-cyclodextrin suspension were also evaluated in experimentally infected neonatal mice. Infection was prevented when the suspension was administered 2 hr before inoculated oocysts and on days 1 and 2 postinoculation. Cryptosporidium spp. are small coccidian parasites that infect the microvillous region of epithelial cells lining the digestive and respiratory organs of vertebrates (Current and Garcı ´a, 1991). Infection with Cryptosporidium parvum causes severe, but self-limiting, diarrhea in immunocompetent hosts that is usually resolved in 15–20 days; diarrhea becomes chronic and often life-threatening in immunocompromised hosts, especially those with AIDS, who can develop severe protracted cryptos- poridial infections resulting in death (Current, 1989). Crypto- sporidium is now thought to be 1 of the 3 most common en- teropathogens causing diarrheal illness found worldwide. This parasite represents a global public health threat, having caused several massive waterborne epidemics (Rose, 1997). Its broad host range, together with its refractoriness to the most common antiparasitic agents and disinfectants, has led to its character- ization as 1 of the more important emerging diseases of the last 2 decades. Cyclodextrins are natural cyclic oligosaccharides, and some of their derivatives have attracted much attention as excipients in the last few years. Cyclodextrins have been extensively ap- plied in combination with lipophilic drugs to optimize biophar- maceutical parameters such as solubility and dissolution rate, stability, permeation across membranes, and absorption, which are often problematic for poorly soluble drugs (El-Gendi et al., 1992). Previous studies in our laboratory on the evaluation of inclusion complexes with diloxanide furoate on mouse experi- mental infections revealed anticryptosporidial activity (E. Ares- Maza ´s, unpubl. obs.). In the present study, we demonstrate in vitro and in vivo efficacies for b-cyclodextrin itself against C. parvum infection. MATERIALS AND METHODS Parasites Oocysts were collected from a naturally infected Friesian calf. Con- centration (distilled water/diethyl ether), purification (discontinuous Per- coll gradient), and quantification (Neubauer hemacytometer) of oocysts were as described previously (Lorenzo et al., 1993). Briefly, fresh fecal material was collected by rectal sampling, suspended immediately 1:1 in cold 5% (w/v) aqueous potassium dichromate solution, and stored at 4 C until purification of oocysts within 48 hr. Feces were washed through sieves (mesh size 45 mm), and the resulting fluid was centri- fuged several times at 1,000 g, for 5 min at 4 C to remove the potassium Received 27 September 1999; revised 10 January 2000 and 29 Feb- ruary 2000; accepted 29 February 2000. dichromate. The sediment was suspended in 20 ml of distilled water and 20 ml of diethyl ether, centrifuged at 1,000 g for 5 min at 4 C, and the top 3 layers were decanted. This step was repeated until the sedi- ment was free of lipids. After several washings, the sediment was sus- pended in distilled water, and 1-ml samples of the resulting suspension were then processed on a discontinuous Percoll gradient (Sigma, St. Louis, Missouri) consisting of 4 2.5-ml layers with densities of 1.13, 1.09, 1.05, and 1.01 g ml 21 and centrifuged at 650 g for 15 min. The band containing purified oocysts was removed, washed 3 times at 1,000 g for 5 min, and suspended in distilled water. The number of oocysts present in 1 ml of suspension was counted in a modified Neubauer hemacytometer after mixing 0.2 ml of suspension with 0.8 ml of mal- achite green solution (malachite green, 0.16 g; sodium dodecyl sulfate, 0.1 g; distilled water, 100 ml).The purified oocysts were employed for in vitro and in vivo assays within 7 days after collection. In vitro viability assay A viability assay for oocysts of C. parvum based on the inclusion or exclusion of 2 fluorogenic vital dyes, 49,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), was used (Campbell et al., 1992). Purified oocysts were exposed to a b-cyclodextrin (2.5%) suspension in 10 ml sterile distilled water, or water without cyclodextrin, at 20 C in a test tube and shaken (70 rpm) for 24 hr; then, the oocysts were washed twice, counted as described previously, and resuspended to 10 ml in sterile distilled water. Approximately 2 3 10 6 treated and nontreated oocysts were resuspended to 100 ml in Hanks’ balanced salt solution (HBSS) and incubated with 10 ml of DAPI working solution (2 mg/ml in absolute methanol) and 10 ml of PI working solution (1 mg/ml in 0.1 M phosphate-buffered saline [PBS], pH 7.2) at 37 C for 2 hr, the length of time optimal for producing maximal dye uptake. Oocysts were washed twice in HBSS before being examined by epifluorescence mi- croscopy. The proportion of ruptured (ghost), PI-positive (PI 1 ; nonvia- ble), DAPI-positive PI-negative (DAPI 1 PI 2 ; viable at assay), and DAPI-negative PI-negative (DAPI 2 PI 2 ; viable after further trigger) oo- cysts were quantified by enumerating more than 100 oocysts in each sample. In vivo viability assays Sixteen coccidium-free litters (6–18 mice/litter) of CD-1 Swiss mice were employed. All litters were maintained separately with their dams at 20 C in plastic cages with wire mesh tops and wood shavings for bedding. The mice received a commercial pelleted feed (Letica, Bar- celona, Spain) with water ad libitum and were maintained under hy- gienic conditions. Mice were weighed just before inoculation and again immediately prior to killing. Two litters were used in each experiment, referred to as Experiment I (mice inoculated with nontreated oocysts), Experiment II (mice inoculated with oocysts exposed to a b-cyclodex- trin 2.5% suspension for 24 hr), experiments III–V (preventive efficacies of b-cyclodextrin 2.5% suspension), and Experiments VI–VIII (curative efficacies of b-cyclodextrin 2.5% suspension). In the experiments on preventive efficacy, the b-cyclodextrin solution was administered 1 hr (Exps. III–IV) or 2 hr (Exp. V) before inoculation of mice with non- treated oocysts, and on days 1 and 2 postinoculation every 24 hr (Exp.
Transcript of UNEXPECTED ACTIVITY OF β-CYCLODEXTRIN AGAINST EXPERIMENTAL INFECTION BY ...
1118
J. Parasitol., 86(5), 2000, p. 1118–1120q American Society of Parasitologists 2000
UNEXPECTED ACTIVITY OF b-CYCLODEXTRIN AGAINST EXPERIMENTAL INFECTIONBY CRYPTOSPORIDIUM PARVUM
Jose A. Castro-Hermida, Francisco Freire-Santos, Angel M. Oteiza-Lopez, and Elvira Ares-MazasLaboratorio de Parasitologıa, Departamento de Microbiologıa y Parasitologıa, Universidad de Santiago de Compostela,15706 Santiago de Compostela, La Coruna, Espana
ABSTRACT: An unexpected activity of b-cyclodextrin, an excipient used in pharmaceutical technology, was observed againstCryptosporidium parvum. The viability and infectivity of purified oocysts, exposed for 24 hr to b-cyclodextrin (2.5% suspension),were evaluated by inclusion/exclusion of 2 fluorogenic vital dyes and a suckling murine model, respectively. Results of theviability assay showed a high proportion of nonviable oocysts (81.5%). The intensity of experimental infection, determined 7days postinoculation by examination of intestinal homogenates, was significantly lower (P , 0.05) than in the control litters.The preventive and curative efficacies of b-cyclodextrin suspension were also evaluated in experimentally infected neonatal mice.Infection was prevented when the suspension was administered 2 hr before inoculated oocysts and on days 1 and 2 postinoculation.
Cryptosporidium spp. are small coccidian parasites that infectthe microvillous region of epithelial cells lining the digestiveand respiratory organs of vertebrates (Current and Garcıa,1991). Infection with Cryptosporidium parvum causes severe,but self-limiting, diarrhea in immunocompetent hosts that isusually resolved in 15–20 days; diarrhea becomes chronic andoften life-threatening in immunocompromised hosts, especiallythose with AIDS, who can develop severe protracted cryptos-poridial infections resulting in death (Current, 1989). Crypto-sporidium is now thought to be 1 of the 3 most common en-teropathogens causing diarrheal illness found worldwide. Thisparasite represents a global public health threat, having causedseveral massive waterborne epidemics (Rose, 1997). Its broadhost range, together with its refractoriness to the most commonantiparasitic agents and disinfectants, has led to its character-ization as 1 of the more important emerging diseases of the last2 decades.
Cyclodextrins are natural cyclic oligosaccharides, and someof their derivatives have attracted much attention as excipientsin the last few years. Cyclodextrins have been extensively ap-plied in combination with lipophilic drugs to optimize biophar-maceutical parameters such as solubility and dissolution rate,stability, permeation across membranes, and absorption, whichare often problematic for poorly soluble drugs (El-Gendi et al.,1992). Previous studies in our laboratory on the evaluation ofinclusion complexes with diloxanide furoate on mouse experi-mental infections revealed anticryptosporidial activity (E. Ares-Mazas, unpubl. obs.). In the present study, we demonstrate invitro and in vivo efficacies for b-cyclodextrin itself against C.parvum infection.
MATERIALS AND METHODS
Parasites
Oocysts were collected from a naturally infected Friesian calf. Con-centration (distilled water/diethyl ether), purification (discontinuous Per-coll gradient), and quantification (Neubauer hemacytometer) of oocystswere as described previously (Lorenzo et al., 1993). Briefly, fresh fecalmaterial was collected by rectal sampling, suspended immediately 1:1in cold 5% (w/v) aqueous potassium dichromate solution, and stored at4 C until purification of oocysts within 48 hr. Feces were washedthrough sieves (mesh size 45 mm), and the resulting fluid was centri-fuged several times at 1,000 g, for 5 min at 4 C to remove the potassium
Received 27 September 1999; revised 10 January 2000 and 29 Feb-ruary 2000; accepted 29 February 2000.
dichromate. The sediment was suspended in 20 ml of distilled waterand 20 ml of diethyl ether, centrifuged at 1,000 g for 5 min at 4 C, andthe top 3 layers were decanted. This step was repeated until the sedi-ment was free of lipids. After several washings, the sediment was sus-pended in distilled water, and 1-ml samples of the resulting suspensionwere then processed on a discontinuous Percoll gradient (Sigma, St.Louis, Missouri) consisting of 4 2.5-ml layers with densities of 1.13,1.09, 1.05, and 1.01 g ml21 and centrifuged at 650 g for 15 min. Theband containing purified oocysts was removed, washed 3 times at 1,000g for 5 min, and suspended in distilled water. The number of oocystspresent in 1 ml of suspension was counted in a modified Neubauerhemacytometer after mixing 0.2 ml of suspension with 0.8 ml of mal-achite green solution (malachite green, 0.16 g; sodium dodecyl sulfate,0.1 g; distilled water, 100 ml).The purified oocysts were employed forin vitro and in vivo assays within 7 days after collection.
In vitro viability assay
A viability assay for oocysts of C. parvum based on the inclusion orexclusion of 2 fluorogenic vital dyes, 49,6-diamidino-2-phenylindole(DAPI) and propidium iodide (PI), was used (Campbell et al., 1992).Purified oocysts were exposed to a b-cyclodextrin (2.5%) suspension in10 ml sterile distilled water, or water without cyclodextrin, at 20 C ina test tube and shaken (70 rpm) for 24 hr; then, the oocysts were washedtwice, counted as described previously, and resuspended to 10 ml insterile distilled water. Approximately 2 3 106 treated and nontreatedoocysts were resuspended to 100 ml in Hanks’ balanced salt solution(HBSS) and incubated with 10 ml of DAPI working solution (2 mg/mlin absolute methanol) and 10 ml of PI working solution (1 mg/ml in0.1 M phosphate-buffered saline [PBS], pH 7.2) at 37 C for 2 hr, thelength of time optimal for producing maximal dye uptake. Oocysts werewashed twice in HBSS before being examined by epifluorescence mi-croscopy. The proportion of ruptured (ghost), PI-positive (PI1; nonvia-ble), DAPI-positive PI-negative (DAPI1 PI2; viable at assay), andDAPI-negative PI-negative (DAPI2 PI2; viable after further trigger) oo-cysts were quantified by enumerating more than 100 oocysts in eachsample.
In vivo viability assays
Sixteen coccidium-free litters (6–18 mice/litter) of CD-1 Swiss micewere employed. All litters were maintained separately with their damsat 20 C in plastic cages with wire mesh tops and wood shavings forbedding. The mice received a commercial pelleted feed (Letica, Bar-celona, Spain) with water ad libitum and were maintained under hy-gienic conditions. Mice were weighed just before inoculation and againimmediately prior to killing. Two litters were used in each experiment,referred to as Experiment I (mice inoculated with nontreated oocysts),Experiment II (mice inoculated with oocysts exposed to a b-cyclodex-trin 2.5% suspension for 24 hr), experiments III–V (preventive efficaciesof b-cyclodextrin 2.5% suspension), and Experiments VI–VIII (curativeefficacies of b-cyclodextrin 2.5% suspension). In the experiments onpreventive efficacy, the b-cyclodextrin solution was administered 1 hr(Exps. III–IV) or 2 hr (Exp. V) before inoculation of mice with non-treated oocysts, and on days 1 and 2 postinoculation every 24 hr (Exp.
CASTRO-HERMIDA ET AL.—b-CYCLODEXTRIN AND C. PARVUM 1119
TABLE I. Activity of b-cyclodextrin 2.5% suspension against Crypto-sporidium parvum infection in neonatal mice.
Exp.no.*
No. infected/no. inoculated
Infectionintensity
(mean 6 oocysts3 106) % Infected
IIIIIIIVVVIVIIVIII
11/1115/1512/1211/110/11
13/134/121/16
2.6 6 1.60.6 6 0.30.3 6 0.10.1 6 0.10.0 6 0.00.2 6 0.10.1 6 0.00.1 6 0.0
100100100100
0100
33.36.2
* Exp. I: control mice. Exp. II: mice inoculated with oocysts exposed to a 2.5%b-cyclodextrin suspension for 24 hr. Exps. III–V: preventive efficacy, the prod-ucts were administered 1 hr (exps. III–IV) or 2 hr (exp. V) before inoculationof mice with nontreated oocysts and on days 1 and 2 postinoculation every 24hr (exp. III) or 12 hr (exp. IV–V) postinfection. Exps. VI–VIII: curative efficacy,the product was administered on days 3, 4, and 5 postinoculation at 24, 12, or8 hr (exps. VI–VIII, respectively) postinfection.
III) or 12 hr (Exps. IV–V) postinfection. In the experiments on curativeefficacy, the treatment was administered on days 3, 4, and 5 postinoc-ulation at 24, 12, or 8 hr (Exps. VI–VIII, respectively) postinfection.The dose of b-cyclodextrin (2.5%) administered in both preventive andcurative treatments was 0.1 ml. In all cases, mice (3 days old) wereinoculated with 2.5 3 104 oocysts and killed 7 days postinoculation.The entire small and large intestines were removed and placed in 5 mlPBS (pH 7.2) containing penicillin (2,000 IU/ml) and gentamycin (10mg/ml), and stored at 4 C for up to 48 hr. Total intestinal oocyst contentwas evaluated following homogenization with an Ultra-Turrax homog-enizer (3 3 10 sec cycles). Oocysts were quantified in a hemacytometeras described above.
Statistical analysis
Differences in infection intensities were compared by a pairwise mul-tiple comparison procedures (Student–Newman–Keuls method), 1-wayANOVA (Sigmastat for Windows version 1.0, 1994).
RESULTS
In vitro viability assay for oocysts of C. parvum treated withb-cyclodextrin (2.5%) suspension for 24 hr versus nontreatedoocysts, based on the inclusion or exclusion of 2 fluorogenicvital dyes, showed the following results: nonviable oocysts(PI1) (81.5% vs. 19%); viables at experiment (DAPI1 PI2) oo-cysts (18% vs. 50%); and viables after further trigger (DAPI2
PI2) oocysts (0.5% vs. 31%). The results of the in vivo exper-iment revealed a prevalence of 100% in control litters, with amean total number of oocysts per animal of 2.6 6 1.6 3 106.The mean number of oocysts per animal in Experiment II wassignificantly lower (P , 0.05) than in the controls (Table I).The experiments on curative efficacy (VII–VIII) also revealeda decrease in the numbers of oocysts per mouse when b-cyclo-dextrin was administered on days 3, 4, and 5 postinoculation(Table I). In the preventive efficacy assay, in which b-cyclo-dextrin was administered 2 hr before oocyst inoculations (Exp.V), and on days 1 and 2 postinoculation, infections were erad-icated (Exp. V) (Table I). Experiments III and IV showed prev-alences of 100%, but the mean numbers of oocysts per animalwere 0.3 6 0.1 3 106 and 0.1 6 0.1 3 106, respectively.
DISCUSSION
To date, more than 120 different compounds have been testedagainst Cryptosporidium spp. without marked success, althoughover 45 preparations have reportedly demonstrated some activ-ity in vivo or in vitro (Blagburn and Soave, 1997). Most at-tempts at conventional chemotherapy with antiparasitic, anti-fungicidal, antibiotic, or antiviral agents have been unsuccess-ful. The b-cyclodextrin tested in our suckling murine modeldoes not appear to be toxic at the level used as indicated bythe lack of noticeable effects on weight gain of mice or sucklingbehavior and by the absence of increased mortality in treatedversus control mice. The results obtained in the present studyshowed that C. parvum oocysts previously exposed to b-cyclo-dextrin were infective, but the intensity of infection was sig-nificantly reduced compared with the controls; moreover, weobserved both preventive (drug administered after entry of spo-rozoites into host cell but prior to complete establishment ofthe infection) and curative (after complete establishment of theinfection) results with this excipient. Surprisingly, the infectionwas eradicated when b-cyclodextrin was administered preven-tively 2 hr before inoculated oocysts and on days 1 and 2 post-inoculation each 12 hr.
Previous studies showed that hemolysis of erythrocytes in-duced by cyclodextrins was probably due to the membrane dis-ruption elicited by the dissolution and removal of membranecomponents such as cholesterol and phospholipids (Ohtani etal., 1989; Arima et al., 1990). Only the basic lipid compositionof sporulated C. parvum oocysts has been documented (Mit-scheler et al., 1994). Of approximately 1.2 3 1029 mmol phos-pholipid per oocyst, phosphatidylcholine (PC) comprised about66%. The only sterol detected was cholesterol, at about 1.7 310210 mol per oocyst. All coccidia have a low cholesterol-to-phospholipid ratio. This, along with the dominance of short-chain fatty acids, significant amounts of unsaturated fatty acids,and high amounts of PC, should contribute significantly to highmembrane fluidity (Gallois et al., 1988). When we quantifiedthe oocysts exposed to b-cyclodextrin using malachite greensolution prior to inoculation of mice, we observed that someoocysts were stained, demonstrating an alteration in the per-meability of the C. parvum oocyst wall that serves as a barrierto the sporozoites within. On the other hand, the results of theviability assay demonstrate that oocyst walls are permeable toPI and less to DAPI after exposure to b-cyclodextrin for 24 hr.Campbell et al. (1992) considered that PI1 oocysts that havesporozoites with disrupted or broken membranes were dead.Horan and Kappler (1977) reported that only cells with dis-rupted or broken membranes can be stained with PI. We suspectthat b-cyclodextrin exposure modified oocyst wall permeability.In conclusion, the results obtained in the present study docu-ment anticryptosporidial activity of b-cyclodextrin and warrantfurther investigation.
ACKNOWLEDGMENTS
This study was supported by the Consellerıa de Educacion eOrdenacion Universitaria da Xunta de Galicia, Spain (XUGA20310B97). We thank Roquette-Laisa (Espana) for graciouslyproviding b-cyclodextrin.
1120 THE JOURNAL OF PARASITOLOGY, VOL. 86, NO. 5, OCTOBER 2000
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