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Erythrocyte drug carriers for babesiosis chemotherapy
Journal
of Controlled
Elsevier
Science
Release, 8 (1988)
Publishers
ERYTHROCYTE
79-83
B.V., Amsterdam
79 -
Printed
DRUG CARRIERS
in The Netherlands
FOR BABESIOSIS
CHEMOTHERAPY*,**
G. Gale Wagner*** Department Station,
of Veterinary
TX 77843
Microbiology
and Parasitology,
College of Veterinary Medicine,
Texas A&M University,
College
(U.S. A.)
Donald E. Corrier and John R. DeLoach L/n/ted States Department Laboratory,
of Agriculture,
College Station,
TX 77840
Agricultural
Research Service,
Veterinary
Toxicology
and Entomology
Research
(U.S.A.)
Imidocarb was encapsulated in murine erythrocytes with a dialysis technique. Carrier erythrocytes were injected intraperitoneally in groups of mice at 2 and at 0.2 mg/kg of body weight dosage. Control groups received free (unencapsulated) drug at the same dose rates mixed with a number of untreated erythrocytes corresponding to the above groups. About 2% of the drug-loaded erythrocytes were detected in the circulation after 24 hours and were still in the circulation after 5 days. The concentration of free drug declined to undetectable levels in both the peritoneum and the circulation before 24 hours. The biological effectiveness of the encapsulated imidocarb was evaluated in mice infected with lo7 Babesia microti infected erythrocytes from donor mice and treated 24 hours later with either encapsulated or free imidocarb at the dosages described above. Intraperitoneal injection of drug-loaded carrier cells at a dose rate of 0.2 mg/kg resulted in a decrease in parasitemia to levels similar to those observed in mice treated with free drug at a 2 mg/kg dosage.
INTRODUCTION
Bovine babesiasis, caused by the protozoan parasite Babesia spp., continues as a major threat to livestock industries throughout the world. Because the parasite is transmitted only through the bite of a tick, the disease can be *Paper presented
at the 14th International
Symposium
on
Controlled Release of Bioactive Materials, August Z-5,1987, Toronto, Ontario, Canada. **This report is based upon work supported by the Texas Agricultural Experiment Station. Any opinions, findings, conclusions or recommendations expressed in this abstract and presentation are those of the authors and do not necessarily reflect the view of the U.S. Department Agriculture. ***To whom correspondence should be addressed.
0168.3659/88/$03.50
0 1988 Elsevier
Science
of
Publishers
readily eradicated by a vigorous tick control program. However, in many areas of the world, effective control programs are difficult and expensive to organize and maintain. When such programs are not well managed, disease incidence increases and both chemotherapy and chemoprophylaxis become reasonable disease control alternatives. Babesiosis can be relatively easily treated if the symptoms are recognized early and chemotherapy initiated. Several babesicidal drugs have been used widelv” for more than two decades. One drue_ in particular, imidocarb (3,3’ - bis- (2.imidazolin2-yl ) carbanilide dihydrochloride ), has marked therapeutic and prophylactic properties [ 11. However, the use of imidocarb is limited be-
B.V.
cause of a narrow toxicity range and it,s persistence in tissues [ 11. We have demonstrated the use of erythrocytes as cellular carriers for several drugs (including imidocarb) t,hat might be useful in the chemotherapy of hemotropic infections of cattle [ 2-41. The earlier work suggested that such carrier cells effectively lower the amount of drug needed by virtue of it,s encapsulation, probably by prolonging the pharmacological lifetime of the drug. Thus, carrier erythrocytes seem to improve drug efficacy. We have also determined that erythrocyte carriers can be effecstudied in tively the mouse, thereby considerably reducing the cost of encapsulated drug testing in experimental animals [ 51. Since Babesia microti infections of mice are being used to model the bovine infection, the current studies were conducted to confirm that imidocarb is effective in murine erythrocyte carriers and t,o determine if t.he encapsulated drug is efficacious at less than the recommended dose.
MATERIALS
AND METHODS
Swiss ICR and BALB/c mice, 4 to 6 months old and weighing 30-35 g, were used. Whole blood was collected in heparinized t,ubes from the retroorbital sinus of normal mice and pooled. The red blood cells (RBC) were washed free of plasma and dialyzed to reduce the osmotic pressure to approximately 130 mOsm/mg. Afterward [“Cl imidocarb dipropionate (Burroughs Wellcome, Research Triangle Park, North Carolina) and [“Hlinulin (New England Nuclear, Boston, Massachusetts ) were mixed with the dialyzed RBC and the cells restored to isotonicity by the addition of NaCl [ 51. Unbound imidocarb and inulin were removed by dialysis against isotonic NaCl. Encapsulated drug and inulin were determined by assaying lo-50 ~1 aliquots of RBC for radioactivity. For comparative purposes, imidocarb compatibility wit,h murine RBC and the in. uiuo survival characteristics of carrier RBC were assessed as pre-
TABLE
1
Percentage
of
carrier
murine
[ ’ ‘C ] imidocarb
and
[“H ] inulin labeled RBC detected 24 hours after intraperitoneal injection Treatment
Encapsulated [ ‘H] inulin [ ’ ‘C ] imidocarb Free [,‘H]inulin [ ’ ‘C ] imidocarb
Peritoneal lavage
Whole blood
4 0.8
20.8 ‘2.2
0.8 0.8
0.6 0.6
viously described [ 21. Various preparations were also evaluated by scanning electron microscopy using methods previously described
[51. Mice (12-20 per group) were randomly allotted into 6 groups, designated as nontreated controls, normal RBC controls, and treatment groups consisting of free drug at 2 mg/kg body weight and at 0.2 mg/kg, and encapsulated drug at 2 mg/kg and at 0.2 mg/kg. The normal RBC, the free drug and the encapsulated imidocarb were all administered to the respective groups by intraperitoneal injection in a volume of 0.44 ml. All mice in each group were infected with B. microti on day 0 by the intraperitoneal inoculation of 0.1 ml of inoculum containing about 1.5 x lo7 B. microti. The normal RBC and drug, both free and encapsulated, were injected intraperitoneally 24 hours later, on day 1. Babe& infections were monitored by examination of Giemsa stained thin blood films prepared from tail blood at selected intervals after exposure to the parasite. The mean parasitemia was determined and compared between the treatment groups.
RESULTS Imidocarb was found to be compatible with mouse erythrocytes in the same way as previous
81
75
. Control o Free
drug,
2 mg
a Encapsulated.
2 mg
60 .m E .z
45
2 2 a 30
15
Days
Fig. 1. Babesia microti parasitemias 2 mg/kg or control RBC.
following infection
post
infection
and treatment
of mice with either encapsulated
or free imidocarb
at
s Control o Free
Drug,
0.2
A Encapsulated,
7
Days
Fig. 2. Babesia microti parasitemias 0.2 mg/kg or control RBC.
following infection
9
post
and treatment
11
mg 0.2
13
mg
15
infection
of mice with either encapsulated
or free imidocarb at
82
work with bovine cells [ 21. Imidocarb encapsulation increased proportionally in the concentration range of 0.1 to 2 mg/ml. No apparent effects on cell parameters such as cell volume, cell number and percentage encapsulation were seen within the concentration range studied. Moreover, up to 2 mg/ml external concentration of the drug did not affect the encapsulation of quality control markers such as [“HI inulin. The disappearance of drug-loaded RBC from the peritoneal cavity and the circulating survival of cells is summarized in Table 1. Less than 1% of either the free imidocarb or inulin could be detected in either the peritoneal cavity or circulating whole blood 24 hours after injection. Less than 1% of the encapsulated imidocarb and 4% of the encapsulated inulin was recovered from the peritoneal cavity after 24 hours. When whole blood was assayed, approximately 2.2% of the encapsulated imidocarb and 20.8% of the encapsulated inulin were detected 24 hours after injection. When carrier erythrocytes were prepared in the presence of varying concentrations of imidocarb, fixed and examined by scanning electron microscopy, pathomorphological cell shapes were readily observed at the 1 mg/ml and 2 mg/ml concentrations. Carrier cells appeared as stomatocytes, echinocytes and a few cells had large holes in their membranes. In contrast, micrographs of carrier erythrocytes containing the lower doses of imidocarb appeared normal. Babesia parasitemias did not differ between the nontreated controls and the RBC controls. The mean parasitemias of the group that received free drug at 2 mg/kg (Fig. 1)) and those that received the encapsulated drug at 0.2 mg/ kg (Fig. 2) were markedly lower than controls for 8-10 days following infection. The parasitemias of the groups that received either encapsulated drug at 2 mg/kg (Fig. 1) or free drug at 0.2 mg/kg (Fig. 2 ) were not markedly different from the controls. By 12-15 days following infection, the parasitemias in all mice in all groups were uniformly low.
DISCUSSION
A standard dialysis procedure, modified for mouse erythrocytes [ 51, was used to encapsulate [ 14C] imidocarb and [“HI inulin to determine certain encapsulation parameters and the effect of drug on the RBC. The kinetic studies indicated that free drug, once in the circulation, apparently went immediately 1J the liver, spleen and kidney, as suggested by the earlier study [ 51. By 24 hours after injection 2-3% of the imidocarb-loaded carrier cells remained in the circulation, and that level was maintained for 5 additional days (data not shown). The kinetic experiments involved RBC encapsulated with both imidocarb and inulin and showed the cells to be “leaky” with respect to imidocarb, but not inulin. At 24 hours more than 20% of the cells in circulation contained inulin while only about 2% of these cells still contained imidocarb. The question at hand is whether 2-3% of the injected dose is sufficient to control babesia parasitemias. A significant lowering of the percent parasitemias was observed in the 0.2 mg/kg imidocarb encapsulated treatment group. Peak parasitemias were about 50% lower than peak parasitemias in the group with no treatment and about 30% lower than that found in the group treated with 0.2 mg/kg free drug. The group that received 2 mg/kg encapsulated imidocarb was not different because the higher concentration of drug used in the encapsulation process was destructive to the cells. Thus, cells with the higher concentration of imidocarb did not circulate well. It remains to be determined whether these damaged cells are more rapidly cleared by phagocytosis, and if drug is then released into the circulation or remains in an organ such as the spleen.
CONCLUSIONS
In those areas of the world where babesiosis is a problem of livestock production, improved
83
drugs for chemotherapy are being sought that will provide the following: prolonged activity in circulation, little or no tissue residue, a low effective dose, and control but not sterilization of the infection. The results of the current study suggest that the use of encapsulated imidocarb provides activity for at least 5 days at one-tenth the dose rate required for free drug, and did not sterilize the infection. Therefore, it would appear that encapsulation of babesicidal drugs offers significant improvement over conventional chemotherapy.
ACKNOWLEDGMENT
The authors thank Jan Johnson, Kate Andrews and Bob Droleskey for expert technical assistance.
REFERENCES
R.A. Todorovic, O.G. Vizcaino, E.F. Gonzalez and L.G. Adams, Chemoprophylaxis (Imidocarb) against Babesia bigemina and Babesia argentina infections, Amer. J. Vet. Res., 34 (1973) 1153-1161. J.R. DeLoach, G.G. Wagner and T.M. Craig, Imidocarb dipropionate encapsulation and binding to resealed carrier bovine erythrocytes for potential babesiosis chemotherapy, J. Appl. Biochem., 3 (1981) 254-262. J.R. DeLoach and G.G. Wagner, Encapsulation of drugs in bovine carrier erythrocytes for treatment of anaplasmosis, 10th Int. Symp. Controlled Release Bioactive Mater., 1982. J.R. DeLoach and G.G. Wagner, Pharmokinetics of tetracycline encapsulated in bovine carrier erythrocytes, Amer. J. Vet. Res., 45 (1984) 640-642. J.R. DeLoach and R. Droleskey, Survival of murine carrier erythrocytes injected via peritoneum, Comp. Biochem. Physiol., 84A (1986) 447-450.
of Controlled
Elsevier
Science
Release, 8 (1988)
Publishers
ERYTHROCYTE
79-83
B.V., Amsterdam
79 -
Printed
DRUG CARRIERS
in The Netherlands
FOR BABESIOSIS
CHEMOTHERAPY*,**
G. Gale Wagner*** Department Station,
of Veterinary
TX 77843
Microbiology
and Parasitology,
College of Veterinary Medicine,
Texas A&M University,
College
(U.S. A.)
Donald E. Corrier and John R. DeLoach L/n/ted States Department Laboratory,
of Agriculture,
College Station,
TX 77840
Agricultural
Research Service,
Veterinary
Toxicology
and Entomology
Research
(U.S.A.)
Imidocarb was encapsulated in murine erythrocytes with a dialysis technique. Carrier erythrocytes were injected intraperitoneally in groups of mice at 2 and at 0.2 mg/kg of body weight dosage. Control groups received free (unencapsulated) drug at the same dose rates mixed with a number of untreated erythrocytes corresponding to the above groups. About 2% of the drug-loaded erythrocytes were detected in the circulation after 24 hours and were still in the circulation after 5 days. The concentration of free drug declined to undetectable levels in both the peritoneum and the circulation before 24 hours. The biological effectiveness of the encapsulated imidocarb was evaluated in mice infected with lo7 Babesia microti infected erythrocytes from donor mice and treated 24 hours later with either encapsulated or free imidocarb at the dosages described above. Intraperitoneal injection of drug-loaded carrier cells at a dose rate of 0.2 mg/kg resulted in a decrease in parasitemia to levels similar to those observed in mice treated with free drug at a 2 mg/kg dosage.
INTRODUCTION
Bovine babesiasis, caused by the protozoan parasite Babesia spp., continues as a major threat to livestock industries throughout the world. Because the parasite is transmitted only through the bite of a tick, the disease can be *Paper presented
at the 14th International
Symposium
on
Controlled Release of Bioactive Materials, August Z-5,1987, Toronto, Ontario, Canada. **This report is based upon work supported by the Texas Agricultural Experiment Station. Any opinions, findings, conclusions or recommendations expressed in this abstract and presentation are those of the authors and do not necessarily reflect the view of the U.S. Department Agriculture. ***To whom correspondence should be addressed.
0168.3659/88/$03.50
0 1988 Elsevier
Science
of
Publishers
readily eradicated by a vigorous tick control program. However, in many areas of the world, effective control programs are difficult and expensive to organize and maintain. When such programs are not well managed, disease incidence increases and both chemotherapy and chemoprophylaxis become reasonable disease control alternatives. Babesiosis can be relatively easily treated if the symptoms are recognized early and chemotherapy initiated. Several babesicidal drugs have been used widelv” for more than two decades. One drue_ in particular, imidocarb (3,3’ - bis- (2.imidazolin2-yl ) carbanilide dihydrochloride ), has marked therapeutic and prophylactic properties [ 11. However, the use of imidocarb is limited be-
B.V.
cause of a narrow toxicity range and it,s persistence in tissues [ 11. We have demonstrated the use of erythrocytes as cellular carriers for several drugs (including imidocarb) t,hat might be useful in the chemotherapy of hemotropic infections of cattle [ 2-41. The earlier work suggested that such carrier cells effectively lower the amount of drug needed by virtue of it,s encapsulation, probably by prolonging the pharmacological lifetime of the drug. Thus, carrier erythrocytes seem to improve drug efficacy. We have also determined that erythrocyte carriers can be effecstudied in tively the mouse, thereby considerably reducing the cost of encapsulated drug testing in experimental animals [ 51. Since Babesia microti infections of mice are being used to model the bovine infection, the current studies were conducted to confirm that imidocarb is effective in murine erythrocyte carriers and t,o determine if t.he encapsulated drug is efficacious at less than the recommended dose.
MATERIALS
AND METHODS
Swiss ICR and BALB/c mice, 4 to 6 months old and weighing 30-35 g, were used. Whole blood was collected in heparinized t,ubes from the retroorbital sinus of normal mice and pooled. The red blood cells (RBC) were washed free of plasma and dialyzed to reduce the osmotic pressure to approximately 130 mOsm/mg. Afterward [“Cl imidocarb dipropionate (Burroughs Wellcome, Research Triangle Park, North Carolina) and [“Hlinulin (New England Nuclear, Boston, Massachusetts ) were mixed with the dialyzed RBC and the cells restored to isotonicity by the addition of NaCl [ 51. Unbound imidocarb and inulin were removed by dialysis against isotonic NaCl. Encapsulated drug and inulin were determined by assaying lo-50 ~1 aliquots of RBC for radioactivity. For comparative purposes, imidocarb compatibility wit,h murine RBC and the in. uiuo survival characteristics of carrier RBC were assessed as pre-
TABLE
1
Percentage
of
carrier
murine
[ ’ ‘C ] imidocarb
and
[“H ] inulin labeled RBC detected 24 hours after intraperitoneal injection Treatment
Encapsulated [ ‘H] inulin [ ’ ‘C ] imidocarb Free [,‘H]inulin [ ’ ‘C ] imidocarb
Peritoneal lavage
Whole blood
4 0.8
20.8 ‘2.2
0.8 0.8
0.6 0.6
viously described [ 21. Various preparations were also evaluated by scanning electron microscopy using methods previously described
[51. Mice (12-20 per group) were randomly allotted into 6 groups, designated as nontreated controls, normal RBC controls, and treatment groups consisting of free drug at 2 mg/kg body weight and at 0.2 mg/kg, and encapsulated drug at 2 mg/kg and at 0.2 mg/kg. The normal RBC, the free drug and the encapsulated imidocarb were all administered to the respective groups by intraperitoneal injection in a volume of 0.44 ml. All mice in each group were infected with B. microti on day 0 by the intraperitoneal inoculation of 0.1 ml of inoculum containing about 1.5 x lo7 B. microti. The normal RBC and drug, both free and encapsulated, were injected intraperitoneally 24 hours later, on day 1. Babe& infections were monitored by examination of Giemsa stained thin blood films prepared from tail blood at selected intervals after exposure to the parasite. The mean parasitemia was determined and compared between the treatment groups.
RESULTS Imidocarb was found to be compatible with mouse erythrocytes in the same way as previous
81
75
. Control o Free
drug,
2 mg
a Encapsulated.
2 mg
60 .m E .z
45
2 2 a 30
15
Days
Fig. 1. Babesia microti parasitemias 2 mg/kg or control RBC.
following infection
post
infection
and treatment
of mice with either encapsulated
or free imidocarb
at
s Control o Free
Drug,
0.2
A Encapsulated,
7
Days
Fig. 2. Babesia microti parasitemias 0.2 mg/kg or control RBC.
following infection
9
post
and treatment
11
mg 0.2
13
mg
15
infection
of mice with either encapsulated
or free imidocarb at
82
work with bovine cells [ 21. Imidocarb encapsulation increased proportionally in the concentration range of 0.1 to 2 mg/ml. No apparent effects on cell parameters such as cell volume, cell number and percentage encapsulation were seen within the concentration range studied. Moreover, up to 2 mg/ml external concentration of the drug did not affect the encapsulation of quality control markers such as [“HI inulin. The disappearance of drug-loaded RBC from the peritoneal cavity and the circulating survival of cells is summarized in Table 1. Less than 1% of either the free imidocarb or inulin could be detected in either the peritoneal cavity or circulating whole blood 24 hours after injection. Less than 1% of the encapsulated imidocarb and 4% of the encapsulated inulin was recovered from the peritoneal cavity after 24 hours. When whole blood was assayed, approximately 2.2% of the encapsulated imidocarb and 20.8% of the encapsulated inulin were detected 24 hours after injection. When carrier erythrocytes were prepared in the presence of varying concentrations of imidocarb, fixed and examined by scanning electron microscopy, pathomorphological cell shapes were readily observed at the 1 mg/ml and 2 mg/ml concentrations. Carrier cells appeared as stomatocytes, echinocytes and a few cells had large holes in their membranes. In contrast, micrographs of carrier erythrocytes containing the lower doses of imidocarb appeared normal. Babesia parasitemias did not differ between the nontreated controls and the RBC controls. The mean parasitemias of the group that received free drug at 2 mg/kg (Fig. 1)) and those that received the encapsulated drug at 0.2 mg/ kg (Fig. 2) were markedly lower than controls for 8-10 days following infection. The parasitemias of the groups that received either encapsulated drug at 2 mg/kg (Fig. 1) or free drug at 0.2 mg/kg (Fig. 2 ) were not markedly different from the controls. By 12-15 days following infection, the parasitemias in all mice in all groups were uniformly low.
DISCUSSION
A standard dialysis procedure, modified for mouse erythrocytes [ 51, was used to encapsulate [ 14C] imidocarb and [“HI inulin to determine certain encapsulation parameters and the effect of drug on the RBC. The kinetic studies indicated that free drug, once in the circulation, apparently went immediately 1J the liver, spleen and kidney, as suggested by the earlier study [ 51. By 24 hours after injection 2-3% of the imidocarb-loaded carrier cells remained in the circulation, and that level was maintained for 5 additional days (data not shown). The kinetic experiments involved RBC encapsulated with both imidocarb and inulin and showed the cells to be “leaky” with respect to imidocarb, but not inulin. At 24 hours more than 20% of the cells in circulation contained inulin while only about 2% of these cells still contained imidocarb. The question at hand is whether 2-3% of the injected dose is sufficient to control babesia parasitemias. A significant lowering of the percent parasitemias was observed in the 0.2 mg/kg imidocarb encapsulated treatment group. Peak parasitemias were about 50% lower than peak parasitemias in the group with no treatment and about 30% lower than that found in the group treated with 0.2 mg/kg free drug. The group that received 2 mg/kg encapsulated imidocarb was not different because the higher concentration of drug used in the encapsulation process was destructive to the cells. Thus, cells with the higher concentration of imidocarb did not circulate well. It remains to be determined whether these damaged cells are more rapidly cleared by phagocytosis, and if drug is then released into the circulation or remains in an organ such as the spleen.
CONCLUSIONS
In those areas of the world where babesiosis is a problem of livestock production, improved
83
drugs for chemotherapy are being sought that will provide the following: prolonged activity in circulation, little or no tissue residue, a low effective dose, and control but not sterilization of the infection. The results of the current study suggest that the use of encapsulated imidocarb provides activity for at least 5 days at one-tenth the dose rate required for free drug, and did not sterilize the infection. Therefore, it would appear that encapsulation of babesicidal drugs offers significant improvement over conventional chemotherapy.
ACKNOWLEDGMENT
The authors thank Jan Johnson, Kate Andrews and Bob Droleskey for expert technical assistance.
REFERENCES
R.A. Todorovic, O.G. Vizcaino, E.F. Gonzalez and L.G. Adams, Chemoprophylaxis (Imidocarb) against Babesia bigemina and Babesia argentina infections, Amer. J. Vet. Res., 34 (1973) 1153-1161. J.R. DeLoach, G.G. Wagner and T.M. Craig, Imidocarb dipropionate encapsulation and binding to resealed carrier bovine erythrocytes for potential babesiosis chemotherapy, J. Appl. Biochem., 3 (1981) 254-262. J.R. DeLoach and G.G. Wagner, Encapsulation of drugs in bovine carrier erythrocytes for treatment of anaplasmosis, 10th Int. Symp. Controlled Release Bioactive Mater., 1982. J.R. DeLoach and G.G. Wagner, Pharmokinetics of tetracycline encapsulated in bovine carrier erythrocytes, Amer. J. Vet. Res., 45 (1984) 640-642. J.R. DeLoach and R. Droleskey, Survival of murine carrier erythrocytes injected via peritoneum, Comp. Biochem. Physiol., 84A (1986) 447-450.