|Year : 2014 | Volume
| Issue : 1 | Page : 31-37
New oral anthelmintic intraruminal delivery device for cattle
Thierry F Vandamme
University of Strasbourg, Faculty of pharmacy, UMR 7199 CNRS, Laboratory of Concept and Application of Bioactive Molecules, Biogalenic team, 74 Route du Rhin, 67400 Illkirch Graffenstaden, France
|Date of Submission||21-Nov-2013|
|Date of Decision||21-Nov-2013|
|Date of Acceptance||21-Nov-2013|
|Date of Web Publication||4-Jan-2014|
Thierry F Vandamme
University of Strasbourg, Faculty of pharmacy, UMR 7199 CNRS, Laboratory of Concept and Application of Bioactive Molecules, Biogalenic team, 74 Route du Rhin, 67400 Illkirch Graffenstaden
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The purpose of this work was to develop a new oral drug delivery system intended for cattle and that enables delayed and pulsed release of an anthelmintic agent. Materials: This new tailored dosage form, also called reticulo-rumen device (RRD) has been evaluated on grazing calves by means of measurements of milliunits of tyrosine concentration, number of eggs per gram of feces, mean number of infective larvae on cattle pasture and increase in mean weight of cattle. Methods: The in vivo evaluation was carried out during two grazing seasons on different groups of dairy cattle. During the first grazing season, Group 1 was designated as an untreated control group. The remaining two were assigned to different treatments as follows: Group 2, early season suppression with a marketed intraruminal slow release bolus (Chronomintic ® , Virbac) administered immediately prior to turn-out and Group 3, mid-season suppression with a new RRD administered immediately prior to turn out. When the cattle were turned out at the start of the second grazing season, they were not given any anthelmintic treatment and were divided into two different groups, corresponding to the previous groups that received an anthelmintic treatment during the first grazing season, on that pasture that they had occupied as separate groups in the previous year. Furthermore, during the second season, samples of feces, blood and herbage were collected every month. Results and Conclusion: During the first grazing season, the results indicated that the fecal egg counts and the number of infective larvae in herbage samples were slightly lower for the group receiving the new RRDs. Regular weighing of the cattle receiving the new RRDs revealed no significant difference with cattle receiving marketed RRDs. Conversely, during the second grazing season, the results for the mean weights of the cattle demonstrated that the weights of animals having been administered new RRDs during the first grazing season were significantly different (P < 0.05) from those in the second group treated with a Chronomintic ® during the first grazing season. A difference in mean weight of 26 kg was observed between these two groups.
Anthelmintic, bolus, cattle, delayed release, eggs, larvae, oral drug delivery device, pulsed release, rumino reticulum device, veterinary parasitology
|How to cite this article:|
Vandamme TF. New oral anthelmintic intraruminal delivery device for cattle. J Pharm Bioall Sci 2014;6:31-7
Over the last few years, a number of different anti-parasite formulas with anthelmintic properties aimed at preventing parasite infestations in ruminants have been developed. Recent advances in the field of pharmaceutical technology have also led to the development of tailored dosage forms, called reticulo-rumen devices (RRDs) or boluses, intended to eradicate these types of infestation. At present, two main types of RRD are available on the market: (i) Matrix systems enabling slow release of the drug and (ii) pulsed-release systems enabling release of the anthelmintic agent every 23 days. Owing to their programmed release, these devices offer the potential for a successful solution to parasite diseases in ruminants. 
Recent progresses made in the field of epidemiology and pharmacology have led to the conclusion that the level and duration of exposure to gastrointestinal nematode infections are of crucial importance for the development of acquired immunity in first-season grazing calves.  An excessive reduction of host-parasite contact by chemoprophylaxis, pasture management or both leads to a reduced level of acquired immunity. Moreover, the level of acquired resistance is negatively correlated with the degree of elimination of host-parasite contact. , Whether or not a reduced resistance against the establishment and development of gastrointestinal nematode infections has a negative effect on weight gain during the second grazing season depends both on the intensity of the prophylaxis used and on the level of the challenge infection. Cross-sectional serological surveys have shown that parasitic nematode control in first-season grazing calves tends to be overprotective. Possible consequences of over-treatment, besides higher treatment costs and a reduced level of acquired immunity, are more drug residues in animal products and in the environment and increased selection for anthelmintic resistance. 
Over the last decade, a significant amount of progress has been made in terms of understanding of the mechanisms involved in cattle immunity to gastrointestinal nematodes and the interactions between gastrointestinal nematodes and the immune system. These previous studies highlighted the fact that most of the common parasites found in cattle are able to stimulate an effective level of protective immunity in most of the herd after the grazing animals have been on pasture for several months. Moreover, some studies demonstrated that reinfection with these parasites led to a significant reduction in the number of worms that become established in the grazing animals and that parasites such as Dictyocaulus viviparus and Oesophagostomum radiatum are extremely effective in eliciting strong protective immune responses. ,, It was also observed that primary exposure of previously naive cattle to infection or even to parasite antigens results in a very significant reduction in the number of parasites that can become established after a subsequent infection. ,, Clearly, from these previous immunological and parasitological studies, it appears that the best anthelmintic delivery system intended to cattle should allow a release of the anthelmintic drug after a period of ~ 3 months of exposure to infected pastures followed by a pulse release each 3-4 weeks.
On the basis of these considerations, we designed new RRDs with the following principles: (i) To be administered to cattle at turnout; (ii) allowing release of the anthelmintic drug at the end of June and continuing during the period from July to August and (iii) preferably ensuring pulsed release of the anthelmintic agent. In order to evaluate these new RRDs in vivo, the performance of the devices were followed during two grazing seasons on different groups of dairy cattle by measurement of serum pepsinogen concentration, number of eggs per gram (EPG) of feces, mean number of infective larvae on cattle pasture and mean weight of cattle.
| Materials and Methods|| |
Vicryl ® , Monocryl ® and PDS II ® monofilaments were supplied by Ethicon (Ethnor, Aulnais Sous Bois, France) and Maxon ® , Dexon ® were supplied by Davis + Geck (Laboratoires Robert et carrier Lederle, Serquigny, France). Microcrystalline cellulose (Avicel PH 101 ® ) and croscarmellose sodium (AC-DI-SOL ® ) were purchased from FMC Europe N.V., Brussels, Belgium. a-lactose monohydrate was obtained from Meggle, Wasserburg, Germany. Starch 1500 was supplied by Colorcon, Orpington, United Kingdom. Aerosil 200 ® and magnesium stearate were obtained respectively from Degussa AG, Frankfurt am Main, Germany and from Mallinkrodt, St Louis, Missouri, USA. Levamisole hydrochloride was supplied by Sigma (Saint Quentin Fallavier, France). The powders were weighed on an analytical balance (Mettler AT 200, Mettler, Greifensee, Switzerland). Chronomintic ® were a gift from Virbac, Carros, France. All other reagents were analytical grade.
Chronomintic® is a system for cattle that releases levamisole hydrochloride (an anthelmintic) for about 90 days. These RRDs are essentially high-density cylinders,  with the active material contained as a cylindrical core in the center. The outside is covered with a waterproof membrane of polyurethane to prevent the release of the drug from the external walls. An internal perforation provides for a radial release of the bioactive, providing release kinetics theoretically close to a zero order. Theoretically, the total payload of the drug (22.05 g of levamisole hydrochloride in Chronomintic®) is dispersed homogeneously in the matrix to provide continuous release of a larvicidal dose for a period of about 90 days.
Manufacture of new RRDs
The RRDs were constructed by assembling various high-density polyethylene elements [Figure 1]a containing levamisole hydrochloride tablets, separated from one another by a cap held in position by a degradable monofilament [Figure 1]b. The first element was covered by a cap, in which holes were pierced, to prevent large particles from the reticulo-rumen of the grazing animal getting into it. At the other end, the last element was made of iron in order to keep the device in the bottom of the reticulo-rumen of the cattle. The RRDs administered to the cattle contained 4 tablets containing each 0.884 g of levamisole hydrochloride and divided into 4 compartments. For the in vivo evaluation, the new RRDs were constructed using a Vicryl ® (2/0) monofilament to join the various compartments except for the first one, which was joined to the second one using a polydioxanone suture thread.
|Figure 1: Cross-section of the new reticulo-rumen device (a) and of a compartment (b) of the device|
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Compression tests were performed as follows. First of all, 88.40 g levamisole hydrochloride, 22.60 g a-lactose monohydrate, 11.31 g starch 1500 and 11.31 g croscarmellose sodium were mixed together using a Turbula ® T2A mixer (Basel, Switzerland) for 10 min at 50 rpm. Then 54.65 g Avicel PH 101 ® was added to this mixture and mixed for a further 10 min at 50 rpm. Lastly, 2.30 g Aerosil 200 ® and 2.30 g magnesium stearate were added and mixed with the previous mixture for 3 min at 50 rpm. The powder was compressed using a Korsch EK0 (Berlin, Germany) eccentric tablet press with flat-faced punches at a constant machine setting to produce tablets (~2.260 g) with an average height of 6.0 mm and a diameter of 20 mm and containing 0.884 g levamisole hydrochloride (750 mg levamisole base). After compression, the tablets were analyzed in accordance with the Pharmaceutical Technical Procedures of the 8.0 th ed.ition of the European Pharmacopoeia. Levamisole hydrochloride was quantified in accordance with the quantification method indicated in the monograph for this drug in the 8.0 th ed.ition of the European Pharmacopoeia.
In vitro degradation studies on biodegradable suture threads
Weighed samples of biodegradable suture threads (~70 mg) were placed in 60 ml screw-capped glass flasks containing 50 ml of a solution with a pH and ionic strength as near as possible to that encountered in the rumen of cattle. The solution for in vitro degradation studies on biodegradable suture threads contained Na 2 HPO 4 12H 2 O, 9.3. g; NaHCO 3 , 9.8 g; NaCl, 4.7 g; KCl, 5.7 g; CaCl 2 2H 2 O, 53.10−3 g; MgCl 2 6H 2 O, 128.10−3 g; FeSO 4 6H 2 O, 75.10−3 g; MnSO 4 H 2 O, 4.10−3 g; Urea, 7.10−2 g; water added to 1000 ml and pH fixed at 6.9 with CO 2 .  This solution was prepared by dissolution of the various chemicals and filtering through a Seitz press-filter with K0 seitz filter sheets. Tests were performed at 39°C in an incubator shaker at 30 cycles/min. Samples were taken at various times (1, 8, 20, 30, 41, 53, 80 and 120 days), depending on their chemical composition. Each time a sample was taken, the flasks were emptied. The biodegradable suture threads were carefully removed from the flasks and placed in a crystallizing dish. They were then rinsed twice with distilled water. The biodegradable suture threads were then placed on an absorbent sheet and put in a desiccator containing P 2 O 5 to remove the remaining traces of water. The tests were repeated 3 times. The degradation process was monitored gravimetrically for weight loss.
The rupture times of the biodegradable suture threads were determined as follows. The tablets were placed in pliable leak-proof sachets, three sides of which were sealed and the fourth side of which was left open. The fourth side of the sachet was sealed with a temporary closure. This closure consisted of a clip made up of two arms and linked at one of its ends by a linking portion forming a hinge to hold the arms of the clip in the open position in the absence of an external force. Each arm had longitudinal grooves on its inner surface. There was a channel along the outer surfaces of the arms. The open end of the pliable leak-proof sachet in which the tablet containing levamisole hydrochloride had been placed, was placed between the open arms of the clip. These arms were then squeezed together, the grooves interlocking and held in this configuration by a biodegradable suture thread located in the channel, the two ends of which were tied together to form a knot. These sachets were placed in 1200 ml screw-capped plastic flasks containing 1000 ml of a solution for in vitro degradation studies reported above. The tests were performed under the same conditions as those described above for determination of weight loss of the biodegradable suture threads. The times taken for a compartment to be released from the device, for the two arms of the clip to open and for the sachet to open were determined for each biodegradable suture thread. The experiments were repeated 3 times.
In vivo degradation studies on biodegradable suture threads
For the in vivo biodegradation tests on the suture threads, the new RRDs [Figure 1] were put into non-degradable plastic envelopes, which were perforated to allow contact between rumen fluids and the RRDs. The envelopes were pooled together in plastic bags, which were also perforated. They were then introduced into the rumen through rumen fistulae. These tests were carried out at a testing farm in the south of Libramont (Belgium). Four cattle were used for these studies. Animals, in their first or second grazing season, were Dutch crossbred cattle and were given feed concentrates, hay and fresh water and straw for bedding. The cattle were housed in cowsheds in floor pens and fed ad libitum. The rumen fistulae were made of polyvinylchloride and silicone and were attached on both sides of the skin. The RRDs were removed periodically to determine the rupture time of the various biodegradable suture threads. All the tests were performed in accordance with the animal ethics regulations indicated in European Directive 86/609/EEC (24 th November 1986) relative on the use of non-anaesthetized animals in research.
In vivo evaluation of the new RRDs
31 st -season grazing calves (Dutch crossbred), aged approximately 5 months, were used in the experiment. They were divided into three groups (1, 2 and 3) on the basis of weight and sex and each individual was identified by a colored, numbered ear-tag. All the animals were vaccinated against D. viviparus 6-2 weeks prior to turn-out. The calves were turned out on 30 April 2009 (Day 0) and the animals were housed on 9 November (Day 193). By the end of September, all the groups were supplemented with hay (35 kg/group/day).
The study was carried out on a commercial dairy cattle farm in the south of Libramont (Belgium). The pasture used had previously been grazed by first-season calves in which parasite control had been kept to the minimum level required to prevent acute disease, but which allowed the build-up of large numbers of parasitic infective larvae. The pasture was divided into three blocks of approximately 0.8 ha, each with its own water supply and crush. Group 1 was designated as an untreated control group and salvage treatment was to be administered only when clinical signs of parasitic gastroenteritis appeared; the remaining two were assigned to different treatments as follows: Group 2, early season suppression with an intraruminal slow release bolus (Chronomintic ® , Virbac) administered using a balling-gun immediately prior to turn-out, Group 3, mid-season suppression with a new RRD administered also by using a balling-gun immediately prior to turn-out. Feces were taken from all the calves on Days 0, 31, 61, 78, 94, 125, 153, 188 and 193 after turn-out. The calves were weighed at the same time and samples of the grass were taken at same dates from each pasture to estimate the number of infective larvae present. Blood samples were taken from all the calves on Days 38, 63, 76, 103, 118, 135, 188 and 193 after turn-out. Plasma was removed from the blood samples and stored at −20°C pending further examination. At housing, all the calves from each group were treated with ivermectin (200 mg/kg) (Ivomec ® 1% Injection, Merial, France). The treated calves were housed during the winter and then put out to graze on 29 April 2010 in two different groups, corresponding to the previous groups, on that pasture that they had occupied as separate groups in the previous year. The cattle grazed throughout the summer until 6 October 2010. During this second season, samples of feces, blood and herbage were collected every month. The cattle were also weighed regularly. On housing, all the calves from each group were again treated with ivermectin (200 mg/kg) (Ivomec ® 1% Injection, Merial, France).
Serum pepsinogen concentrations were determined according to Berghen et al. and expressed in milliunits of tyrosine (mU tyr).  Ostertagia and Cooperia Immunoglobulin G (IgG) antibodies against crude L3, L4 and adult antigens (4 mg/ml) were determined by enzyme linked immunosorbent assay. Only one serum dilution was used in duplicate (1/400). Anti-bovine IgG coupled to horseradish peroxidase was used as a conjugate and 0-phenylenediamine served as a substrate. The values were expressed as optical densities.
Fecal egg counts were carried out using the McMaster technique and are expressed as geometric group means. For larval determination, third stage larvae were collected after 10 days of incubation at 25°C from a pooled faeces culture for each group. Changes in the output of eggs per animal throughout the year were measured using the method of Rickard et al. 
Herbage samples were collected from each pasture throughout 2003 and 2004 using a W-transept  and avoiding grass close to fecal matter. Larvae counts were made using a sedimentation/flotation technique  and expressed as L 3 /kg dried herbage.
Data were statistically analyzed by Mann-Whitney U-test. Data were considered to be significantly different if P < 0.05. Results of in vitro and in vivo degradation of biodegradable suture threads are presented as means ± SEM. Statistical differences between the degradation times were analyzed by analysis of variance followed by Student's t-test. Data were considered to be significantly different if P < 0.05.
| Results and Discussion|| |
In vitro and in vivo degradation of biodegradable suture threads
The weight losses of the biodegradable suture threads are reported in [Table 1] and lead to the conclusion that their biodegradation depends on their physicochemical characteristics. The suture threads (Vicryl ® , Monocryl ® , Dexon ® ) made of lactic acid or glycolic acid polymers or copolymers hydrolyzed quickly. When the number of methylene groups between the two ester groups is higher (Maxon ® , PDS II ® ), the hydrolysis phenomena are much slower and consequently so are their rates of biodegradation. After 120 days, the weight losses for polyglyconate (Maxon ® ) and polydioxanone (PDS II ® ) monofilaments were 56% and 23% respectively. From a chemical point of view, in vivo and in vitro biodegradation occurred as described by Lin et al., Mehta et al. and Lindhart. ,, Indeed, according to these authors, polyesters degrade in four main stages: (1) Polymer hydration causing disruption of the primary and secondary structure due to hydrogen bonding and van der Walls forces; (2) loss of mechanical strength caused by the rupture of covalent bonds forming the polymer backbone; (3) loss of mass integrity resulting in accelerated water absorption; and (4) polymer dissolution and/or phagocytosis.
|Table 1: In vitro degradation studies on biodegradable suture threads (n=10)|
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Examination of the biodegradable suture threads indicated in [Table 2] reveals that they had different rupture times, which were in vitro and in vivo reproducible (P < 0.05) from one sample to another with the same physicochemical characteristics. For example, a monofilament made of lactic acid and ε-caprolactone copolymer (Monocryl ® ) and a monofilament made of PDS II ® had in vivo rupture times of 22 ± 1 days and 86 ± 1 days, respectively. It was also interesting to observe a good correlation between the in vitro and in vivo rupture times. According to these results, the new RRDs have been constructed as followed. Biodegradable suture threads made of PDS have been fixed between the first and the second compartments, the rumen liquid will only reach the second compartment after 86 days and therefore the drug will only be released after this period of time has elapsed. Conversely, by choosing a different one, such as a Vicryl ® (2/0) monofilament, the rupture time will be only 23 days and the drug will be released after this period of time. With this construction, we obtained new RRDs which met the strategic requirements defined before the start of these studies, namely the absence of a drug release during ~ 3 months after the administration of the new RRDs to the cattle followed by a release of the anthelmintic drug each 3 weeks.
|Table 2: In vitro and in vivo breaking times of the biodegradable monofilaments (n=10)|
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In vivo evaluation of the efficacy of the new RRDs
[Figure 2] shows that the values for mU tyr during the 1 st year were much higher for the control group and for the group receiving the new RRD, indicating the high level of exposure to parasites of these two groups at the start of the grazing season. Conversely, during the 2 nd year, the values for mU tyr were lower for the group which received the new RRDs during the 1 st year compared with the group administered conventional RRDs (Chronomintic ® ) [Figure 3]. These results indicate that the animals' exposure to parasites during the first part of the grazing season was beneficial since it increased their immunity. The second test performed concerned fecal egg counts that were conducted using the McMaster Technique. The results indicated in [Figure 4] show that the fecal egg counts were slightly lower for the group receiving the new RRDs than for the other two groups (P < 0.05). The fecal egg count results during the 2 nd year were broadly similar. Indeed, during the 2 nd year, the fecal egg counts were lower for the cattle having received new RRDs during the 1 st year [Figure 5]. The results for the herbage samples [Figure 6] and [Figure 7] were relatively similar to the results for the fecal egg counts. These results confirm the fecal egg count results since they are a direct consequence of them. For the fourth and the most important test, i.e. the mean weight of the cattle [Figure 8], regular weighing of the cattle during the first grazing season revealed no significant difference at Day 193 (P > 0.05) between the two groups receiving anthelmintic treatment with either a Chronomintic ® or a new RRD and a significant difference (P < 0.05) in comparison with the untreated group. After housing, the cattle were weighed regularly [Figure 9] and no significant difference (P > 0.05) was observed over this period between the two groups treated with anthelmintic agents during the first grazing season. When the same cattle were turned out at the start of the second grazing season [symbolized by the arrow in [Figure 9], they were not given any anthelmintic treatment. The results for the mean weights of the cattle demonstrate that the weights of animals having been administered new RRDs during the first grazing season were significantly different (P < 0.05) at Day 359 from those in the second group treated with a Chronomintic ® during the first grazing season. These results were also attributed to the development of a better immunity during the first grazing season for the group that received the new RRDs. At the end of the second grazing season, a difference in mean weight of 26 kg was observed between these two groups [Figure 9].
| Conclusion|| |
From our experimental studies, it can be concluded that we have been able to design a new RRD that enables delayed and pulsed release of an anthelmintic agent during the first grazing season and ensures optimum release of the anthelmintic agent at the period when larval infestation is very high (July-August). The in vivo evaluation of these new RRDs has demonstrated the potential of this optimized dosage form with regard to required tyrosine concentration, number of EPG of feces, mean number of infective larvae on cattle pasture and increasing in mean weight of cattle.
| References|| |
|1.||Vandamme TF, Ellis KJ. Issues and challenges in developing ruminal drug delivery systems. Adv Drug Deliv Rev 2004;56:1415-36. |
|2.||Houdijk JG, Kyriazakis I, Kidane A, Athanasiadou S. Manipulating small ruminant parasite epidemiology through the combination of nutritional strategies. Vet Parasitol 2012;186:38-50. |
|3.||Claerebout E. The effects of chemoprophylaxis on acquired immunity against gastrointestinal nematodes in cattle. Verh K Acad Geneeskd Belg 2002;64:137-49. |
|4.||Vercruysse J, Claerebout E. Treatment vs non-treatment of helminth infections in cattle: Defining the threshold. Vet Parasitol 2001;98:195-214. |
|5.||Gasbarre LC, Leighton EA, Sonstegard T. Role of the bovine immune system and genome in resistance to gastrointestinal nematodes. Vet Parasitol 2001;98:51-64. |
|6.||Stromberg BE, Gasbarre LC. Gastrointestinal nematode control programs with an emphasis on cattle. Vet Clin North Am Food Anim Pract 2006;22:543-65. |
|7.||Li RW, Sonstegard TS, Van Tassell CP, Gasbarre LC. Local inflammation as a possible mechanism of resistance to gastrointestinal nematodes in Angus heifers. Vet Parasitol 2007;145:100-7. |
|8.||Hou Y, Liu GE, Bickhart DM, Matukumalli LK, Li C, Song J, et al. Genomic regions showing copy number variations associate with resistance or susceptibility to gastrointestinal nematodes in Angus cattle. Funct Integr Genomics 2012;12:81-92. |
|9.||Rubin R, Lucker JT. Acquired resistance to Dictyocaulus viviparus, the lungworm of cattle. Cornell Vet 1956;46:88-96. |
|10.||Weber TB, Lucker JT. Immunity against the cattle lungworm: Resistance resulting from initial infection with small numbers of larvae. Proc Helminthol Soc Wash 1959;26:132. |
|11.||Gasbarre LC, Canals A. Induction of protective immunity in calves immunized with adult Oesophagostomum radiatum somatic antigens. Vet Parasitol 1989;34:223-38. |
|12.||Duncan R, Symour LW. Controlled Release Technologies. Amsterdam, New York: Elsevier; 1989. |
|13.||Vandamme TF, Legras R. Physico-mechanical properties of poly (epsilon-caprolactone) for the construction of rumino-reticulum devices for grazing animals. Biomaterials 1995;16:1395-400. |
|14.||Berghen P, Dorny P, Vercruysse J. Evaluation of a simplified blood pepsinogen assay. Am J Vet Res 1987;48:664-9. |
|15.||Rickard LG, Zimmerman GL, Hoberg EP, Lockwood PW, Weber DW, Miller R. Efficacy of the morantel sustained release trilaminate matrix against gastrointestinal nematodes in beef calves. Vet Parasitol 1989;33:125-33. |
|16.||Taylor EL. Technique for the estimation of pasture infestation by strongyloid larvae. Parasitology 1939;31:473. |
|17.||Ministry of Agriculture, Fisheries and Food. Manual of Veterinary Parasitological Laboratory Techniques, Ref. Book 418. London: HMSO; 1986. |
|18.||Lin WJ, Flanagan DR, Linhardt RJ. Accelerated degradation of poly (epsilon-caprolactone) by organic amines. Pharm Res 1994;11:1030-4. |
|19.||Mehta RC, Jeyanthi R, Calis S, Thanoo BC, Burton KW, Deluca PP. Biodegradable microspheres as depot system for parenteral delivery of peptide drugs. J Control Release 1994;29:375. |
|20.||Lindhardt RJ. Biodegradable polymers for controlled release of drugs. In: Roscoff M, editor. Controlled Release of Drugs: Polymers and Aggregate Systems. New York: VCH; 1988. p. 53-96. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2]