Co-polymerised peptide particles II: Oral uptake of a novel co-polymeric nanoparticulate delivery system for peptides

journal of

ELSEVIER

Journal of Controlled Release 42 (1996) 65-73

controlled release

Co-polymerised peptide particles II: Oral uptake of a novel co-polymeric nanoparticulate delivery system for peptides Anya M. Hillery l'a, I s t v a n T o t h b, Alexander T. Florence a'* "Centre for Drug Delivery Research, School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N lAX, UK bDepartment of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N lAX, UK Received 4 July 1995; revised 30 January 1996; accepted 13 February 1996

Abstract A novel co-polymeric nanoparticulate drug delivery system (the co-polymerised peptide particle, CPP) has been developed [1], in which a derivative of the peptide luteinizing hormone releasing hormone (LHRH) was co-polymerised with n-butylcyanoacrylate (n-BCA) and a radiolabel, the co-polymer being formulated as nanoparticles. These particles thus contain covalently bound LHRH molecules as constituents of the oligomeric chains. The CPP system was orally dosed to male Wistar rats. A double antibody radioimmunoassay (RIA) demonstrated that LHRH was present in the plasma over a 12 h period, whereas the free peptide did not show detectable absorption. Maximum plasma uptake, amounting to 1.6% of the administered dose of LHRH, was detected 3 h after dosing, but the total cumulative uptake of LHRH was significantly higher, as demonstrated by the plasma area under the curve (AUC) determined over a 12 h post-dosing period. Non-encapsulated LHRH, i.e., LHRH dissolved in the continuous phase of a suspension of n-BCA particles, but not entrapped within the particulate matrix, was not absorbed after oral dosing, confirming the protective effect of CPP encapsulation. RIA may have under-estimated the extent of oral uptake of LHRH due to 'shielding effects' of the CPP particulate matrix as, in vitro, only 10% of the peptide in the particles could be detected by RIA. Multiple (2 or 5 days) dosing led to higher LHRH uptake. The particles were broken down in the blood, as shown by gel filtration chromatography. The results demonstrated that chemical conjugation of a peptide within a protective particulate matrix was a viable approach for enhancing oral peptide delivery, presumably utilising the gut-associated lymphoid tissue as a route delivery. Keywords: Nanoparticles; Oral drug delivery; Luteinizing hormone releasing hormone; Radioimmunoassay; Gel filtration chromatography

1. Introduction

g,

Correspondingauthor. Tel.: +44 171 7535819; Fax: +44 171 8375092. ~Current address: Department of Pharmacy, University of Brighton, Moulsecoomb, Brighton BN2 4GJ.

Although significant advances in the areas of biotechnology, biochemistry, molecular biology and peptide synthesis have led to the availability of large quantities of pure, potent and highly specific peptide and protein drugs, this has not been matched by

0168-3659/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0168-3659(96)01368-5

66

A.M. Hillery et al. / Journal of Controlled Release 42 (1996) 65- 73

similar progress in the area of peptide delivery. The challenge of the oral delivery of peptides and proteins remains. Strategies to enhance oral peptide and protein absorption include the use of colloidal particles as drug carriers, exploiting the phenomenon of particulate uptake across the gut-associated lymphoid tissue [2-4], and the use of drug-polymer conjugates, such as the conjugation of labile peptides with hydroxypropylmethacrylamide and other polymers [5-8]. We have employed an approach that combines both methods of oral absorption enhancement, i.e., the use of particulate carriers and the use of drugpolymer conjugates, by synthesizing a novel drugpolymer conjugate that formed its own nanoparticulate delivery system (the co-polymerised peptide particle system, CPP) [1]. Using the peptide luteinizing hormone releasing hormone (LHRH) as a model drug to investigate the viability of the approach, a polymerizable derivative of LHRH was prepared by conjugating the peptide with vinylacetic acid. The LHRH-vinylacetate conjugate was then co-polymerised with n-butylcyanoacrylate (n-BCA) and a radiolabel (methyl-2-vinylacetylarnido-3-L-[2.6- 3HIphenylpropionate) and the reaction conditions were manipulated to exploit the particle-forming properties of alkyl-2-cyanoacrylates [9]. The co-polymeric system was formulated as particles of average diameter 100 nm, in which LHRH molecules were covalently linked under controlled conditions, as constituents of the oligomeric chains which formed the particles. The particles demonstrated stability in vitro when incubated in intestinal contents, mucosal scrapings and serum [1]. In vitro transport studies using the Caco-2 cell line suggested that absorption in vivo might take place [1]. For the in vivo quantitation of intact, biologically active peptides, methods must be both highly sensitive, due to the low concentrations of the molecules, and also highly specific, due to the similarity in structure between the peptide analyte and its degradation products [10]. Interference from endogenous components, their metabolites and impurities can also arise. Further problems for analysis are due to the inherent instability of peptides, which results in various degradative reactions and a tendency for these moieties to adsorb, aggregate and denature [11]. Analytical methods must also take into account

the relationship between purity and actual activity as, although the chemical composition of the analyte may be established, changes which affect biological potency, such as in conformation or aggregation, may go undetected. Although use of a radiolabel is a convenient and sensitive method to study the uptake and translocation of a bioactive peptide, there is a lack of specificity in the method and the radioactivity determined in vivo may be due to radiolabelled peptide fragments, rather than intact peptide. Radiochemical analysis does not give any information on the extent, if any, of degradation, or on the nature of the degradation products. A more sensitive and specific analytical technique involves the radioimmunoassay (RIA), which exploits the high affinity and specificity of binding between an antibody and an antigenic determinant, to detect very low concentrations of peptide antigens in a variety of biological matrices [11]. Here we describe the analysis of LHRH uptake in rats after oral dosing with the CPP system. Two different moieties - - the peptide and its particulate carrier - - were subject to degradation in vivo. The techniques of gel filtration chromatography, in conjunction with radioactivity analysis and RIA, were used to examine the in vivo integrity of both the biodegradable nanoparticulate carrier and the active drug moiety it contained.

2. Materials and methods 2.1. CPP synthesis and characterisation

The CPP system was synthesized as described previously [1] using LHRH-(PyGlu was replaced by Glu)-vinylacetate (2 mg, 0.0015 mmol), methyl-2vinylacetylamido-3-L-[2.6- 3H]phenylpropionate (2 mg, 0.007 mmol) and n-BCA monomer (0.5 MI) in the co-polymerization reaction. The precipitated copolymer was dried, dissolved in dimethylformamide (DMF, 2 M1) and co-polymeric particles were precipitated by the addition, under vigorous stirring, of an aqueous medium (48 ml) containing poloxamer 188 (0.2%), adjusted to pH 3.5 using 1 N HC1. The CPP system was characterised by laser desorption mass spectrometry, ~H nuclear magnetic resonance

A.M. Hillery et al. / Journal of Controlled Release 42 (1996) 65-73

(NMR), transmission electron microscopy and photon correlation spectrometry, as described previously [1]. A suspension of peptide-free n-BCA nanoparticles of average diameter 80 nm was also prepared and characterised as described previously [1], using nBCA monomer (0.5 ml), then DMF (2 ml) and LHRH (2 mg) was added to the polymeric suspension, so that the peptide was present with n-BCA particles as a physical mixture, but not entrapped within the particulate matrix. 2.2. Animals and dosing

Male Wistar rats (average weight 250 g, 8 weeks old) were housed in metabolic cages to prevent coprophagia and to allow growth monitoring, as described previously [12]. The rats were fed maintenance expanded rat and mouse pellet diet (Bantin and Kingman, Hull) and maintained in an air conditioned environment at 20°C under a schedule of 12 h light/12 h darkness. Animals were fasted for 14 h prior to dosing, but free access to water was given at all times. Dosing was via gavage (1 ml), using a blunt tipped feeding needle inserted into the stomach. Heparinized blood samples (5 ml) were collected via the abdominal aorta under halothane anaesthesia at certain time intervals after dosing, after which the animals were sacrificed, with 3 animals sacrificed at each time interval. Control groups (3 animals per group) were dosed with saline, a control group was also sacrificed at each time interval. 2.3. Scintillation counting

Blood samples collected 3 h, 6 h, 12 h, 24 h and 48 h after oral dosing, were solubilized in Scintran T M (BDH, UK), aliquots were heated at 50°C for 20 min, followed by decolorization using 30% H20 2 solution. The samples were evaporated to dryness using N 2 flow at 50°C, dissolved in Optiphase scintillation fluid and counted for 15 min in a Beckman LS5000CE liquid scintillation system. Stomach, small intestine, large intestine, liver, kidney and spleen were also analyzed, and the gut tissues were washed thoroughly prior to analysis, as described previously [12]. The tissue samples were

67

homogenized in buffer, followed by solubilization, decolorization, evaporation and counting. Results were corrected for background counts, control counts, the various dilutions of the samples and, for the blood samples, extrapolation to the total blood volume, i.e., 20 ml for a 250 g rat [13]. 2.4. Radioimmunoassay o f L H R H

LHRH concentration was measured in duplicate using a double antibody RIA with a polyethylene glycol (PEG) precipitation step, following a protocol supplied by Sigma (UK) for the RIA of human chorionic gonadotrophin. Anti-LHRH whole antiserum (Sigma, UK) was used as the primary antiserum. The antiserum had been developed in rabbit using LHRH conjugated to bovine serum albumin (BSA) as the immunogen. Goat anti-rabbit IgG (whole molecule) whole antiserum (Sigma, UK) was used as the precipitation antiserum. (3[125I]iodotyrosylS)LHRH with a specific activity of 2000 Ci/mmol (Amersham, UK) was used as the radioactive tracer, at a concentration of 23 000 counts/min/ml at the reference date. Standard curves were constructed by plotting the percentage of tracer which bound to antibody versus the logarithm of the LHRH-vinylacetate concentration. The limit of detection for the assay was determined as l0 ng/ml and the non-specific binding as 3%. The concentration of peptide in unknown samples was determined by interpolation from the standard curve. The RIA was carried out in vitro on diluted particle samples. Blood samples were collected 40 min, 1 h, 1.5 h, 2 h, 3 h, 6 h and 12 h after single dosing with either (i) the CPP system (0.04 mg LHRH), (ii) saline, (iii) LHRH or LHRH-vinylacetate (2 mg) dissolved in RIA buffer (50 ml), (iv) a suspension of n-BCA nanoparticles containing LHRH dissolved in the continuous phase (see above). In a multiple dose study, rats were dosed for 2 or 5 successive days with the CPP system or saline. Food was returned 3 h after dosing, except after the final (i.e., second or fifth) dose. Blood samples were collected 3 h after the final dose. Blood samples taken after oral dosing were collected into cooled 5 ml ethylenediamine tetraacetic acid (EDTA) tubes (LIP Equipment Services, UK)

AdV1. Hillery et al. / Journal of Controlled Release 42 (1996) 65-73

68

containing 0.25 ml Trasylol (15 trypsin inhibitor units/ml, Sigma, UK), and centrifuged at 3000 × g for 5 min at 4°C. The plasma was stored at - 2 0 ° C until assay. Aliquots (0.25 ml) were extracted with cooled absolute ethanol (1 ml). The protein precipitate was removed by centrifugation at 2500 × g for 15 min at 4°C. The supernatant was decanted, evaporated to dryness using N 2 flow and re-constituted in assay buffer (0.2 ml). Standard curves to quantify LHRH uptake in vivo were prepared by vortexing LHRH-vinylacetate standards with blood samples, followed by plasma preparation and extraction as described above. The efficiency of the extraction procedure was determined by vortexing an aliquot of [~25I]LHRH with blood samples, preparing and extracting the plasma, and calculating the percentage recovery: (%) Recovery = 100

(200 /xl) and rat plasma (200 /zl) were mixed, an aliquot of the mixture (200 /~1) was applied to the column and the resulting fractions were analyzed for the presence of radioactivity by scintillation counting and for the presence of protein using the Bradford assay [14].

2.5.2. In vivo experiments Plasma (200 /xl) from blood samples taken 3 h after dosing with the CPP system was applied to the column, fractions (1 ml) were collected and analyzed for radioactivity by scintillation counting and for the presence of protein using the Bradford assay. The fractions corresponding to the radioactive peak (fractions 5 - 1 0 ) were pooled and diluted with buffer prior to RIA.

3. R e s u l t s a n d d i s c u s s i o n

( Total cpm after extraction × \ Total cpm before extraction £ The extraction recovery was determined as 85% -+ 2%

2.5. Gel filtration chromatography 2.5.1. In vitro experiments CPP particles (200/xl) were eluted on a Sepharose CL-2B column (Pharmacia, Herts) using phosphate buffered saline (PBS, 0.15 M, pH 7.4) as the eluent and an elution volume of 20 ml. The resulting fractions (1 ml) were analyzed for the presence of radioactivity by scintillation counting. CPP particles

3.1. Radioactivity analysis Approximately 9% of the administered dose of radiolabel was detected in the blood, and 25% in the analyzed organs, 3 h after oral dosing (Table 1). The detected uptake gradually decreased to 5.6% in the blood and 13.2% in the organs over a 48 h analysis period (Table 1). The detected uptake of radiolabel in blood and organs was relatively high and difficult to reconcile with other reports on the low oral uptake of either LHRH [15] or particulates [16,17]. Results are likely to be overestimates of absorption, measuring the radiolabel as opposed to the uptake of intact peptide or particles. This lack of specificity, coupled

Table 1 Percentage uptake of radiolabel detected in blood and tissue samples after single dosing with CPP particles" Sample

2h

6h

12 h

24 h

Stomach S.I. L.I. Liver Spleen Kidneys Uptake Blood Total

2.4 _+0.4 7.6 -+ 0.8 1.0 +_0.1 7.2 -+ 0.3 0.6 +- 0.1 5.7 -+ 0.3 24.5 -+ 2.0 8.7 -+ 0.4 33.2 -+ 2.4

1.8 _+0.7 6.2 -+ 0.5 1.7 -+ 0.1 6.9 + 0.2 0.6 + 0.1 5.1 -+ 0.2 22.3 -+ 1.8 9.0 -+ 0.3 31.3 -+ 2.1

1.2 _+0.4 5.1 _+0.5 0.8 -+ 0.2 6.8 -+ 0.3 0.6 -+ 0.2 5.1 -+ 0.2 19.6 -+ 1.8 6.4 -+ 0.4 26.0 -+ 2.2

1.0 -+ 0.7 4.7 -+ 0.6 0.9 _+0.2 5.6 -+ 0.4 0.5 -+ 0.1 3.8 -+ 0.3 16.5 -+ 2.3 5.4 -+ 0.3 21.9 -+ 2.6

"Results are expressed as mean values _+ SD (n = 3), blood results include extrapolation to whole blood volume.

48 h 0.7 -+ 0.3 3.0 -+ 0.5 0.9 -+ 0.3 5.5 -+ 0.3 0.5 -+ 0.1 3.1 -+0.2 13.2 -+ 1.7

5.6 -+ 0.3 18.8 + 2

A.M. Hillery et al. / Journal of Controlled Release 42 (1996) 6 5 - 73

with the possibility of tritium exchange, prompted the use of the more sensitive and specific analytical technique of RIA to investigate the potential of the CPP system.

3.2. Radioimmunoassay for LHRH In vitro, RIA could detect approximately 10% of the peptide present in the CPP system. This suggested that 10% of the peptide was accessible to antiserum binding, the remainder of the peptide being trapped within the polymeric matrix and physically shielded from interaction with antibody. A shielding effect caused by a polyalkylcyanoacrylate (PACA) particulate matrix was also demonstrated when a RIA was used to detect growth hormone releasing factor (GRF) adsorbed to polyhexylcyanoacrylate (PHCA) nanoparticles [18], the antiserum being unable to detect encapsulated GRF, but able to bind unabsorbed GRF in solution. After oral dosing with the CPP system, LHRH was detected in the blood over a 12 h period (Fig. 1). No LHRH was detected in the blood of animals

0 a

0

1 ~ $ 4

S 6

T 8 9 10 11 12 13

Time (h) Fig. I. Percentage uptake of immuno-reactive LHRH in plasma after single dosing with the CPP system, determined using RIA.

69

dosed with saline. The non-detection of peptide in the saline control samples confirmed the efficiency of the extraction procedure in removing interfering substances from the assay, and also demonstrated the specificity of the antiserum for LHRH. Oral dosing with the free peptide or LHRH-vinylacetate in a buffer vehicle also did not produce detectable levels of LHRH in the blood, confirming the ability of the CPP particles to promote the oral uptake of LHRH. These results also confirmed the specificity of the antiserum for the recognition of intact LHRH. In order to assess the effect of the presence of nanoparticles, n-BCA particles of 100 nm were synthesized and LHRH was dissolved in the continuous phase of the suspension, which was subsequently orally dosed. No peptide was detected in the blood. In this case, although LHRH was in the presence of particles as a physical mixture, it was not encapsulated within the nanoparticulate matrix. Maximum plasma uptake, amounting to 1.6% of the administered dose of LHRH, was detected 3 h after single oral dosing with the CPP system (Fig. 1). The total cumulative uptake of LHRH was significantly higher, as demonstrated by the area under the plasma-time curve (AUC) determined over a 12 h post-dosing period (Fig. 1). Although the half life of LHRH in the blood is normally approximately 2 - 8 min [19], dosing with the CPP system meant that immuno-reactive peptide was detected up to 12 h after dosing. The 'shielding effects' of the CPP particulate matrix may have resulted in an under-estimation of peptide uptake using RIA, if particles exist in the blood. Furthermore, large molecular species in blood samples interfere with LHRH binding to specific antibodies [20,21], and plasma proteases rapidly degrade LHRH [22,23]. Therefore, untreated plasma samples cannot be used for LHRH determination [19,24] and immediate chilling and extraction with ethanol [25] is essential. The concentration of LHRH in vivo may also have been under-estimated because intact CPP particles or their break-down products may not have been recovered entirely in the postsampling ethanol extraction. Because of the limitations associated with the RIA, which include shielding effects from the matrix and the possibility of incomplete extraction of LHRH, the results for LHRH uptake in vivo may

70

A.M. Hillery et al. / Journal of Controlled Release 42 (1996) 65-73

only represent the minimum values of uptake. However, the RIA clearly demonstrated that oral dosing with the CPP system was capable of promoting the oral uptake of LHRH, resulting in a plasma AUC for the peptide, as well as sustained plasma levels. In contrast, Lowe and Temple [26] demonstrated that no significant overall enhancement of the oral delivery of calcitonin was achieved using polyisobutylcyanoacrylate (PIBCA) nanoparticles. Intragastric administration of PIBCA nanocapsules loaded with insulin to fasted diabetic rats resulted in prolonged hypoglycaemic effects; however, the pharmacokinetics were rather unusual in that hypoglycaemia was detected after a 2-day delay, but persisted for up to 20 days [27]. The use of particulates as oral carriers has been previously demonstrated for nonffpeptide drugs including vincamine [28], Lipiodol [29] and indomethacin [30]. The biological activity of the peptide after oral dosing in the CPP system was confirmed in rats using a bioassay which investigated the paradoxical anti-reproductive effects on spermatogenesis, serum testosterone levels and weight of the male accessory sex organs, caused by chronic exposure to LHRH [31].

3.3. Multiple oral dosing with CPP particles and detection of LHRH 0.64/zg of LHRH was detected in the blood after a single dose of CPP particles, which rose to 2.5 times this concentration (1.6/xg) after multiple (2 or 5 days) dosing (Table 2). Interestingly, the same concentration of LHRH was detected in the plasma Table 2 Concentration of LHRH in blood after 1, 2, or 5 doses of CPP particles, determined by RIAa Dosing scheduleb

Concentration (ng)~

(%) Uptake

Single dose Dosing for 2 days Dosing for 5 days

640 _+ 100 1600 _+ 160 1600 + 160

1.6 -+ 0.25 4.0 _+ 0.4d 4.0 _+ 0.4d

aResults are expressed as mean values + SD. hEM. 3 h after the final (i.e. first, second or fifth) dose. CAftercorrecting for dilution factors and extrapolatingto the total blood volume. dApparent percentage uptake.

after 2 or 5 days dosing with the CPP system, suggesting saturation of the uptake process. This hypothesis is currently under investigation. The detection of 1.6/zg LHRH in the blood after multiple dosing further demonstrated the potential of the delivery system for allowing the oral absorption of LHRH. The threshold dose for LHRH therapeutic activity in man is 10 /zg [32]. For multiple dosing, the percentage uptake was calculated with respect to the concentration of a single administered dose (0.04 mg). This gives an apparent uptake of approximately 4% after 2 days dosing, significant uptake for a peptide which is not normally absorbed, and significantly higher than the determination of 1.6% uptake after single dosing. 3.4. Determination of CPP particle integrity using gel filtration chromatography In vitro, the CPP particles eluted in the void volume of a Sepharose CL-2B column as determined by scintillation counting of the eluted fractions (Fig. 2, panel A). When CPP particles and plasma were mixed in vitro, the particles eluted prior to the plasma proteins (Fig. 2, panels A and C). For plasma samples taken 3 h after oral dosing with the CPP system, the radioactivity was detected in the fractionation range of the column (Fig. 2, panel B), suggesting that the particles had broken down in the blood in vivo. Nevertheless, these fractions also contained intact immuno-reactive LHRH, as determined by carrying out the RIA on the fractions corresponding to the radioactive peak. The CPP particles may have degraded by surface erosion, in a similar manner to the in vivo degradation of PACA nanoparticles [33,34]. Surface bio-erosion resulted in smaller diameter, but still intact particles, which eluted later from the column than the original CPP particles. Interestingly, LHRH co-eluted with the plasma proteins after oral dosing with the CPP system (Fig. 2, panels B and C). However, LHRH is not normally associated with plasma proteins [35,36] and, as described above, intact CPP particles do not associate with plasma proteins (Fig. 2, panels A and C). This suggested that the CPP system degraded in vivo and that CPP metabolites containing LHRH were subsequently associated with plasma proteins.

71

A.M. Hillery et al. / Journal of Controlled Release 42 (1996) 6 5 - 7 3 O0 -

SO00

• (e) SO

V

|. a

n,., 10

'---:f:---'--::T

0

0

10

Elutlon V o l u m e

'

I

lO

o

30

:==-J, 0

I

--T . . . . T

10

2O

'

I ao

Elutlon V o l u m e (mL)

(mL) OJ

• (c) E

c ur) 0.2 a ~D

o u c gl

0.1 o JD

4C

0.0

m - ,

.

0

.

.

.

'

I ....

T ....

10

Elutlon V o l u m e

T

20

'

I

30

(mL)

Fig. 2. Elution profile on Sepharose CL-2B of CPP particles in vitro (panel A), plasma samples taken 3 h after oral dosing with CPP particles, analyzed by either scintillation counting (panel B) or the Bradford assay for protein determination (panel C).

72

A.M. Hillery et al. / Journal of Controlled Release 42 (1996) 65-73

Protein-bound L H R H may have been inaccessible to antiserum binding sites and this shielding effect may also have resulted in an under-estimation in the amount of peptide present in the samples. The binding of CPP metabolites containing L H R H to plasma proteins may be responsible for the apparent prolonged half-life of the peptide. The extended half-life of the L H R H analogue, naferelin acetate, has been attributed to its increased hydrophobicity, which resulted in greater plasma binding and protection of the molecule against enzymatic degradation [30]. Protein binding may also have reduced glomerular filtration of the peptide, a process that causes extensive degradation of L H R H by the brush border enzymes of the proximal tubule [37]. Although the GPC results demonstrated that the CPP particles were at least partly broken down in the blood, the measured oral delivery of immuno-reactive L H R H using this system, taken in conjunction with the results of various other studies which have demonstrated the phenomenon of particulate uptake [2-4] and our own findings that non-encapsulated L H R H was not detectable after oral dosing, suggest that at least some of the CPP particles remained intact in the gastrointestinal (GI) tract to protect the peptide from degradation. It is also obvious that biologically active L H R H is released from the particles after oral absorption of the intact LHRHCPP complex.

4. Conclusions Initial studies using a radiolabelled complex to assess uptake of L H R H in vivo after oral dosing with the CPP system were ambiguous, but measurement of immuno-reactive peptide absorbed after delivery in the system was shown by RIA, whereas the free peptide showed no detectable absorption. The R I A may have under-estimated the extent of oral uptake of LHRH, because of incomplete extraction of L H R H and also due to the shielding effects of the intact particles, CPP degradation products, and plasma proteins (which might be associated with CPP metabolites in vivo). The CPP particles were capable of delivering L H R H to the blood, whereas free L H R H or nonencapsulated LHRH, i.e., L H R H dissolved in the

continuous phase of a suspension of particles, were not absorbed. It was concluded that at least some of the CPP particles remained intact in the GI tract to protect the peptide, and that it was this proportion of intact particles which were responsible for the detected uptake of LHRH. The chemical conjugation of L H R H within a protective particulate matrix proved a viable approach to promoting oral delivery, although there is, of course, a need to optimize the parameters of the construct.

Acknowledgments We thank Professor Calum B. Macfarlane, formerly of the Syntex Research Centre, Edinburgh, for his interest and for the provision of support for A.M.H.

References [1] A.M. Hillery, I. Toth, A.M. Shaw and A.T. Florence, Copolymerised peptide particles: synthesis, characterisation and in vitro studies on a novel oral nanoparticulate delivery system, J. Control. Rel. (1996) in press. [2] D.T. O'Hagan, Intestinal translocation of particulates: implications for drug and antigen delivery, Adv. Drug Del. Rev. 5 (1990) 265-285. [3] A.T. Florence and P.U. Jani, Particulate delivery: the challenge of the oral route, in: A. Rolland (Ed.) Pharmaceutical Particulate Carriers, Therapeutic Applications, Marcel Dekker, New York, 1993, pp. 65-107. [4] J. Kreuter, Peroral administration of nanoparticles, Adv. Drug Del. Rev. 7 (1991) 71 86. [5] P.A. Flanagan, P. Kopeckova, J. Kopecek and R. Duncan, Evaluation of protein-N-(2-hydroxypropyl)methacrylamide copolymer conjugates as targetable drug carriers. 1. Binding, pinocytic uptake and intracellular distribution of transferrin and anti-transferrin receptor antibody conjugates, Biochim. Biophys. Acta. 933 (1989) 83-91. [6] L.M. Sanders, Controlled delivery systems for peptides, in: V.H.L. Lee (Ed.) Peptide and Protein Drug Delivery, Marcel Dekker, New York, 1991, pp. 785-806. [7] J. Kopecek, P. Kopecova, H. BrCnsted, R. Rathi, B. Rihova, P.Y. Yeh and K. Ikesue, Polymers for colon-specific drug delivery, J. Control. Rel. 19 (1992) 121-130. [8] P. Kopeckova, R. Rathi, S. Takada, B. Rihova, M.M. Berenson and J. Kopecek, Bioadhesive N-(2-hydroxypropyl)methacrylamide copolymers for colon-specific drug delivery, J. Control. Rel. 28 (1994) 211-222. [9] P. Couvreur, B. Kante, M. Roland, P. Guiot, P. Bauduin and P. Speiser, Polycyanoacrylate nanocapsules as potential lysomotropic carriers: preparation, morphological and sorptive properties, J. Pharm. Pharmacol. 31 (1979) 331-332.

A.M. Hillery et al. / Journal of Controlled Release 42 (1996) 6 5 - 7 3

[10] C.S. Randall, T.R. Malefyt and L.A. Sternson, Approaches to the analysis of peptides, in: V.H.L. Lee (Ed.) Peptide and Protein Drug Delivery, Marcel Dekker, New York, 1991, pp. 203-246. [11] J.M. Samanen, Physical biochemistry of peptide drugs: structure, properties, and stabilities of peptides compared with proteins, in: V.H.L. Lee (Ed.) Peptide and Protein Drug Delivery, Marcel Dekker, New York, 1991, pp. 137-166. [12] A.M. Hillery, P.U. Jani and A.T. Florence, Comparative quantitative study of lymphoid and non-lymphoid uptake of 50 nm polystyrene particles, J. Drug Target. 2 (1994) 151156. [13] B.M. Mitruka and H.M. Rawnsley (Eds.) Clinical biochemical and hematological reference values in normal experimental animals, Masson, New York, 1977 pp. 14. [14] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein binding, Anal. Biochem. 72 (1976) 248-254. [15] B.H. Vickery, Comparison of the potential for therapeutic utilities with gonadotropin-releasing hormone agonists and antagonists, Endocr. Rev. 7 (1986) 115-124. [16] P.G. Jenkins, K.A. Howard, N.W. Blackhall, N.W. Thomas, S.S. Davis and D.T. O'Hagan, The quantitation of the absorption of microparticles into the intestinal lymph of Wistar rats, Int. J. Pharm. 102, 261-266. [17] D.T. O'Hagan, N.M. Christy and S.S. Davis, Particulates and lymphatic drug delivery, in: W.N. Charman and V.J. Stella (Eds.) Lymphatic Transport of Drugs, CRC Press, Boca Raton 1992, pp. 279-315. [18] J.C. Gautier, J.L. Grangier, A. Barbier, P. Dupont, D. Dussossoy, G. Pastor and P. Couvreur, Biodegradable nanoparticles for subcutaneous administration of growth hormone releasing factor (hGRF), J. Control. Rel. 20 (1992) 67-78. [19] D.J. Handelsman and R.S. Swerdloff, Pharmacokinetics of gonadotropin-releasing hormone and its analogues, Endocr. Rev. 7 (1986) 95-105. [20] C.P. Fawcett, E.A. Beezley and J.E. Wheaton, Detection of a second form of luteinizing hormone releasing factor, Int. Res. Commun. Sy. Libr. Compend. 2 (1974) 1663. [21] K.G. de la Cruz and A. Arimura, Evidence for the presence of immunoreactive plasma LH-RH which is unrelated to LH-RH decapeptide, Proc. 57th Ann. Meeting Endocrine Soc, New York, 1975, p. 103 (abstract). [22] K. Elkind-Hirsch, V. Ravnikar, I. Schiff, D. Tulchinsky and K.J. Ryan, Determination of endogenous immunoreactive luteinizing hormone-releasing hormone in human plasma, J. Clin. Endocrinol. Metab. 54 (1982) 602-607. [23] A. Arimura, A.J. Kastin, D. Gonzales-Barcena, D.H. Coy, E.J. Coy, D.S. Schaich and A.V. Schally, Disappearance of LH-releasing hormone in man as determined by radio-immunoassay, Clin. Endocrinol. 3 (1974) 421-425. [24] A. Amiura, A.J. Kastin, A.V. Schally, M. Salto, T. Kumasada, N. Nishi and K. Okhura, Immunoreactive LHreleasing hormone in plasma: Mid-cycle elevation in women, J. Clin. Endocrinol. Metab. 38 (1974) 510-513.

73

[25] T.M. Nett and G.D. Niswender, Gonadotropin-releasing hormone, in: B.W. Jaffe and H.R. Behrman (Eds.) Methods of Hormone Radioimmunoassay, 2nd Edition, Academic Press, New York, 1979, pp. 57-75. [26] P.J. Lowe and C.S. Temple, Calcitonin and insulin in isobutylcyanoacrylate nanocapsules: protection against proteases and effect on intestinal absorption in rats, J. Pharm. Pharmacol. 46 (1994) 547-552. [27] C. Damgr, C. Michel, M. Aprahamian and P. Couvreur, New approach for oral administration of insulin with polyalkylcyanoacrylate nanocapsules as drug carrier, Diabetes 37 (1988) 246-251. [28] P. Maincent, R. Le Verge, P. Sado, P. Couvreur and J.P. Devissaguet, Disposition kinetics and oral bioavailability of vincamine-loaded polyalkylcyanoacrylate nanoparticles, J. Pharm. Sci. 10 (1986) 965-968. [29] M. Aprahamian, C. Michel, W. Humbert, J.P. Devissaguet and C. Damgr, Transmucosal passage of polyalkylcyanoacrylate nanocapsules as a new drug carrier in the small intestine, Biol. Cell. 61 (1987) 69-76. [30] N. Ammoury, H. Fessi, J.P. Devissaguet, M. Allix, M. Plotkine and R.G. Boulu, Effect on cerebral blood flow of orally administered indomethacin-loaded poly(isobutylcyanoacrylate) and poly (DL-lactide) nanocapsules, J. Pharm. Pharmacol. 42 (1990) 558-561. [31] A.M. Hillery, I. Toth and A.T. Florence, Co-polymerised peptide particles (CPP) III: Biological evaluation of a novel nanoparticulate peptide delivery system using luteinizing hormone releasing hormone (LHRH), Pharm. Sci. (1996) in press. [32] C. Manieri, A.R. Marolda, M. Musso, R. Pastorino and M. Messina, The GnRH 10 micrograms test promotes a maximal gonadotropic response in normal women, Gynecol. Endocrinol. 6 (1992) 167-170. [33] R.H. Mfiller, C. Lherm, J. Herbort and P. Couvreur, In vitro model for the degradation of alkylcyanoacrylate nanoparticles, Biomaterials 11 (1990) 590-595. [34] J.L. Grangier, M. Puygrenier, J.C. Gautier and P. Couvreur, Nanoparticles as carriers for growth hormone releasing factor, J. Control. Rel. 15 (1991) 3-13. [35] L. Tharandt, H. Schulte, G. Benker, K. Hackenburg and D. Reinwein, Binding of luteinizing hormone releasing hormone to human serum proteins - - influence of a chronic treatment with a more potent analogue of LHRH, Horm. Metab. Res. 11 (1979) 391-394. [36] R.L. Chart and M.D. Chaplin, Plasma binding of LHRH and nafarelin acetate, a highly potent LHRH agonist, Biochem. Biophys. Res. Commun. 127 (1985) 673-679. [37] F.A. Carone, D.R. Peterson and G. Flouret, Renal tubular processing of small peptide hormones, J. Lab. Clin. Med. 100 (1982) 1-14.