Enzyme Inhibitory Effects of Polyacrylates

LueBen and coworkers (1996a) have studied the potency of mucoadhesive excipients to inhibit intestinal proteases. Among the different mucoadhesive polymers investigated, uniquely the poly(acrylates) polycarbophil and carbomer 934P were able to inhibit the activities of trypsin, a-chymotrypsin, carboxypeptidase A, and cytosolic leucine aminopeptidase. However, they failed to inhibit microso-mal leucine aminopeptidase and pyroglutamyl aminopeptidase. Carbomer was found to be more efficient to reduce proteolytic activity than polycarbophil (most probably due to various flexibilities of the polymer chains). The authors also could demonstrate the pronounced binding properties of polycarbophil and car-bomer for bivalent cations such as zinc and calcium, being a major reason for the observed inhibitory effect. These polymers were able to deprive Ca2+ and Zn2+, respectively, from the enzyme structures, thereby inactivating their activities. Carboxypeptidase A and a-chymotrypsin activities were observed to be reversible upon addition of Zn2+ and Ca2+ ions, respectively. Table 6.2 shows some of the results of luminal enzyme inhibition by polyacrylates. However, the poly(acrylic acid) derivatives polycarbophil and carbomer showed rather weak inhibitory effects on enzymes of the intestinal brush border cell membranes responsible for DGAVP and metkephamid degradation (LueBen et al., 1996b).

In another study, LueBen and coworkers (1996c) compared the enhanced intestinal absorption of carbomer, the neutralized carbomer-sodium salt (NaC934P), and chitosan hydrochloride for the peptide drug buserelin and concluded that the higher bioavailability with chitosan hydrochloride compared to carbomer and NaC934P is an indication that for buserelin the intestinal transmucosal transport enhancing effect of the polymer plays a more dominant role than the protection against proteases such as a-chymotrypsin.

It is emphasized again that the decrease of enzyme activity is time dependent (i.e., after 10-20 min maximum deactivation is achieved). Although enzyme inhibition by these polyacrylates is not an absorption-enhancing effect per se, especially in the intestinal peptide and protein absorption, it is a more than favorable effect to increase the amount of peptide drug absorbed (LueBen et al., 1996b, 1997).

Table 6.2. Effects of Polycarbophil® (PCP) and Carbomer 934P® (C934P) on protease activities (Lueßen et al., 1995, 1996a)

Enzyme

Polymer

Concentration (%)

Inhibitiona

Trypsin

PCP

0.35

+

0.25

±

0.15

-

C934P

0.25

++

0.15

+

0.1

±

a-Chymotrypsin

PCP

0.5

±

0.25

-

C934P

0.5

+

0.25

-

Carboxypeptidase A

PCP

0.1

+

0.05

±

C934P

0.1

++

0.05

+

Leucine

PCP

0.5

-

Aminopeptidase M

C934P

0.5

-

Leucine

PCP

0.5

++

Aminopeptidase C

0.25

+

C934P

0.5

++

0.25

+

Pyroglutamyl

PCP

0.5

-

Aminopeptidase

C934P

0.5

-

a ++, strong inhibition; +, inhibition; ±, slightly reduced enzyme activity; —, no inhibition a ++, strong inhibition; +, inhibition; ±, slightly reduced enzyme activity; —, no inhibition

Removal of endogeneous Ca2+ from the intestinal epithelial cells by the formation of poly(acrylic acid)-Ca2+ complexes loosens the cellular barrier, especially by triggering the (reversible) opening of the tight junctions. This effect has been demonstrated by Borchardt et al. (1996) by flux studies of mannitol and dextran fluorescently labeled fluorescein isothiocyanate (FITC-dextran, MW 4,400 Da) across intestinal Caco-2 cell monolayers after incubation with poly(acrylic acid) derivatives and by visualization studies using CLSM. It should be noted that the observed reversibility in tight junction opening was gradual only because of the high viscosity and adhesive character of the polymer solutions to the cell layer.

Carbopol 934P was also used by Thanou and collaborators (2001b) for the enhancement of the intestinal absorption of low molecular weight heparin (LMWH) in rats and pigs. LMWH is a polyanion and does not interact with polycarbophil 934P. To both animal species LMWH was administered intraduo-denally and the antiXa levels were measured. Both studies showed a remarkably enhanced LMWH uptake after about 1 h and the effect for providing sufficient antithrombotic effect lasted for both animal species about 7 h showing that this polyacrylate may be a good absorption enhancer for LMWH, provided a good delivery system can be developed based on polycarbophil 934P.

6.3.3.2 Chitosan

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