Human plasma enzymatic hydrolysis

The stability of these compounds is maximal at pH 4 - 6, and decreases very sharply at lower and higher pH values, and the mechanism and products of the reaction differed with pH. In the neutral range, hydrolysis yielded the aromatic sulfonamide and the ester, whereas, under acid catalysis in the low pH range, the products were the AT-acyl sulfonamide and an alcohol (R OH, Fig. 11.9). Of particular interest is that the tm values for hydrolysis of the N-sulfonyl imidates in 80% human plasma were 3-150 times lower than in buffer solution at identical pH and temperature. This was taken as evidence for enzymatic hydrolysis by human plasma hydrolases. Hydrolysis under these conditions yielded the sulfonamide and the ester in quantitative amounts. 

The next example is 1,3-dibenzoylurea (4.222), a diacetylated linear urea. The hydrolysis kinetics and enzymatic cleavage of 1,3-dibenzoylurea together with various other /V-acylbenzamides were studied to assess the suitability of these compounds as prodrugs for the amido group [113]. At pH 7.4 and 37°, 1,3-dibenzoylurea was hydrolyzed quantitatively to benzoic acid and N-benzoylurea (4.223) with a tm value of 39 h. Since the hydrolysis of 1,3-di-benzoylurea was not catalyzed by human plasma, it was concluded that the acyclic diureide structure is not appropriate in prodrug design. 

We have included in this Section the presentation of aminoacyltriazenes since they can be formally regarded as imino derivatives of hydrazide. Aminoacyltriazenes have been developed as chemically stable triazene prodrugs capable of enzymatic hydrolysis under physiological conditions to liberate cytotoxic monomethyltriazene antitumor agents 4.276 [189], The aminoacyl-triazene prodrugs were found to undergo hydrolysis in isotonic phosphate buffer and in human plasma. A /3-alanyl derivative (4.274) was more stable... 

The L-dopa esters were also examined for their enzymatic hydrolysis in human plasma and/or by purified pig liver carboxylesterase (EC 3.ELI Table 8.1). In human plasma under the conditions of study, hydrolysis again followed pseudo-first-order kinetics. In all but two cases examined, enzymatic hydrolysis was slightly faster than chemical hydrolysis. For the methyl ester, rates of chemical and enzymatic hydrolysis were comparable, whereas the /erf-butyl ester was not hydrolyzed in plasma and was protected from chemical hydrolysis presumably by becoming bound to plasma proteins. 

A well-designed series of AA-disubstituted 2-aminoethyl and 3-amino-propyl esters of indomethacin (8.9, R = OH) was examined for its chemical and enzymatic (human plasma) rates of hydrolysis [40]. The 3-(diethylami-no)propyl ester had a transcutaneous permeability coefficient ca. 100-times... 

R = Me, Et, and PhCH2, respectively Fig. 8.1). In 80% human plasma at pH 7.4 and 37°, these model prodrugs were hydrolyzed with tm values of 3.5, 16, and 2.6 min, respectively [59]. Such rates of enzymatic hydrolysis are comparable to those of various carbamoylmethyl esters of benzoic acid (Table 8.2). It is important to note that the direct liberation of benzoic acid by Reaction a (Fig. 8.1) was severalfold faster than the competitive Reaction b. Reaction c was very slow in human plasma (tm > 100 h). In HO -catalyzed hydrolysis, the opposite regioselectivity was seen, with the terminal ester bridge being cleaved markedly faster than the central one. No data appears to be available on chemical hydrolysis at neutral pH. 

Compared to the carbamoylmethyl esters discussed in Sect. 8.2.3, glycolic acid esters appear somewhat disadvantageous because of the possibility of a parasitic reaction. The fact that the chemical hydrolysis of glycolic acid esters is faster while their enzymatic hydrolysis in human plasma is slower may not necessarily be detrimental. 

A number of prodrugs in clinical use are esters of fatty acids. For example, haloperidol decanoate is of interest in slow-release preparations. This compound was hydrolyzed by such hydrolases as purified carboxylesterase but was reported to be stable in human blood or plasma and in a variety of rat tissue homogenates [107], The source of this apparent lack of reactivity was competitive binding to blood and tissue proteins. In other words, protein binding sequesters this very lipophilic prodrug and prevents enzymatic hydrolysis, thereby slowing its activation and prolonging its in vivo effects. 

In a series of ten morphine 3-benzoates, large differences in rates of enzymatic hydrolysis were seen [122], In 80% human plasma at pH 7.4 and 37°, the unsubstituted 3-benzoate had a tm value of 0.6 h, whereas esters of 2,6-disubstituted benzoic acid were much more resistant to enzymatic attack (t1/2 ranging from 60 h for the dimethylbenzoate to 300 h for the dichloro-and dimethoxybenzoates). Although these results point to marked steric hindrance, electronic effects cannot be excluded but escape characterization because of the limited series. Furthermore, and as mentioned repeatedly in this text, the possibility of binding to plasma proteins is a complicating factor that should be kept in mind. 

Benzoic acids substituted with a basic side chain also are also of interest as pro-moieties whose physicochemical properties and rates of enzymatic hydrolysis can readily be modulated. A number of drugs have been converted to prodrugs with this type of pro-moiety, e.g., hydrocortisone, prednisolone, acyclovir, chloramphenicol, and paracetamol [148] [149], These prodrugs appear well suited as parenteral formulations, being water-soluble, stable in slightly acidic solution, and readily hydrolyzed enzymatically. As examples, we consider here the hydrolysis in human plasma of a number of (aminomethyl)ben-zoates of metronidazole (8.109-8.115, Sect. 8.5.5.1, Table 8.9) [138], These prodrugs are very rapidly activated, which may be beneficial for parenteral administration. However, this type of pro-moiety may be cleaved too rapidly after oral administration to be of interest for poorly absorbed drugs. 

Most of the hemiesters 8.136 underwent no or little enzymatic degradation in human plasma, in agreement with the known inertness of hemiesters toward cholinesterase (see Chapt. 7). In contrast, very rapid hydrolysis was usually seen in pig and rat liver preparations, indicating the involvement of carboxylesterases. The only inert compound was the 3,3-dimethylglutarate hemiester of paracetamol (8.136, X = C(CH3)2CH2, Fig. 8.12). Data on the hydrolysis of such prodrugs by human hepatic enzymes will be welcome. 

The stability of some prodrugs and mutual prodrugs (8.137 and 8.139, Fig. 13) in human and rat plasma was also examined [175], An approximately tenfold acceleration was noted for the mutual prodrug 8.139 at pH 7.4 and 37° for human plasma (71/2 ca. 45 s) compared to buffer (t1/2 ca. 400 s). This and other evidence indicated that cleavage of these carbamates is enzymatic. In contrast, the A-(2-hydroxyphenyl)carbamates (8.137, Fig. 13) showed two- to threefold increases in t1/2 values in human and rat plasma compared to buffer, indicating the absence of an enzymatic hydrolysis, and modest stabilization due to binding to plasma proteins. 

Four/V-[(acyloxy)methyl] derivatives of 5-FU (8.152, R = AcOCH2, Et-COOCH2, PrCOOCH2, and (7-Bu)COOCH2) were very stable in buffer at pH 7.4 and 37° (t1/2 70, 90, 140, and 700 h, respectively) [219], In 80% human plasma under the same conditions of pH and temperature, the f1/2 values were 14, 9.6, 2.3, and 40 h, respectively, i.e., a marked substrate-dependent acceleration. In rat liver homogenates, the tm values for decomposition were in the order of a few minutes or less. Clearly enzymatic hydrolysis is possible for such compounds and may even be quite significant. Additional data on A-[(acyloxy)methyl] prodrugs have confirmed their potential for topical delivery of 5-FU [220],... 

A- [(Acy loxy )methyl] derivatization was also examined for its potential to improve the biological stability of peptides. For example, the peptide-like model A-[(benzyloxy )carbonyl]glycine benzylamide (8.171, R = H) was de-rivatized to a few N-/Yacyloxy)methyl] derivatives whose chemical and enzymatic hydrolysis was investigated [225], The results compiled in Table 8.13 indicate a fast chemical hydrolysis, the mechanism of which is depicted as Reaction b in Fig. 8.21. Enzymatic hydrolysis also occurs in human plasma, resulting in short half-lives, with the exception of the pivaloyl analogue. 

N. Mprk, H. Bundgaard, Stereoselective Enzymatic Hydrolysis of Various Ester Prodrugs of Ibuprofen and Flurbiprofen in Human Plasma , Pharm. Res. 1992, 9, 492 - 496. 

Human and rat serum, plasma, and urine Q Extraction with EtAc, centrifugation, derivatization with BSTFA, enzymatic hydrolysis for conjugates DB-5 He GC/MS/SIM 0.01 (g/L) 104... 

Human plasma D, DHD, G, ODMA Acidification, enzymatic hydrolysis, extraction with C18 SPE or diethyl ether, partitioning C-8 (Phenolphthalein glucuronide, 4-methylumbelliferone glucuronide, BCA) ACN-H20- ammonium acetate HPLC/MS/MS/ APCI(-) 24... 

VX appears to follow a similar pathway, the major metabolite being ethyl MPA (EMPA). An additional metabolite, derived from the diisopropy-laminoethyl substituent, was identified in human plasma following an assassination with VX (45). The sulfide (17), derived from enzymatic S-methylation of the hydrolysis product HSCH2CH2N(i-Pr)2, was identified in human serum by GC/MS after simple extraction. Experiments in rats confirmed the rapid metabolic formation of (17) from HSCH2CH2N(i-Pr)2 (46). Identification of this metabolite distinguishes VX from the O-ethyl analogue of sarin. 

In human plasma, two groups of compounds were seen. In most cases, the reaction in plasma (i.e., chemical plus enzymatic activation) was approximately twofold faster than in buffer. This indicates that enzymatic hydrolysis in human plasma, if any, was modest at best. Only for the three prodrugs with an unsubstituted carboxamido group (R = H) was the enzymatic reaction several-fold faster than was intramolecular catalysis, suggesting these compounds to be substrates of plasmatic hydrolases. Thus, the A -(2-carbamoylphenyl)carbamate promoiety allowed a highly modulatable intramolecular activation with little enzymatic activation. 

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