Clearance of oxytocin and its potentially enzyme resistant analogues in the OXT‐receptor compartment of the potassium depolarized rat myometrium

1 INTRODUCTION

Neurohypophyseal hormones—oxytocin and vasopressin—are rather short-acting peptides in current pharmacological in vivo experiments. Their half-life in the blood plasma lies by human and animal species so far investigated between 1.5 and 8 min.1 Regardless of the gender and the reproductive circle by female animals, the half-life of oxytocin was estimated in rats to 1.65 min2; the related elimination rate constant is 0.6 min−1. However, estimates of the so-called overall decay rate constants (kρ)3 for its uterotonic and antidiuretic responses are considerably lower, 0.18 to 0.25 min−1. In general, low kρ values were reported in a number of reviews also for other neurohypophyseal peptide analogues3-7; those for uterotonic response to peptides used in this communication are outlined in Table 1.

TABLE 1. Peptides used as enzyme probes: Uterotonic activities and decay rates in vivo (rat) Symbol Substancea Common name Uterotonic activity (rat uterus)b Response decay rate kρ (min−1)c In vitro (IU) In situ (IU) OXT image Oxytocin

450  486  507  546

490

468

0.250 0.237

0.173 ± 0.023

DOT [1-β-mercaptopropionic acid]-OXT Deaminooxytocin

368  551  803

677

476 0.148 HOT [1-(2-Hydroxy-3-β-mercaptopropanoic acid)]-OXT Hydroxyoxytocin

1607  1641

1624

874 C1OT [6,1-Cystathionine]-OXT Carba1-oxytocin

368  743

743

120d

973

0.277 DC1OT [1,6-(2-Amino-4-thiasuberic acid)]-OXT Deamino-carba1-oxytocin 1899

1206  1251

1229

0.172 C1,6OT [1,6-α′,α-diaminosuberic acid]-OXT

Dicarba-oxytocin

Carba1,6-oxytocin

5.4 DC1,6OTe [6,1-(α-aminosuberic acid)]-OXT Deamino-dicarba-oxytocin 93 95 0.079 DC6OT [6,1-(2-Amino-4-thiasuberic acid)]-OXT Deamino-carba6-oxytocin 929

2792

746

0.041?

0.127 (interpolated)

AOT [9-Azaglycine]-OXT Azaglycine-oxytocin ≈700 DAOT [1-β-mercaptopropionic acid, 9-azaglycine]-OXT Deamino-azagylcine-oxytocin 1099 GOT [4-Glutamic acid-δ-methylester]-OXT 10.2f  18g DGOT [1-β-mercaptopropionic acid, 4-glutamic acid-δ-methylester]-OXT 21.4f  43g a Synthesis, properties and detailed nomenclature are summarized in K. Jošt, M. Lebl and F. Brtník (eds): CRC Handbook of Neurohypophyseal Hormone Analogs, vol. II, CRC Press, Inc., Bota Racon, FL, 1987 (pp. 127–267). b Uterotonic activities international units defined by The Third International Standard for Oxytocic, Vasopressor, and Antidiuretic Substances8 in international units (IU) per mg peptide. Data by various authors are shown in upper rows, adjusted values (see Section 3) in fat italics. In vitro activities relate to estimates in Mg2+-free tissue medium. Oxytocin (adjusted value) was used as a local reference peptide. c Rate constant of response decay (earlier formal elimination constant).9 Data: Barth et al.,4 Pliška3 (in italics), values denoted by a question mark (?) are outliers (corresponding values corrected by interpolation are specified; see text). d Value quoted in the thesis of O. Keller (Diss. ETH 5325, 1974) considered as a preliminary estimate. Adjusted value was attained by interpolation (see Result section). e Not used in the oil-immersion experiments presented here. f Pliška and Rudinger10 g Photaki et al.11

The time span of a response to neurohypophyseal peptides became an important factor in the clinical pharmacology. Structural changes potentially resulting in its prolongation were already subjects of early studies,12 and several prolonged analogues found their place in medicine. Thus, long-acting 1-deamino-8-D-arginine-vasopressin (dDAVP, Desmopressin INN) is currently utilized as the preferential drug in the substitution therapy of the central form of diabetes insipidus13 and/or as a haemostatic in mild forms of haemophilia A14; 1-triglycyl-8-lysin-vasopressin (Terlipressin INN) is occasionally used as a vasoconstrictor drug in various forms of cardiovacsular collapse and shock.15 Carbetocin (INN), 1-deamino-2-O-methyltyrosin-carba1-oxytocin,16 a long-acting partial agonist of oxytocin, found a prominent place in emergency obstetrics as a uterotonic and haemostatic drug in critical states of postnatal hemorrhagy17 or after caesarian sections.18

Earlier results of the pharmacokinetic analysis19, 20 point out that the response dynamics is predominantly controlled by the drug concentration in the immediate vicinity of the corresponding receptor sites, the so-called receptor compartment.12, 21-24 Thus, kinetics of clearance and drug transport processes are limiting factors in the response duration.

In order to investigate them more directly and to segregate the irreversible clearance from the peptide transport processes, we used a combination of the ex vivo1 washout experiments and the oil-immersion method (assigned here as a “stopped-transport” procedure), introduced by Kalsner and Nickerson.25, 26 In our modification,10, 22, 27 it consists in eliciting a steady-state tonic contraction of an isotonically or isometrically suspended muscle strip ex vivo by a uterotonic stimulant in an aqueous medium, and successively exchanging this medium for a mineral oil. The extreme hydrophobic barrier around the tissue prevents the reverse diffusion of the stimulating agents from the receptor compartment and hence allows following solely irreversible clearance in the response relaxation phase.

The peptide chain of oxytocin carries several potential sites exposed to enzymatic attacks reported in a number of reviews,28-31 and outlined in Figure 1. Splitting the Ν-terminal peptide bond without opening the -S-S- bridge by the so-called oxytocinase,32, 33 a placental leucine aminopeptidase of the P-LAP family, was initially detected in the blood serum during pregnancy. Action of other aminopeptidases after enzymatic or nonenzymatic (thiol-disulphide interchange) reduction of the disulphide bond appears as a likely inactivation step but has not been proved experimentally. The C-terminal hydrolysis of the Leu8-GlyNH29 bond by a carboxypeptidase-type enzyme,34-36 and the Pro7-Leu8 bond splitting37 were identified in homogenates of rat and human uteri.

image

Oxytocin analogues used as enzyme probes: schematic sequences. Arrows indicate the site of potential splitting at sequence sites 1, 6, 9 and at intramolecular -S-S- bond, grey areas mark protected sites

The so-called “enzyme probes,”10, 12, 38 analogues with potentially enhanced enzyme resistance of individual peptide chain sites, still possessing biological activity, were used to detect sites of oxytocin molecule critical for its clearance in the myometrial oxytocin receptor compartment (OXTR).2 Table 1 summarizes those currently employed in our oil-immersion experiments. Since most of the investigated peptides carry a combination of several protective modifications, the molecular segment contributions analysis according to Free and Wilson39 was applied to estimate site contributions of individual structural features to clearance and transport rate constants.

2 MATERIALS AND METHODS 2.1 Substances The peptides used here as enzyme probes and their abbreviations are listed in Table 1. Analogues modified on the N-terminal by removing, or altering, the amino group of 1-cystein are expected to assess sensitivity against aminopeptidase splitting. Substitution of one or both sulphur atoms in the -S-S- bridge by a CH2 group,40 the so-called carba-analogues, may clarify the potential clearance role of disulphide reduction. Analogues with a Leu8-Gly9 peptide bond (see Figure 1) modified by insertion of aza-glycine in position 941 were introduced as probes of potential C-terminal cleavage. Furthermore, two analogues esterified in position 4, [4-glutamic acid-δ-methylester]-oxytocin and its Ν-deaminated counterpart (GOT and DGOT, see Table 1), were included in our investigations as a proof of concept5, 10 as analogues eliciting short-lasting responses.

Oxytocin was donated by FERRING AB, Malmö, Sweden. The commercial products were purified by counter-current distribution and stored in lyophilized form. HOT42 was supplied by Dr. D. Hope, Oxford, England; AOT41 by Dr. H. Niedrich, Berlin-Friedrichsfelde, Germany; DC1OT and DC6OT by Drs. K. Jošt and T. Barth, Praha, Czech Republic; GOT and DGOT11 by Dr. I. Photaki, Athens, Greece. Deaminooxytocin and DAOT were prepared by Dr. M. Mühlemann, ClOT and Cl,6OT by Dr. O. Keller at the Department of Molecular Biology and Biophysics, ETH Zürich. Uterotonic activities of these analogues were assayed ex vivo on an isolated isometric rat uterus taken from females in natural oestrus, in the bath medium according to Munsick.43, 44 Activities in IU3 per mg peptide were collected from literature sources (standard: 3rd International Standard for Oxytocic, Vasopressor and Antidiuretic Substances8). Activities assessed in our laboratory (Table 1) were evaluated from dose–response curves according to the scheme suitable for nonparallel peptide/standard dose–response curves.9, 45 (details in Pliška and Krejčí45 accessible via ResearchGate portal, https://www.researchgate.net/publication/17259066).

2.2 Animals and tissue preparations

Female virgin Sprague–Dawley rats (body weight approximately 200 g) in natural oestrus (detected by vaginal smears) were sacrificed by decapitation, both uterus horns were dissected and the perimetrium was pulled off. Their middle sections (approximately 15 mm) were longitudinally cut into 3- to 4-mm strips; the endometrium (the inward layer of the horns) was removed by scraping. In average, three strips of each animal (31 rats in total) were used in the experiments.

2.3 Experimental procedure

A stripe was mounted in an adapted annealed organ chamber (17 mm × 65 mm) with a ventilation inlet tube containing 10-ml Munsick medium (see above) and attached to an isometric force transducer. The basic tension was adjusted to 10 ± 0.2 mN (1 gf, gram-force). Measurements were carried out at 30°C under continuous ventilation with Carbogen gas (25% O2 + 5% CO2). The protocol of time–response measurements was presented in detail in our precedent communication.22 Each strip used for stimulation by a single peptide (for concentration of stimulating agents, cf. Table 2). Oil-immersion and washout experiments were carried out on different strips from the same animal. Stimulation runs (two to four per strip) for each peptide were repeated on strips of different rat uteri. In order to avoid a presumed effect of tachyphylaxis, the duration of an experiment did not exceed 5 h. Number of strips used in oil-immersion experiments is indicated in Table 2.

TABLE 2. Oxytocin analogues: Estimated rate constants kr, κ (min−1) and affinities in K+-depolarizer rat myometrium Peptide Groupa No. of strips Runsb κ κ(N) − κ(D)e krc

Cumulative clearance

kr + κc

f

K+-depolarized myometrium

pCEg

Observedc Predicted d OXT N 6 35 0.137 ± 0.025 0.133 0.088 0.137 ± 0.030 0.254 ± 0.028 0.250 7.25 ± 0.45 DOT D 9 18 0.049 ± 0.021 0.037 0.149 ± 0.026 0.176 ± 0.024 0.148 8.28 ± 0.16 C1OT N 2 8 0.162 ± 0.032 0.181 0.060 0.116 ± 0.041 0.272 ± 0.037 0.277 7.43 ± 0.41 DC1OT D 4 6 0.102 ± 0.016 0.089 0.097 ± 0.025 0.160 ± 0.021 0.172 7.62 ± 0.24 C1,6OT N 2 3 0.076 ± 0.005 0.096 0.076 0.194 ± 0.011 0.263 ± 0.009 8.76 ± 0.11 DC1,6OTh D ≈0 0.226 4.94 AOT N 2 5 0.118 ± 0.008 0.115 0.076 0.158 ± 0.020 0.278 ± 0.015 DAOT D 7 11 0.042 ± 0.015 0.029 0.139 ± 0.044 0.194 ± 0.033 6.68 ± 0.50 GOT N 1 2 0.166 0.169 0.092 0.108 0.267 7.44 ± 0.44 DGOT D 2 6 0.076 ± 0.018 0.084 0.107 ± 0.034 0.211 ± 0.027 8.36 ± 0.20 HOT N 2 12 0.141 ± 0.008 0.137 0.139 ± 0.012 0.289 ± 0.010 5.41 ± 0.77 DC6OT D 6 7 0.105 ± 0.008 0.108 0.080 ± 0.020 0.147 ± 0.015 0.127 5.41 ± 0.77 Pooledi N 29 0.136 ± 0.028 0.079 ± 0.014 0.139 ± 0.031 0.266 ± 0.013 7.25 ± 0.45 D 40 0.073 ± 0.039 0.126 ± 0.041 0.172 ± 0.026 8.28 ± 0.16 a N: peptides carrying hydrophilic N-terminal group NH2 or OH, D: deamino-analogues. b Total number of computation runs (various estimation procedures and initial parameter estimates). c Arithmetic mean ± standard deviation (when no. of runs > 2). Dimension min−1. d Values recomputed from substituent contributions obtained by Free–Wilson analysis. e κ-difference of coherent N & D pairs κ(N),κ(D) (reflects a fraction of the overall inactivation rate equivalent to the splitting of C-terminal peptide bond). In italics: DC1,6OT estimate (cf. footnote d), not included in the mean value (i). f Affinity constants pCE ≡ −log CE estimated in this project. Arithmetic means ± standard deviations of (number of computed values: 3 to 15). Data obtained by Equation (12). g Not used in the present oil-immersion experiments (presented are solely predicted values; cf. footnote d). h Mean ± standard deviation of values of N and D groups. i Means ± standard deviations of values in groups N & D resulting in individual computation runs (value in italics deleted)

Figure 2 shows in a synoptic form the experimental setup. Tonic contraction of the depolarized myometrium strips in a high potassium Ringer organ bath medium46 (concentration of Ca2+ and of Mg2+: 0.5 mM) was elicited by roughly equipotent concentrations of single peptides around their D2 values (the concentration causes the half of the maximally attainable contraction in oestrogen-dominated myometrium). After reaching a steady-state level (stimulation phase), the medium was exchanged by a low viscosity paraffin oil (oil-immersion phase) or alternatively by the peptide-free bath medium (washout phase). The time-tension profiles of the strip were recorded by an isometric force transducer (Statham strain gauge UC3).

image

Phases of oil-immersion experiment in the oxytocin stimulated myometrium: compartment model and clearance kinetics (upper panels). The compartment system of peptide distribution (upper panels) consists of the receptor compartment as a part of the interstitial space (white background) and the aqueous tissue medium (light grey), suppressed in the oil-immersion phase by the mineral oil (dark grey). Upper part: stimulation phase (middle block), washout (left-hand block), insertion of oil (right-hand block; aqueous medium reduced from Vw to vw). The inserted block in the right-hand panel depicts a magnified membrane section with the potential remnant of the aqueous medium after the displacement by oil. Vertical arrows between two compartments indicate directions of the peptide transport, horizontal arrows its irreversible clearance. State (and steady state) equations for the receptor compartment (time change of the peptide concentration cr) are indicated. Lower part: response profiles (isometric contraction) of the depolarized myometrium strip to oxytocin in the respective phase. E(t) is a (here nonspecified) time–response function Φ of cr(t)

2.4 Evaluation routines

The software package Wolfram Mathematica™ (version 11.3) was employed for integration of differential equation systems describing the compartmented model (see below). Time-tension profiles were digitized in regular time intervals (0.3 to 2 min, according to the total length of the decay record). SYSTAT (version 13.2, NONLIN, regression, descriptive statistics and testing routines), GraphPad Prism (version 8.3) and in part also MATLAB routines (version R2021a: interpolation, interp1) were used for parameter estimates by nonlinear regression routines, numerical operations in the Free–Wilson analysis (see below), descriptive statistics and statistical testing.

3 RESULTS 3.1 Kinetic analysis 3.1.1 “Stopped-transport” by oil-immersion The structure of the compartment system employed in oil-immersion experiments was described in a block box form in our recent communication.22 We employed here its modified version focused on the comparison of clearance and transport rate constants (Figure 2). It consists virtually of two distribution spaces: the receptor compartment (subscript r; see Section 4) and the external aqueous tissue medium (w). The rate of mass transport of agents through the tissue-medium interface (transport constants kr, kw) is directly proportional to the rate constant of diffusion k (dimension: volume/time) and inversely proportional to the respective compartment volume Vr, Vw: for the receptor compartment kr = k/Vr, for the medium kw = k/Vw. The constant κ relates to the (irreversible) clearance rate from the receptor compartment (dimension: time−1). The rate equations determining the time response of the peptide concentrations in the corresponding compartments (cr, cw) are then urn:x-wiley:10752617:media:psc3372:psc3372-math-0001(1a)

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