Riku Niemi

An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A1 and cannabinoid CB1 receptors

At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB1 receptor. In the micromolar range, these compounds were shown to inhibit basal G‐protein activity, and this is often interpreted to implicate constitutive activity of the CB1 receptors in native tissue. We show here, using [35S]GTPγS binding techniques, that micromolar concentrations of SR141716 and AM251 inhibit basal G‐protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A1 receptor signaling is not eliminated. Unlike lipophilic A1 receptor antagonists (potency order DPCPX≫N‐0840 ∼cirsimarin>caffeine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 receptor‐dependent G‐protein activity. Importantly, all antagonists reduced basal signal to the same extent (20%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N‐0840. These data indicate that rat brain A1 receptors are not constitutively active, but that an ADA‐resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. SR141716 and AM251, at concentrations fully effective in reversing CB1‐mediated responses (10−6 M), did not reduce basal G‐protein activity, indicating that CB1 receptors are not constitutively active in these preparations. At higher concentrations (1–2.5 × 10−5 M), both antagonists reduced basal G‐protein activity in control and ADA‐treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right‐shifted A1 agonist dose–response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. When further optimizing G‐protein activation assay for the labile endocannabinoid 2‐arachidonoylglycerol (2‐AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of 2‐AG and concomitantly enhanced the potency of 2‐AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB1 receptor. The findings establish an optimized method with improved signal‐to‐noise ratio to assess endocannabinoid‐dependent G‐protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of 2‐AG are fully eliminated.

Bisphosphonate prodrugs : synthesis and in vitro evaluation of alkyl and acyloxymethyl esters of etidronic acid as bioreversible prodrugs of etidronate

The synthesis and preliminary evaluation of novel alkyl and acyloxymethyl esters of etidronic acid as etidronate prodrugs is reported. Tetramethyl ester of etidronic acid was found be isomerized at pH 7.4 and P-C-P bridge was rearranged to P-C-O-P. This unwanted process was prevented via acylation of the bridging carbons alcohol group. Acylation showed to be stable if one or more phosphonic OH- groups were substituted. However, when none of the phosphonic OH- groups were substituted, the acylation was chemically hydrolysed and the parent drug was released. This finding was successfully applied in the design of tetrapivaloyloxymethyl ester of acetylated etidronic acid which released etidronic acid via enzymatic (first step) and chemical (second step) hydrolysis in liver homogenate. However, the corresponding tri-substituted pivaloyloxymethyl ester having adequate water-solubility and lipophilicity (logP(app) 0.6 at pH 7.4), is probably the most potential prodrug candidate reported to enhance the oral bioavailability of etidronate.

Simultaneous determination of clodronate and its partial ester derivatives by ion-pair reversed-phase high-performance liquid chromatography coupled with evaporative light-scattering detection

A new ion-pair HPLC method coupled with evaporative light-scattering detection (ELSD) for the simultaneous determination of clodronate and its partial esters has been developed. The simultaneous chromatographic separation was achieved on a reversed-phase C8 column with a gradient system and butylamine as an ion-pair reagent. This method provides good enough reproducibility and sensitivity for in vitro determinations of clodronate and its ester derivatives. The method is applied for hydrolysis studies of clodronate monoesters which have been described as possible prodrugs of clodronate.

In-vitro corneal permeation of cannabinoids and their water-soluble phosphate ester prodrugs.

Topically administered cannabinoids have been shown to reduce intraocular pressure by interacting with the ocular cannabinoid receptor. Most cannabinoids have very poor aqueous solubility, which limits their pharmaceutical development and usefulness. In this study, permeation of three cannabinoids (arachidonylethanolamide, R‐methanandamide and noladin ether) and their water‐soluble phosphate ester prodrugs across isolated rabbit cornea was investigated in vitro. Hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) was used to solubilize the parent cannabinoids in permeation studies to achieve the required concentration in donor and receiving cells. Highest fluxes were obtained with lipophilic parent compounds administered with HP‐β‐CD, and the fluxes of phosphate esters were 45–70% that of their corresponding parent compounds. Phosphate esters hydrolysed on the surface of the cornea or during the permeation to release the lipophilic parent compound, which further permeated the cornea. No phosphate esters were detected on the endothelial side of the cornea. Although the phosphate esters had lower fluxes than their corresponding parent compounds in these HP‐β‐CD formulations, the results are promising and the fluxes of phosphate esters are significantly higher than the fluxes of parent compounds administered as a suspension (due to their low aqueous solubility) without HP‐β‐CD.

Anandamide prodrugs. 1. Water-soluble phosphate esters of arachidonylethanolamide and R-methanandamide.

Phosphate esters of arachidonylethanolamide (AEA) and R-methanandamide were synthesized and evaluated as water-soluble prodrugs. Various physicochemical properties (pK(a), partition coefficient, aqueous solubility) were determined for the synthesized phosphate esters. The chemical stability of phosphate esters was determined at pH 7.4. In vitro enzymatic hydrolysis rates were determined in 10% liver homogenate, and in a pure enzyme-containing (alkaline phosphatase) solution at pH 7.4. The intraocular pressure (IOP) lowering properties of R-methanandamide phosphate ester were tested on normotensive rabbits. The phosphate promoiety increased the aqueous solubility of the parent compounds by more than 16500-fold at pH 7.4. Phosphate esters were stable in buffer solutions, but rapidly hydrolyzed to their parent compounds in alkaline phosphatase solution (t(1/2)<<15 s) and liver homogenate (t(1/2)=8-9 min). The phosphate ester of R-methanandamide reduced IOP in rabbits. These results indicate that the phosphate esters of AEA and R-methanandamide are useful water-soluble prodrugs.

Cellular uptake and metabolism of clodronate and its derivatives in Caco-2 cells: a possible correlation with bisphosphonate-induced gastrointestinal side-effects

PURPOSE To investigate possible reasons for the low frequency of GI side-effects of clodronate, even though clodronate is known to be metabolised into a cytotoxic nucleotide analogue (AppCCl(2)p) by many cell types. The effects of some lipophilic prodrugs of clodronate were also studied. METHODS The effects of clodronate and its lipophilic derivatives on the proliferation and viability of Caco-2 cells were examined using an MTT assay. The intracellular uptake of 14C-clodronate and the accumulation of a clodronate metabolite (AppCCl(2)p) in Caco-2 cells were evaluated using ion-pairing HPLC-ESI-MS. RESULTS Clodronate had little effect on growth of proliferating, or the viability of confluent, Caco-2 cells. The uptake of clodronate by Caco-2 cells was only about 0.04% of total clodronate. The potentially cytotoxic clodronate metabolite, AppCCl(2)p, was detected in Caco-2 cell extracts after 3 h of exposure. Dianhydride- and triPOM-clodronate were metabolised to AppCCl(2)p more efficiently and also affected the viability of Caco-2 cells more than clodronate. CONCLUSIONS Clodronate appears to be metabolised into a cytotoxic ATP-analogue (AppCCl(2)p) by any cell type capable of internalising the drug. However, the cytotoxicity depends on the degree of uptake of clodronate. Due to the very low initial uptake of clodronate by epithelial Caco-2 cells, they do not accumulate sufficient intracellular concentrations of AppCCl(2)p to affect cell function. This explains the low frequency of gastrointestinal side-effects caused by oral clodronate therapy.

Bisphosphonate prodrugs : synthesis and in vitro evaluation of novel partial amides of clodronic acid

Abstract Novel partial amides of clodronic acid were synthesized and evaluated in vitro for their properties as bioreversible prodrugs of clodronate. The hydrolysis studies indicated that these derivatives release the parent drug via chemical hydrolysis. Monoamides were hydrolysed rapidly ( t 1/2 =16–19 min at pH 7.4) to clodronic acid, which suggests that they are useful intermediates in the design of enzymatically labile double prodrugs of clodronate.

First synthesis of etidronate partial amides starting from PCl3.

Methods for the preparation of mixed tetra-amide esters 1 and 2, the partial amide ester 3, and tri- and P,P-diamides 4 and 5 from monophosphorus spieces 12, 8 and 9, respectively, were developed. Compounds 8 and 9 were obtained from phosphorus trichloride via MeOPCl2, which was treated with 2 eq. and 4 eq. of piperidine, followed by water or acetyl chloride, respectively. Tetrasubstituted amide bisphosphonates 1 and 2 were selectively dealkylated with lithium or silyl halide to achieve target compounds 3-5. Piperidine was found to be a good desilylation reagent. Quantum mechanical calculations illustrate why derivative 2 was produced in low yield. The usefulness of compounds 1, 3 and 4 as prodrugs of etidronate was determined in aqueous buffer and human serum.

Fadolmidine‐induced ocular hypotension in normotensive rabbits

Fadolmidine, a novel selective α‐adrenoceptor agonist, was evaluated for its efficacy to lower intraocular pressure in normotensive rabbits (n = 5–6). The dose‐response profile between 0.004 μg and 12.5 μg of fadolmidine was determined. The effect of pH on the partition of fadolmidine was studied in order to select an optimal pH for topical fadolmidine administration. After topical administration, fadolmidine significantly lowered the intraocular pressure in normotensive rabbits. The onset of action was immediate, with no initial increase in intraocular pressure. A significant decrease in intraocular pressure was already observed at 1 h after dosing. The maximum decrease in intraocular pressure was observed after a 2.5 μg dose of fadolmidine in both eyes at 2 h after dosing. The mean maximum decrease in the treated and untreated eye was 6.4 mmHg and 3.9 mmHg, respectively. In conclusion, fadolmidine is a potent intraocular pressure lowering agent. In addition, fadolmidine does not cause a significant initial increase in intraocular pressure. Because of the strong dependence of the distribution coefficient on pH, the pH of the administered solution is important, with physiological pH being optimal in this respect.

Bisphosphonate prodrugs: unusual dimerisation of clodronic acid trimethyl ester to a cyclic bis(bisphosphonate)

Cl2C[P(O)(OMe)2P(O)(OMe)(O− Z+)] selectively reacts with acetyl chloride to provide a new enzymatically stable heterocyclic bis(bisphosphonate); the structure is confirmed by X-ray crystallography.