Wolfgang Sadée

5′-Amino Acid Esters of Antiviral Nucleosides, Acyclovir, and AZT Are Absorbed by the Intestinal PEPT1 Peptide Transporter

AbstractPurpose. General use of nucleoside analogues in the treatment of viral infections and cancer is often limited by poor oral absorption. Valacyclovir, a water soluble amino acid ester prodrug of acyclovir has been reported to increase the oral bioavailability of acyclovir but its absorption mechanism is unknown. This study characterized the intestinal absorption mechanism of 5′-amino acid ester prodrugs of the antiviral drugs and examined the potential of amino acid esters as an effective strategy for improving oral drug absorption. Methods. Acyclovir (ACV) and Zidovudine (AZT) were selected as the different sugar-modified nucleo-side antiviral agents and synthesized to L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-Val-ACV) and glycyl ester of ACV (Gly-ACV). The intestinal absorption mechanism of these 5′-amino acid ester prodrugs was characterized in three different experimental systems; in siturat perfusion model, CHO/hPEPTl cells and Caco-2 cells. Results. Testing 5′-amino acid ester prodrugs of acyclovir and AZT, we found that the prodrugs increased the intestinal permeability of the parent nucleoside analogue 3- to 10-fold. The dose- dependent permeation enhancement was selective for the L-amino acid esters. Competitive inhibition studies in rats and in CHO cells transfected with the human peptide transporter, hPEPTl, demonstrated that membrane transport of the prodrugs was mediated predominantly by the PEPT1 H+/dipeptide cotransporter even though these prodrugs did not possess a peptide bond. Finally, transport studies in Caco-2 cells confirmed that the 5′-amino acid ester prodrugs enhanced the transcellular transport of the parent drug. Conclusions. This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.

Pharmacogenomics: the promise of personalized medicine.

Pharmacogenetics and pharmacogenomics deal with the genetic basis underlying variable drug response in individual patients. The traditional pharmacogenetic approach relies on studying sequence variations in candidate genes suspected of affecting drug response. On the other hand, pharmacogenomic studies encompass the sum of all genes, i.e., the genome. Numerous genes may play a role in drug response and toxicity, introducing a daunting level of complexity into the search for candidate genes. The high speed and specificity associated with newly emerging genomic technologies enable the search for relevant genes and their variants to include the entire genome. These new technologies have essentially spawned a new discipline, termed pharmacogenomics, which seeks to identify the variant genes affecting the response to drugs in individual patients. Moreover, pharmacogenomic analysis can identify disease susceptibility genes representing potential new drug targets. All of this will lead to novel approaches in drug discovery, an individualized application of drug therapy, and new insights into disease prevention. Current concepts in drug therapy often attempt treatment of large patient populations as groups, irrespective of the potential for individual, genetically-based differences in drug response. In contrast, pharmacogenomics may help focus effective therapy on smaller patient subpopulations which although demonstrating the same disease phenotype are characterized by distinct genetic profiles. Whether and to what extent this individual, genetics-based approach to medicine results in improved, economically feasible therapy remain to be seen. To exploit these opportunities in genetic medicine, novel technologies will be needed, legal and ethical questions must be clarified, health care professionals must be educated, and the public must be informed about the implications of genetic testing in drug therapy and disease management.

Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence

The µ opioid receptor, MOR, displays spontaneous agonist‐independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPγS binding to cell membranes and cAMP levels in intact cells. β‐CNA, C‐CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6α‐ and 6β‐naltrexol and ‐naloxol, and 6β‐naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine‐induced dependence, 6β‐naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone‐induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine‐dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.

Calmodulin Binding to G Protein-coupling Domain of Opioid Receptors

The ubiquitous intracellular Ca2+ sensor calmodulin (CaM) regulates numerous proteins involved in cellular signaling of G protein-coupled receptors, but most known interactions between GPCRs and CaM occur downstream of the receptor. Using a sequence-based motif search, we have identified the third intracellular loop of the opioid receptor family as a possible direct contact point for interaction with CaM, in addition to its established role in G protein activation. Peptides derived from the third intracellular loop of the μ-opioid (OP3) receptor strongly bound CaM and were able to reduce binding interactions observed between CaM and immunopurified OP3 receptor. Functionally, CaM reduced basal and agonist-stimulated35S-labeled guanosine 5′-3-O-(thio)triphosphate incorporation, a measure of G protein activation, in membranes containing recombinant OP3receptor. Changes in CaM membrane levels as a result of overexpression or antisense CaM suppression inversely affected basal and agonist-induced G protein activation. The ability of CaM to abolish high affinity binding sites of an agonist at OP3 further supports the hypothesis of a direct interaction between CaM and opioid receptors. An OP3 receptor mutant with a Lys273→ Ala substitution (K273A-OP3), an amino acid predicted to play a critical role in CaM binding based on motif structure, was found to be unaffected by changes in CaM levels but coupled more efficiently to G proteins than the wild-type receptor. Stimulation of both the OP1 (δ-opioid) and OP3 wild-type receptors, but not the K273A-OP3 mutant, induced release of CaM from the plasma membrane. These results suggest that CaM directly competes with G proteins for binding to opioid receptors and that CaM may itself serve as an independent second messenger molecule that is released upon receptor stimulation.

Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective μ or δ opioid agonists in mice

&NA; N‐Methyl‐ d‐aspartate (NMDA) receptor antagonists have been shown to block the development of antinociceptive tolerance to morphine. Assessment of the effects of NMDA antagonists on development of antinociceptive tolerance to selective opioid mu (&mgr;) and delta (&dgr;) agonists, however, has not been reported. In these experiments, selective &mgr; and &dgr; receptor agonists, and morphine, were repeatedly administered to mice either supraspinally (i.c.v.) or systemically (s.c.), alone or after pretreatment with systemic NMDA antagonists. Antinociception was evaluated using a warm‐water tail‐flick test. Repeated i.c.v. injections of &mgr; agonists including morphine, fentanyl, [d‐Ala2, NMePhe4, Gly‐ol]enkephalin (DAMGO) and Tyr‐Pro‐NMePhe‐ d‐Pro‐NH2 (PL017) or [d‐Ala2, Glu4]deltorphin, a &dgr; agonist, or s.c. injections of morphine or fentanyl, produced antinociceptive tolerance as shown by a significant rightward displacement of the agonist dose‐response curves compared to controls. Single injections or repeated administration of MK801 (a non‐competitive NMDA antagonist) or LY235959 (a competitive NMDA antagonist) at the doses employed in this study did not produce behavioral toxicity, antinociception or alter the acute antinociceptive effects of the tested opioid agonists. Consistent with previous reports, pretreatment with MK801 or LY235959 (30 min prior to agonist administration throughout the tolerance regimen) prevented the development of antinociceptive tolerance to i.c.v. or s.c. morphine. Neither NMDA antagonist, however, affected the development of antinociceptive tolerance to i.c.v. fentanyl, DAMGO, or [d‐Ala2, Glu4]deltorphin. Additionally, MK801 pretreatment did not affect the development of antinociceptive tolerance to i.c.v. PL017 or to s.c. fentanyl. Further, MK801 pretreatment also did not affect the development of tolerance to the antinociception resulting from a cold‐water swim‐stress episode, previously shown to be a &dgr;‐opioid mediated effect. These data lead to the suggestion that the mechanisms of tolerance to receptor selective &mgr; and &dgr; opioids may be regulated differently from those associated with morphine. Additionally, these findings emphasize that conclusions reached with studies employing morphine cannot always be extended to ‘opiates’ in general.

Specific G protein activation and μ-opioid receptor internalization caused by morphine, DAMGO and endomorphin I

Previous studies have shown that the agonist [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO) but not morphine induces mu-opioid receptor internalization [Arden, J.R., Segredo, V., Wang, Z., Lameh, J., Sadee, W., 1995. J. Neurochem. 65, 1636-1645]. In the present study we investigated the relationship between internalization of the mu-opioid receptor and the specific G proteins activated following treatment with morphine, DAMGO and endomorphin I (Tyr-Pro-Trp-Phe-NH2) (a putative endogenous mu-opioid receptor agonist) in human embryonic kidney (HEK) cells. Endomorphin I and DAMGO, but not morphine, caused mu-opioid receptor internalization. Morphine, DAMGO and endomorphin I each activated Gi1 alpha/Gi2 alpha, Go alpha and Gi3 alpha to a similar extent, but not Gq alpha/G11 alpha or Gs alpha in HEK membranes. Therefore, the three ligands tested differed in their ability to internalize mu-opioid receptors even though they were similar in activating individual G proteins.

Initial United States clinical and pharmacologic evaluation of misonidazole (Ro-07-0582), an hypoxic cell radiosensitizer

Abstract The hypoxic cell radiosensitizer, misonidazole, is in a Phase I clinical pharmacology and toxicology trial. Forty patients were evaluated on a weekly dose schedule. Misonidazole was given on the dose schedule of a single oral dose weekly for 3 or 6 weeks in separate patient groups. Radiation therapy, of a palliative nature, was given 4–6 hr after drug administration to allow for maximal tumor levels. The initial dose level was 1 gm/m 2 and escalated to 3 gm/m 2 weekly × 6 weeks and 5 gm/m 2 weekly × 3 weeks. Toxicologic evaluation included serial clinical and laboratory evaluations. The principle toxicities observed were nausea and vomiting and neurotoxicity. The nausea and vomiting were acute, and dose limiting at 5 gm/m 2 . The neurotoxicity was primarily a peripheral sensory polyneuropathy, either seen on objective exam only or with mild to moderate paresthesias. Three patients manifested motor neuropathy with moderate weakness. Some ototoxicity and encephalopathy was observed. All patients had reversible, non-progressive neuropathy. The development of neuropathy was not related to the pharmacologic parameters of serum level or half-life. The incidence of neuropathy was related to total drug dose. Patients receiving less than or equal to 10 gms/m 2 had an incidence of 4 of 19 (21%) compared to patients receiving more than 10 gms/m 2 who had an incidence of 15 of 19 (79%). Pharmacologic evaluation included ultraviolet (UV) or high pressure liquid chromatography (HPLC) measurements of blood, urine, stool, and tumor biopsies. The HPLC identified Ro-05-9963, the desmethylated compound, as the principal metabolite in both blood and urine. Comparison of sera levels at the time of radiation (4–6 hr) with oral drug dose showed a good correlation, with maximum oral drug absorption not reached. Clinically significant blood level of 100 μg/ml could be achieved by a dose of about 2.5 gm/m 2 or 65–80 mg/kg. The pharmacology yielded peak sera levels at 2–4 hr with a mean half life of 15 hr and median of 14 hr; the half life was not related to drug dose. Urinary excretion was principally of the active metabolite (9963); total per cent excretion of both compounds in 24 hr ranged from 12–65% with a mean of 28% and a median of 23%. Tumor levels in 2 patients were 80 and 90% of peak sera levels. No increased toxicity of normal tissues in the radiation field was observed. Some interesting patient responses were observed. Further toxicologic, pharmacologic, and efficacy studies are underway.

Opiate antagonist receptor binding in vivo: evidence for a new receptor binding model.

The in vivo accumulation and retention of the opiate antagonist tracers [3H]diprenorphine and [3H]naloxone at cerebral opiate receptor sites in rats exceed that expected from their known in vitro receptor affinities. The [3H]diprenorphine serum and brain levels can be stimulated with a pharmacokinetic model that contains the receptors in a micro-compartment. The receptor micro-compartment consists of a population of binding sites next to a diffusion boundary which restricts ligand diffusion away from the receptor. Such an arrangement introduces a delay in the binding equilibrium of potent antagonists with the receptor sites and an increase in the apparent in vivo receptor affinity at subsaturating doses of the ligand; at saturating ligand concentrations these functions of the receptor micro-compartment are abolished. A physiological interpretation of the receptor micro-compartment could be the location of clustered opiate receptor sites on the exterior cell surface next to the synaptic cleft as the diffusion boundary. This kinetic approach involving a combination of pharmacokinetics and drug-receptor interactions permits the quantitative analysis of receptor site availability in the intact animal. Our results support the hypothesis that only one receptor population affects the in vivo disposition of the antagonist tracers, while they do not exclude the presence of low affinity binding sites that have been observed with the use of [3H]naloxone in vitro. Moreover, the binding site population observed in vivo may be responsible for mediating opiate agonist analgesia.

Regulation of Cyclic AMP by the μ‐Opioid Receptor in Human Neuroblastoma SH‐SY5Y Cells

Abstract: The human neuroblastoma clonal cell line SH‐SY5Y expresses both μ‐ and δ‐opioid receptors (ratio ∼4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by μ‐opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE,), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (∼65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphines inhibition of the PGE, and VIP cAMP response from ∼50 to ∼80%. The use of highly μ‐ and δ‐selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly μ, and not δ, receptor mediated in SH‐SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE,‐stimulated, RA‐differentiated SHSY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over 12h, a fourfold shift of the PGE,‐morphine dose‐response curve was observed, whether or not IBMX was added. However, μ‐opioid receptor number and affinity to the μ‐selective [D‐Ala2, N‐Me‐Phe4, Gly5‐ol]enkephalin were largely unaffected, and Na+‐ and guanyl nucleotide‐induced shifts of morphine‐[3H]naloxone competition curves were unchanged. Further, morphine pretreatment elicited an AC rebound effect, causing higher cAMP accumulation after the drug was removed from the medium or acutely antagonized by naloxone. These results document biochemical correlates of μ‐opiate tolerance and dependence in SH‐SY5Y cells.

A synthetic human Agouti-related protein-(83–132)-NH2 fragment is a potent inhibitor of melanocortin receptor function

Chemical synthesis of Agouti proteins – Agouti and Agouti‐related proteins – is complicated by their large size and by multiple cysteine residues located in the carboxyl terminal regions. Three human Agouti‐related protein (AGRP) fragments, two of which correspond to a proposed endoprotease cleavage site between amino acids 82 and 83, were synthesized and tested for anti‐melanotropic activity using Xenopus laevis dermal melanophores. Amino‐terminal fragments AGRP(25–51) and (54–82) were devoid of significant antagonist activity, whereas the amidated carboxyl‐terminal AGRP fragment (83–132)‐NH2 was potently active with an inhibitory equilibrium dissociation constant (K i) of 0.7 nM. The ability to synthesize functionally active AGRP should help unravel its role in the central nervous system and its unusual properties with respect to interaction with the melanocortin family of G‐protein coupled receptors.