A Recombinant Adenovirus Expressing β-EP(Ad-NEP) In Treating Acute Morphine Withdrawal Syndrome In Rats

BACKGROUND AND OBJECTIVEThe problem of narcotics is a grobal troublesome topic. In addition to its heavy burden on the economy, drug abuse also accelerates the epidemic of AIDS and other diseases, thus leads to serious social and ethical and even political problems.Opioid addiction is considered as a chronic brain disease by some scholars. The strong addictiveness and extreme difficulty of opiate drugs in withdrawal further aggravate the abuse. The concomitant acute syndrome during withdrawal brings such agony to the patient that makes physical de-addiction the hardest first step in the whole procedure and requires coerced execution. Vigorous negative emotions and counteractions are common among the patients receiving physical withdrawal therapy. Therefore, a long term syndrome relieving solution is needed to ensure a successful withdrawal.So far, a world-wide routine in treating drug addiction is to substitute with synthesized opioid analgesics or low addictive methadone along with decrement therapy. Some so-called"mild"drugs are legitimated in order to reduce social problems. A more radical opinion suggested permanently damaging those addiction related encephalic nuclei. Some other methods temporarily suppress the acute withdrawal symptoms, but the significantly altered body homeostasis hardly reinstates after prolonged exposure to the drugs, turning robust acute withdrawal symptoms into chronic protracted abstinence syndrome. Further investigation in physiological addictive mechanism indicated that, insufficient endogenousβ-EP, especially in those addictions related encephalic nuclei, is one of the most important neurobiological mechanisms underlying opioid withdrawal syndrome and emotional psychological dependence. It is reported that direct replenishment of centralβ-EP was enable to relieve withdrawal symptoms. Nevertheless, the duration of effect was short in direct replenishment and there is a higher risk of infection and injury after repeated central injection. Moreover, the cost of such therapy is prohibitive. Adenovirus vector is very effective in transfecting and expressing genes. Its major drawback is triggering immuno-reaction of the host and being virtually eliminated after 3-4 weeks. However, it is the natural clearance of the adenovirus and the gradual decrease of the target gene that provide a self-adjusting time window for the recovery of endogenous endorphin level. Using Ad-NEP, a recombinant adenovirus expressingβ-EP, in treating opioid addiction can simultaneously suffice as pharmacological substitution and decremental therapy. Meanwhile, the immune system of the host will prevent the exogenous gene from having long term effect. This design take charge of a defult of adenovirus,and make it utilitive.In present study, we tested the Ad-NEP therapy on the acute morphine withdrawal syndrome in SD rats, investigating the effect of virus products on isolated organ, the dose and administration pathway of vector, target gene expression level in rat C.S.F, as well as withdrawal behavior and body weight change over time, thus primarily explore the efficiency and safety profile of this gene therapy.【Methods】1. Rats received subcutaneous morphine for 5 days with dose progressively increase to establish morphine addiction model;2. 2 groups of HEK293 cells are respectively infected by recombinant adenovirus Ad-NEP and empty adenovirus vector (MOI=100).β-EP concentration in the supernatant is determined through radioimmunoassay (RIA) after 5 days of routine culture. 20 times Krebs solution diluted supernatant from each group (with or withoutβ-EP) is used as the bath for isolated ileum preparation. The other negative control group and normal control group used Krebs solution as bath.3. Rats were sacrificed by cervical dislocation. The terminal ileum was then isolated for about 1.5 cm as testing preparation. These preparations were bathed in 37℃Krebs solution or Krebs solution diluted supernatant saturated with mixture of 95%O2+5%CO2. The tension and contracting power of the isolated ileum is measure by a tension transducer. After balanced with a tension of 1 g for 30 min, naloxone was added in bath solution (final concentration 4μmol/L) to induce withdrawal contraction. The tension change and number of withdrawal contractions were compared across the groups.4. The original titer of Ad-NEP is 5×1011 PFU. It was then diluted with normal saline into 30μl of 5 different titers (5×107, 5×108, 5×109, 5×1010 and 5×1011PFU). These virus solutions were then injected into the paracele of the rats. The normal control group received normal saline instead of virus solution. Rat C.S.F from different groups were collected after 4 days and the concentration ofβ-EP was determined. An analysis on the relationship of the logarithm of titer withβ-EP concentration in C.S.F was performed to find out the most appropriate virus dose. 5. According to previous result, the later experiments went on with 2 different virus titers (5×1010 PFU and 5×109 PFU). While establishing acute morphine withdrawal syndrome model, the rats were randomly divided into 4 groups: the first group received 5×1010 PFU Ad-NEP, the second received 5×109 PFU Ad-NEP, and the other 2 were sham group and natural withdrawal group. All the rats received Ad-NEP or normal saline injection into the paracele in the night of the 5th day except for the natural withdrawal group. All the rats then received naloxone (2 mg/kg, i.p.) on day 1, day 4, day 8 and day 12 after intra-paracele injection to induce withdrawal symptoms. The withdrawal behavior was scored according to modified Maldonado's evaluation of withdrawal symptoms. The C.S.F was immediately collected after the evaluation and the concentration ofβ-EP was determined. The change ofβ-EP concentration and behavioral score across the observatory period were recorded and the relativity was determined. The body weight was recorded daily throughout the procedure. The trends of change and the body weight loss at the end of observation were compared among the groups. The mortality was also compared using chi-square test.【Results】1. We successfully established the acute morphine addiction model of rats and the isolated ileum preparation of the acute morphine addiction rats. By shorten the acute experimental time, we managed to reduce the impact of the experimental results by time factor;2. Before naloxone application, there is a significant heterogeneity in contracting strength of ileum preparation from addicted rats and normal rats (P<0.05). This result is consistent with the fact that intestinal tension increases after morphine addiction. Naloxone markedly altered contracting strength of ileum preparation from negative control group and empty vector infected supernatant group (P<0.01) but neither Ad-NEP infected supernatant group nor normal control group exhibited a similar change (P>0.05). Naloxone induced withdrawal contraction in ileum preparation from all 3 addicted rat groups but no withdrawal contraction was observed in that of normal rats. Preparations of Ad-NEP infected supernatant group had significantly less contraction than that of other 2 groups (P<0.01). These data suggest thatβ-EP is effective in reducing the number and extent of the naloxone-induced withdrawal contraction on isolated ileum preparation in rats.3. 5×1011 PFU viral solution was associated with high mortality in normal rats. 4 days after 5×109 or 5×1010 PFU viral solution injection,β-EP level in C.S.F is statistically higher than that from other groups.4. Performing ONE-WAY-ANOVA on the data ofβ-EP level at previous described 4 time points after i.c.v injection, we found thatβ-EP level reached peak at Day 4 in 2 groups receiving Ad-NEP. Whereasβ-EP level continued to rise and reached maximum at Day 12 in sham group and natural withdrawal group. There is a statistical difference in the maxβ-EP concentration between Ad-NEP groups and non- Ad-NEP groups (P<0.01). Intra-paracele Ad-NEP significantly reduced withdrawal behavioral score as early as Day 4 (P<0.01), however, withdrawal behavioral score only significantly lower at Day 12 (P<0.01) in the other 2 groups.β-EP level is negatively related to the behavioral score. This relativity reached peak at Day 4 and then diminished over time. At the end of the study, the lossed body weight percentage was compared across the groups via paired t-test and the result indicated that Ad-NEP significantly rescued body weight loss in virus injected rats compared with sham and natural withdrawal group rats (P<0.01). The chi-square test performed on mortality data also suggested that Ad-NEP can reduce death in virus injected rats compared with natural withdrawal group rats (P<0.01).Conclusionβ-EP can counteract the withdrawal contraction which induced by naloxone on the isolated ileum from acute morphine addicted rats and can alleviate the opioid withdrawal syndrome at organ level.The suitable virus dose for i.c.v. injection in rats is 30μl with a titer of 5×109-1010 PFU. This dose can get a high concentration ofβ-EP in the C.S.F of the experimental rats without causing a large number of deaths in rats.β-EP level is positively related to the titer of viral solution.I.C.V. injection of recombinant adenovirus Ad-NEP can alleviate the naloxone induced acute morphine addiction withdrawal symptoms in rats. In the early phase of the withdrawal, higher C.S.Fβ-EP concentration is correlated with lower withdrawal behavioral score. However, such relativity diminished with time. These data indicated thatβ-EP plays an important role in the earlier phase of acute withdrawal syndrome and it is not that crucial in the later phase.