Preparation And Evaluation On 5-hydroxymethyl Tolterodine Hydrogels For The Treatment Of Overactive Bladder

Tolterodine is widely used for the treatment of urinary incontinence and other overactive bladder?OAB?symptoms.5-hydroxymethyl tolterodine?5-HMT?is the major pharmacologically active metabolite of tolterodine and exhibits the anti-muscarinic activity 10 times higher than tolterodine.However,approximately7%of patients can be classified as poor metabolisers?PMs?because of the lack of CYP 2D6 enzyme.Among the PMs,only a little part of tolterodine are metabolized to 5-HMT,leading to great individual differences.Meanwhile,both tolterodine and 5-HMT lack functional selectivity and have systemic side effects,which limit their therapeutic effects.5-HMT is not suitable for oral administration due to increasing hydrophilicity compared with tolterodine and reduced absorption in gastrointestinal tract.Therefore,there is a great interest to develop a suitable dosage form of 5-HMT to provide relatively consistent drug levels at the application site and avoid the oral side effects.In this study,5-HMT was synthesized and characterized.An in vitro analysis method of 5-HMT was established.The effects of gel matrix,enhancers,drugs,ethanol and glycerol on the skin permeation of 5-HMT were evaluated.The optimized hydrogels formulation was based on the maximum skin penetration,which was selected for the stability,pharmacokinetics,pharmacodynamics,skin irritation and transdermal mechanism studies.1.Synthesis and determination of in vitro assays of 5-HMT5-HMT was synthesized according to the method of Teng et al.Nuclear Magnetic Resonance Spectroscopy?¹H NMR?,Electrospray Ionization Mass Spectrometry?ESI-MS?,Differential Scanning Calorimetry?DSC?,X-ray diffraction?XRD?,Fourier Transform infrared spectroscopy?FTIR?have been used to characterize 5-HMT.The results showed that the synthesized white compound was 5-HMT with a melting point of 106.44°C and existed as a crystalline state.In this paper,a 5-HMT in vitro analysis method was established.The analytical method has good specificity,accuracy and precision.2.Formulation optimization and evaluation of 5-HMT hydrogelsIn this study,the effects of gel matrix,enhancers,drugs content,ethanol and glycerol on the skin permeation of 5-HMT were evaluated.Carbopol 940 based hydrogels revealed steady release for 12 h.The results indicated that the gel matrix Carbopol 940 was a good candidate for the delivery of 5-HMT,showing a better drug transdermal penetration than Carbopol 934 and 980.The 5-HMT hydrogels with 10%NMP as enhancer displayed the maximum accumulative percutaneous amount and permeated significantly.Stratum corneum of skin was the greatest barrier against drug transdermal penetration.The polar molecule of NMP might competitively combine hydrogen bond,destruct ordered structure of stratum corneum,and thus reduce the diffusion resistance of drug in the skin.We found that 20%ethanol can increase the drug solubility,and 10%glycerol could adjust the viscosity of hydrogels,thus decreased diffusion resistance and promoted drug release.The hydrogels with 3%drug loading could attain higher transdermal delivery rate.Thus,1.5%Carbopol 940,10%NMP,20%ethanol,10%glycerol and 3%5-HMT were chosen as the optimized hydrogels formulation.We randomly selected three batch?D,H and L?to investigate their stability at room temperature and with extreme environmental conditions.The drug loading and pH value were monitored before and after the experiments.The pH value of three batches was almost unchanged no matter in cold-resistant or heat-resistant experiments.However,in heat-resistant test,the drug loading significantly increased with a p-value<0.01 which might attribute to the evaporation of water and ethanol within hydrogels.After 3 months of storage at room temperature,the appearance of 5-HMT hydrogels was still transparent with no stratification and the drug loading was almost unchanged.3.Pharmacokinetic and skin irritation studie of 5-HMT hydrogelsThe pharmacokinetic studies were carried out on New Zealand white rabbits using 5-HMT hydrogels.Compared with intravenous injection?i.v.?of 5-HMT,5-HMT hydrogels can sustained release and keep stable plasma concentration in rabbits.After i.v.administration of 5-HMT,the C_maxax of 5-HMT was 2703±625ng/mL and the drugs were exhausted after 12 h.While,the C_maxax for 5-HMT hydrogels was 45±41 ng/mL and 5-HMT released from hydrogels lasted over 24h.It indicated that topical administration of 5-HMT prolonged the treatment time.The absolute bioavailability for 5-HMT hydrogels reached 20.7%.Erythema and edema were checked on test areas of three rabbits after applying with 5-HMT hydrogels at the seven days.And no erythema and no edema were observed on the skin of the rabbits.No inflammatory cell infiltration was found in the vertical and horizontal skin sections.The results indicated that 5-HMT hydrogels had no skin irritation.4.Pharmacodynamics studie of 5-HMT hydrogelsPilocarpine?150?g/kg,i.p.?increased appreciable overactivity of the rat urinary bladder.The cumulative urinary output?8.16±1.57?within 12 h was higher than that of the control group?5.15±0.83?,which indicated that pilocarpine-induced bladder overactivity rat model was established successfully.After p.o.administration of tolterodine tablets?0.4 mg/kg?,the contractions of the rat urinary bladder were improved.The cumulative urinary output was reduced from 6.08±0.73 to 4.11±1.00?p<0.05?within 8 h and from 8.16±1.57 to 4.90±0.92?p<0.05?within 12 h.After topical administration of 5-HMT hydrogels at a dose of 1.5 mg/kg,the cumulative urinary output was reduced to 3.12±1.45?p<0.01?within 12 h.5-HMT hydrogels displayed better pharmacological responses than tolterodine tablets,which was identical with pharmacokinetic studies.The histological examination results of bladder were studied.The normal bladder tissue contained 4-5 layers of urothelium cells,a layer of lamina propria mononuclear cells and smooth muscle cells full of longitudinal and transversal fibers.There were no pathological changes in bladder biopsies after treatment with and without hydrogels.The treatment of bladder overactivity of 5-HMT hydrogels is associated with neurotransmitter Norepinephrine?NE?in rats,but the specific mechanism of its action needs to be further studied.5.Transdermal mechanism studyIn this study,5-HMT transdermal capacity was studied preliminary.The results showed that 5-HMT was very easy to detained in the epidermis but not absorb in the lipids among epidermal cells.The percutaneous absorption pathway was independent of the intercellular lipid channels.After treatment with azone,the distribution of 5-HMT in the epidermis decreased,indicating that azone can promote 5-HMT through the epidermis.However,oleic acid and NMP increased the oil-water partition coefficients of 5-HMT in the epidermal tissue and increased skin retention.The transdermal mechanism of 5-HMT at the stratum corneum was investigated by DSC and FTIR.In the DSC experiment,the stratum corneum was treated with ethanol,triethanolamine,NMP,and 5-HMT hydrogels,and the melting peak shifted to a lower melting point and decreased.The results indicate that5-HMT hydrogels increase the transdermal delivery of drugs by affecting the ceramide and cholesterol status in the stratum corneum.The mouse stratum corneum was investigated by FTIR.It could be seen that there were obvious differences in the stratum corneum of mice before and after treatment with triethanolamine and 5-HMT hydrogels.DSC together with the FTIR results suggest that 5-HMT hydrogels increase transdermal flux of drug through altering lipid status in stratum corneum.Diffusional parameters about activation energy were studied to evaluate transdermal mechanism.The activation energy was 68.64 kcal/mol?conversion into human epidermis 7.63 kcal/mol?for 5-HMT hydrogels,which was lower than activation energy for ion transport across human epidermis?10.7 kcal/mol?.It proved that the rate of transdermal delivery depended on rate of delivery lipophilic part.The drug permeating might disturb the hydrogen bonds of tight ceramides structures,which was in accordance with DSC and FTIR results.