Comparative antinociceptive effect of aspirin and aspirin nano particles in semisolid formulae in mice

1. This article enhanced in formulation of new formula of aspirin.
2. Evaluate the median effective dose of new formulated aspirin and aspirin nanoparticles.
3. Evaluate the analgesic effects of newly formed preparation in mice.

Comparative antinociceptive effect of aspirin and aspirin nano particles in semisolid formulae in mice

Labeeb H. Al- Alsadoon¹, Maha T.AL-Saffar² and Ghada A. Taqa²

¹Mosul Technical Institute, Northern Technical University, ²Departmentof Dental Basic Sciences, College of Dentistry, University of Mosul, Mosul, Iraq

labeebhasson@ntu.edu.iq, 0000-0002-5831-9722

mahatala72@yahoo.com, 0000-0003-1319-1785

ghadataqa@uomosul.edu.iq, 0000-0001-9683-7881, Corresponding author

2020-05-17

2020-06-26

Abstract

Aspirin are commonly used analgesic, anti-inflammatory, and anti-pyretic drug in medicine, oral route is the most common one for drug administration as a result it will produce different adverse effects like peptic ulcer, nephropathy, and thrombocytopenia even with low and continuous therapeutic dose, so the alternative topical route is preferable with minimal adverse effects and effective concentration.Therefore, in the present study was to investigate whether the antinociceptive property of aspirin would enhance if used aspirin as nanoparticles after preparing it in several forms (gel, cream and ointment). Thirty-two healthy male mice weighing 30-35 gm. were used in the present study. The animals were divided as a randomized design. Each mouse was treated topically. All drug concentration of aspirin was prepared using gel, cream and ointment as vehicle and topically application on fore and hind paw of experimental animals. Pain was induced by application of hot plate for assessment of latency of pain stimulus. Time from placement to jumping or hind paw licking was recorded as latency of response. The result showed that the median effective concentration (EC₅₀) for analgesic effect of aspirin (gel, cream, and ointment) were 0.848, 0.958 and 1.00% respectively while these EC₅₀s were decrease when used nanoparticles aspirin (gel, cream and ointment) to 0.72, 0.657, and 0.701% respectively. In conclusion, topical applied of aspirin will produce effective therapeutic antinociceptive effects in mice although gel preparation produce a better response followed by cream, then ointment due to pharmacokinetic properties. Also nanoparticle preparation will produce superior response in all forms, whether Nano aspirin is prepared in gel form, cream or ointment.

Keywords: Aspirin gel, Aspirin cream, Aspirin ointment, Nanoparticles, Hot plate technique

مقارنة التأثیر المسکن للأسبرین والجسیمات النانویةللأسبرین فی التراکیب شبه الصلبة فی الفئران

 لبیب حسون عبد الله¹ مها طلال فتاح² غادة عبد الرحمن طاقة²

¹الجامعة التقنیة الشمالیة، المعهد التقنی بالموصل، ²فرع العلوم الأساسیة، کلیة طب الأسنان، جامعة الموصل، الموصل، العراق

الخلاصة

یستخدم الأسبرین بشکل شائع فی الطب کمسکن ومضاد للحرارة ومضاد للالتهاب، والإعطاء الفموی هو الأکثر شیوعاً، ونتیجة لذلک سوف ینتج آثاراً ضارة مختلفة مثل القرحة المعدیة واعتلال الکلیة ونقص الصفیحات حتى مع جرعة علاجیة صغیرة، لذلک فإن الاستخدام الموضعی هو الطریقة البدیلة والمفضلة مع الحد الأدنى من الآثار الجانبیة والاستجابة العلاجیة الفعالة. لذلک، فی هذه الدراسة، تم التحقق مما إذا کانت الخاصیة المضادة للمسببات للأسبرین ستعزز إذا استخدمت الأسبرین کجزیئات نانویة بعد تحضیره بعدة أشکال (هلام، کریم ومرهم). اثنان وثلاثون فأرا ذکرا صحیا تزن 30-35 غرام استخدمت فی الدراسة الحالیة. تم تقسیم الحیوانات عشوائیا وتم علاج کل فأر موضعیا. تم تحضیر تراکیز الأسبرین باستخدام الجل والکریم والمرهم کوسیط ناقل واستخدموا موضعیاً على القدم الأمامیة والخلفیة للحیوانات التجریبیة. تم تحفیز الألم عن طریق اختبار الصفیحة الساخنة لتقییم مدة الألم. تم تسجیل الوقت من وضع الحیوان على الصفیحة إلى أن یقفز أو یلعق أقدامه وسجل کفترة استجابة. أظهرت النتائج أن الترکیز الوسطی للتأثیر المسکن للأسبرین بشکل (هلام، کریم ومرهم) کان 0.848، 0.958، 1.00% على التوالی بینما انخفض الترکیز الوسطی للتأثیر المسکن عند استخدام جزیئات النانوللاسبرین بشکل (هلام، کریم ومرهم) إلى 0.72، 0.657 و 0.701٪ على التوالی. نستنتج أن لاستخدام الأسبرین عبر الجلد تأثیرات علاجیة فعالة مضادة للألم فی الفئران على الرغم من أن تحضیر الأسبرین مع الهلام ینتج استجابة أفضل یتبعها الأسبرین مع الکریم، ثم الأسبرین مع المرهم ویرجع ذلک بسبب الاختلاف بالخصائص الحرکیة الدوائیة بین (الهلام، الکریم والمرهم). کما أن تحضیر الجسیمات النانویة ینتج استجابة فائقة فی جمیع الأشکال سوآءا أکان النانواسبرین محضر بشکل هلام، کریم أو مرهم.

Introduction

Acetylsalicylic acid (aspirin) is one of the most common drug used in medicine and it have been used since Hippocrates time for their analgesic, antipyretic, and anti-inflammatory effects (1). The analgesic property was produced by mechanism similar to other non-steroidal anti-inflammatory drugs by inhibition of prostaglandin synthesis through different steps which results finally to the inhibition of cyclooxygenase enzymes (COX) (2,3). These enzymes enhance the production of prostaglandin E2 from arachidonic acid usually COX present in two form, COX-1 which is the constitutive form and COX-2 which is the inducible form, aspirin inhibit both enzymes irreversibly, the COX-2 produce therapeutic effects while COX-1 inhibition leading to irritation of gastric mucosa and affect kidney functions (4). When aspirin administrated orally they are rapidly and completely absorbing in a large degree in small intestine and to lesser degree in stomach, and are accompanied by different side effects like gastric upset, blood loss, and to lesser extent Reys syndrome which is rare but fetal adverse effect (5) As a consequence of various therapeutic effects of aspirin, it presents in different pharmaceutical form tablet, cream, powder (6). Transdermal delivery system provides an alternative method of administrating that bypasses the gastrointestinal tract and its more acceptable by the patients, non- invasive and safe particularly in long term uses of aspirin. The dermal aspirin provide low bioavailability in addition to the avoidance of COX-1 direct contact which present in gastric mucosa (7). Nanotechnology in pharmacology has been of great interest in current time, for the delivery of different drugs (8,9). The ability of these Nano size range small particle to deliver the drug in faster way has been described previously (10,11). EC₅₀ is the concentration required to produce 50% of the wanted response in 50% of population, in addition to LD₅₀ is required in monitoring submission of new drug (12 ,13). EC₅₀ seems to be a helpful guide in for physician in selecting starting concentration (14), it is safe to start concentration of the drug under the concentration of EC50 due to the fact that different drug can cause morbidity specially when given to the patient with old age and it is dose related, so close monitoring of the concentration is of great important to assess the adverse effects early (12, 15). This study aims to prepare normal aspirin and nano aspirin in several forms (gel, cream and ointment) and determine the median concentration of analgesic effect (EC₅₀) for them and then compare them in better analgesic after using them locally through the skin in mice.

After preparing it in several forms (gel, cream and ointment), the mean concentration of analgesic effect (EC₅₀) was determined for them, and then compared them in terms of analgesia from pain after using them locally through the skin in mice.

Materials and methods

Animals

The study was carried out in the pharmacology lab after approved by scientific committee in depart. of dental basic science, College of Dentistry, University of Mosul.Thirty-two male albino mice weighing 30-35 gm were used in the present study. Mice were housed at 23±2ºC and take enough cycle of light / dark and nourished with good diet (standard) and water ad libitum. Mice were brought to laboratory before 3 hours of experiment to adaptation for new environment. The animal obtained from special animal house in dentistry collage, of Mosul university Iraq.

The drug used was aspirin pure (SDI) The animals were randomly divided to treat topically by different formulation aspirin drug. Mouse was treated with topically. all drug concentration of aspirin was prepared using gel, cream and ointment as vehicle and topically application on fore and hind paw of experimental animals. Pain was induced by application of (Heidolph, MR Hei-standard type, Germany) hot plate. To measurement of latency of pain stimulus. Mice were put on a Hot-plate at constant temperature 55±1ºC. The reaction time determine from putting mouse on hot-plate and to licking or hold of the hind paws or fore. Time of acute off 30 seconds is followed to stop any paws thermal injury (16). Time between putting mouse and hind paw licking was considered as response time.

Formulation of aspirin, aspirin nanoparticles Semisolid preparation

All the raw materials used of aspirin pure powder, aspirin nanoparticles (prepared by attrition method that describe by Rajput (17) base and vehicles were supply from commercial sources of analytical grade for research preparation. The concentration prepared as 0.25, 0.5, 0.75, 1, 1.25, and 1.5% by add 0.25, 0.5, 0.75, 1, 1.25, and 1.5 g of active ingredients in total 100 g of formulation respectively, and stirred to confirm homogeneity and contact uniformity.

Ointment

Was prepared by Fusion methods, via melted the desired amount of the active ingredients together with base and additive (PEG 4000,6000, Tween 80, Propylene glycol and distilled water) in two steps of preparation of oil and liquid phase.

Cream

Prepared as oil in water cream, by dissolve desired amount of the active ingredients in water phase then mixed with oil phase mineral oil, stearic acid and lanolin.

Gels

Were prepared by mechanical cold method. by dissolve desired amount of the active ingredients in water then mixed with glycerin and propylene glycol. Evaluation of The prepared semisolid preparations by various specifications of formulations as pH (pH meter), viscosities(viscometer), spread ability (spreading block) and content uniformity (titration and spectrophotometry) according to manufacture quality control instruction (18)

Evaluation of Median analgesic concentration (EC₅₀) of aspirin (gel, cream and ointment)

Fifteen mice were used in our study to evaluate median analgesic concentration (EC₅₀) of aspirin (gel, cream, ointment), by using up and down method (19). The pain threshold was determining for each animal before treatment and after 2 minutes (time determine according to pilot study) of aspirin (gel, cream, ointment) topical application on fore and back paw starting by 1% reliant on previous study. The change of increase dose and decrease dose is 0.5%. The analgesic EC₅₀ for aspirin (gel, cream and ointment) were determined according to (19).

Evaluation of (EC₅₀) of aspirin nanoparticles (gel, cream and ointment)

Seventeen mice were used in our study to evaluate the median analgesic concentration (EC₅₀) of aspirin nanoparticles (gel, cream and ointment), by using up and down method (19). The pain threshold was determining for each animal before treatment and after two minutes of aspirin nanoparticles gel topical application on fore and back paw starting by 1%. The change of the dose increasing and decreasing were 0.25% for aspirin nanoparticles (gel, cream and ointment). The EC₅₀ for aspirin nanoparticle was determined according to (19).

Result

Aspirin gel

The Median Effective Concentration (EC₅₀) of aspirin gel after topical applied in mice is 0.848 % (Table 1).

Table 1: Evaluation of median analgesic concentration (EC₅₀) of aspirin gel

Aspirin cream

The median effective concentration (EC₅₀) of aspirin cream topical application in mice is 0.958% (Table 2).

Table 2: Evaluation of median analgesic concentration (EC₅₀) of aspirin cream

Aspirin ointment

The median effective doses (EC₅₀) of aspirin ointment topical application in mice is 0.958% (Table 3).

Table 3: Evaluation of median analgesic concentration (EC₅₀) of aspirin ointment

Aspirin nanoparticles gel

The median effective concentration (EC₅₀) of aspirin nanoparticles gel topical application in mice is 0.72% (Table 4).

Table 4: evaluation of median analgesic concentration (EC₅₀) of aspirin nanoparticles gel

Aspirin nanoparticles cream

The median effective concentration (EC₅₀) of aspirin nanoparticles cream topical application in mice is 0.657% (Table 5).

Table 5: Evaluation of median analgesic concentration (EC₅₀) of aspirin nanoparticles cream

Aspirin nanoparticles ointment

The median effective concentration (EC₅₀) of aspirin nanoparticles ointment topical application in mice 0.657% (Table 6).

Table 6: Evaluation of median analgesic concentration (EC₅₀) of aspirin nanoparticles ointment

Discussion

Oral analgesics are usually given for treatment of pain (acute and chronic), aspirin which is one of the most common one although it is old and in expensive drug it usually showed several adverse effects even with small dose like gastrointestinal disorder, urticaria, asthma exacerbation, angioedema, in addition to more sever and rare adverse effects like thrombocytopenia, interstitial nephritis, and prolongation of prothrombine time (20,21).

Topical analgesic provides a promising route in giving the same analgesic effects provided by oral analgesic but with negligible adverse effects (22). In our study we tried to use a transdermal delivery system (by formulation of aspirin gel, ointment and cream in normal and nanostructure) as alternative route of administration which bypass the gastrointestinal tract in addition it is more safe and convenient for patients in prolong use. According to the result of our study better analgesic effects was demonstrated with aspirin gel (EC₅₀=0.848%) compared with equal analgesic effects of aspirin cream and ointment (EC₅₀=0.958%). this is disagreement with the study of Ravindra et al who showed that ointment give a better result (18). Aspirin delivery in our study was greater from gel formulation than from ointment and cream which may demonstrate that physical factor in the donor vehicle are more effective in controlling the release of the active ingredient from gel than are chemical factors (1).

Aspirin is slightly water soluble so it has a law affinity to the gel formulation, so it partitions to large extent into the lipophilic situation of the membrane of mice skin, the rapid flux rate seen in this process are concentration gradient dependence (23). on the contrary its suggested that ointment has relatively smaller affinity for the skin mice than for gel and therefore showed a lower partition and flux parameter in addition to lower flux rate also observed (24). The cream is lipophilic hydrophilic blend so the partition between two phases so slower flux rate are showed compared with ointment and gel (25)

The results obtained from this study showed that nanoparticles produce a better antinociceptive effects than normal size particles in all the three topical form (gel, ointment, and cream), this partially in agreement with study of Subramanian et al, who showed that nano emulsion preparation of aspirin will enhance the anti-inflammatory effects of aspirin in mouse model (26), this may be due to increase bioavailability of aspirin in nanostructure as it reported by other studies (27,28). The other evidence which support this is the increased bioavailability of paclitaxel transdermal system showed by pharmacokinetic analysis (29).

Conclusion

This study formulates a transdermal delivery system (topical form) for aspirin (gel, ointment and cream) and demonstrate that the analgesic effect of gel is better compared with ointment and cream in addition the antinociceptive effects of aspirin are enhance by Nano formulation of all three vehicles in mice model.

Acknowledgment

The authors are very grateful to Dentistry College, University of Mosul for their help to get better excellence of this research.

Conflict of interest

Authors states that she has no known rival monetary attention or private dealings that could affect the work of this manuscript