ROLE OF OPIORPHINS IN THE MANAGEMENT OF PAIN

What is pain: pain is any noxious, unpleasant, subjective and emotional experience associated with actual or potential tissue damage.

Based on the mechanism, pain is of different types

1.     Nociceptive pain

2.     Neuropathic pain

3.     Functional pain

Based on the duration, pain is either acute or chronic.

Physiological mechanism in the body to painful stimuli: stimulation of the nerve endings known as nociceptors is the 1^st step leading to the sensation of pain. These receptors are found in both visceral and somatic structures and are due to mechanical, chemical and electrical factors. The action potential that is produced due to the stimuli travels along the spinal cord and reaches higher cortical centers where the pain is processed.

In some cases pain is produced due to nerve damage as in diabetic neuropathy, neuralgia etc., is such situations due to abnormal operation of the nervous system pain circuits may rewire themselves and produces spontaneous nerve stimulation causing pain.

When our CNS processes the pain it activates endogenous opiate system and causes the release of certain pain inhibiting neurotransmitters like enkephalins, dynorphins and beta endorphins which binds to their receptors and inhibits the transmission of pain impulses.

But the drawback is that these pain inhibiting neurotransmitters can’t survive in the body for long periods because our body contains certain proteases (ecto- aminopeptidases, ecto- endopeptisases) which causes the disintegration of pain inhibiting neurotransmitters (enkephalins, dynorphins and beta endorphins). As a result this endogenous opiate system is not effective in the management of pain.

 

So we are using different classes of drugs (NSAID’S and Opiates) for treating the pain, but these drugs have a potential to cause many side effects such as gastric irritation, renal and hepatic impairment, heart burn, constipation, bleeding, ulcers, nausea vomiting, drowsiness, visual changes, hallucinations, mental disturbances and are highly addictive (opioids).

Morphine is the most commonly used opioid for the treatment of chronic pain but it has many side effects.

What is opiorphin:

Opiorphin is an endogenous chemical compound found in the human saliva. Researchers found that painkilling effect of opiorphin is 3-6 times greater than that of morphine. It works by stopping the normal breakdown of  natural pain inhibiting neurotransmitters ( Enkephalins, Endorphins and Dynorphins) in the CNS. It is a relatively simple molecule consisting of a five-amino acid polypeptide, Gln-Arg-Phe-Ser-Arg (QRFSR).

Mechanism of action of opiorphin:

Opiorphin inhibits three proteases namely neutral ecto-endopeptidase (MME), ecto-aminopeptidase  (ANPEP) and perhaps also a dipeptidyl peptidase (DPP3) there by permitting the enkephalins to stay in the body for longer durations to show their action.

Therapeutic application of opiorphin in humans would require modifying the molecule to avoid its rapid degradation in the intestine and its poor penetration of the blood–brain barrier. This modification is done in the body by transformation of N-terminal glutamine into pyroglutamate. This form preserves the analgesic properties of opiorphin but with increased pharmaceutical stability.

 

Protein extraction:

The process of protein extraction starts with very crude samples that are cleaned up through filtration, centrifugation, solubilization and precipitation, and refined with techniques such as affinity columns and immunoprecipitation. A general workflow proceeds as follows:

1.     Lyse cells by sonication or homogenize tissue by freezing and grinding or homogenizing. This process breaks cell walls and frees proteins into the sample. Tissue samples can then be filtered to remove large debris.

2.     Centrifuge sample to remove cellular debris. This is usually performed in an ultracentrifuge via differential centrifugation or density gradient centrifugation. This step can be particularly useful for extracting specific compartmental proteins such as exosomal proteins

3.     For protein that will be used for a denatured gel electrophoresis or a western blot—use acetone to precipitate proteins.

4.     Resuspend sample in an appropriate protein extraction buffer. This buffer will contain an appropriate amount of salt, will contain a buffering agent to maintain pH 6-8, and will contain any necessary detergents, reducing agents, denaturants, or protease inhibitors. Detergents help to solubilize poorly soluble proteins such as integral membrane proteins that are used to a hydrophobic environment and to prevent non specific interactions. Reducing agents decrease oxidative damage. Denaturants are used only when the goal is to denature proteins for analysis of molecular weight such as SDS Page. Protease inhibitors prevent degradation by proteases once the membranes are damaged and everything is suddenly all mixed up together.

5.     Biomagnetic separation may be used as an optional step for further refinement of target protein solution. This technique is able to specifically enrich target protein due to a “lock-and-key” match between the target protein and the capture protein conjugated to magnetic nanoparticles. Once the capture protein and the target protein are bound together they are retained by a magnetic separation rack while the buffer is exchanged. The result is a highly enriched population of extracted target protein.

6.     Measure protein concentration via absorbance at 280 nm, Lowry Assay, Bradford Assay, or Bicinchoninic Assay (BCA)

7.     Store protein extract at -80ºC or -20ºC until needed for further analysis.

Conclusion:

So using opiorphins in place of morphine or any other pain killer can help to achieve many positive outcomes and reduces the negative outcomes normally caused by opioids and other pain killers. As opiorphin is a natural chemical present in the biological fluid (saliva) it can be made available at very low cost, which is highly beneficial for the poor and those requiring pain killers for longer durations.