As described above, the authors have developed two forms of a new preparation: an injection solution and an ointment. The analgesic and anti-inflammatory effects studies of both forms have shown promising results. Toxicological studies of the liquid combined preparation, in case of IP injection, have shown that the range between the toxic and the therapeutic doses is quite narrow, so the ointment version of the combined preparation was chosen as a new, effective analgesic and anti-inflammatory remedy.

Upon IP injection, viper venom components (e.g., proteins, enzymes, peptides) are absorbed into the peritoneal cavity and enter systemic circulation via the extensive capillary network of the peritoneum. The venom contains enzymes like PLA2, metalloproteinases, and serine proteases, which can interact with local tissues, causing inflammation and cytotoxicity. The smaller amount of venom results in reduced local damage, potentially slowing absorption as tissue inflammation and vascular permeability are less pronounced compared to higher doses. Venom proteins and peptides bind to plasma proteins and are distributed throughout the body. Specific components of the venom preferentially target endothelial cells leading to mild endothelial disruption, platelets, and clotting factors, and may trigger subclinical coagulation disturbances. Low-dose neurotoxins may cause subtle changes in nerve signaling without overt paralysis, as the venom quantity is insufficient to overwhelm physiological detoxification pathways. At low doses, many venom components may not reach the threshold needed to activate their specific toxic effects.

The analgesic effect of low doses of viper venom may seem paradoxical, given the pain-inducing properties of venom at higher doses. However, there are plausible mechanisms, supported by scientific observations, that explain how low doses of viper venom could exert an analgesic effect: activation of endogenous pain inhibition pathways such as endorphin release or descending pain modulation, desensitization of nociceptors such as TRPV1, and Nav channels. In addition, peripheral adaptation may develop, such as receptor downregulation, as well as local neurotoxicity at low levels of venom doses. Venom-induced hormesis can be manifested as activation of protective or adaptive mechanisms that suppress nociception or inflammation [25]. In cases of cytokine modulation, venom can influence the production of cytokines. At low doses, it might stimulate the release of anti-inflammatory cytokines (e.g., interleukin-10) or inhibit pro-inflammatory cytokines (e.g., tumor necrosis factor-α), contributing to analgesia.

So, there is sufficient information about the analgesic effect of viper venom PLA2 both in the scientific literature and the authors’ studies [4,26,27]. Although the specific mechanism of action of blunt-nosed viper’s venom has not been shown at the molecular level yet, the most likely mechanism is desensitization of the sensory endings of pain receptors when different voltage-gated channels of the neuronal membrane are modified by the PLAA2 of the venom. This effect has been shown e.g., for the PLA2 from the viperid venom (Agkistrodon blomhoffii ussurensis), which contains Gln49 in its active center [3]. Today, different modifications and isoforms of venom phospholipases A2 have been identified, which contain different amino acid residues in the active center, including Asp49, Lys49, Gln49, and some others. PLA2 containing Lys49 is considered to lack enzymatic activity, and Asp49 (which is characteristically proved for a lot of vipers’ venoms and particularly for MLO [9]) has pronounced enzymatic activity. Moreover, many isoforms of phospholipases, in addition to enzymatic activity, exhibit activity as toxins and signaling molecules. Particularly Gln49-PLA2, very similar to Asp49-PLA2, markedly affects the function of voltage-gated ion channels and blocks neuronal signal transduction in the nerve terminal. This is the main mechanism of analgesic action in Gln49-PLA2 [3]. The analgesic effect of MLO venom is also thought to be related to increased activity of sodium and the blockade of potassium voltage-gated channels.

At low doses of venom, when the synthesis of prostaglandins is limited due to small amounts of arachidonic acid release, prostaglandin receptors may lead to the activation of anti-inflammatory processes [28]. This fact has been described for prostaglandin D2 [29]. In addition, low amounts of PLA2 can be rapidly bound by the trans-membrane glycoprotein and undergo internalization. Then phospholipase inactivation and cleavage in lysosomes take place. Low doses of venom can lead to marked desensitization of afferent pain nerves without significant inflammatory processes. The authors have shown that it is the enzymatic activity of Asp49-PLA2 that is responsible for the analgesic effect of viper venom, used in small doses (about 1/20 LD50, which is about 90 µg/kg body weight for mice when administered IP) [4]. However, with other routes of administration—intravenous, intramuscular, or for example, subcutaneous—effective analgesic doses vary greatly. When viper venom, as part of a classic lanolin ointment, is spread on the skin in the area of inflammation, its components are deposited in the subcutaneous fatty tissue and its effect is delayed compared to IP administration.

Another active component of the combined preparation, OVEO, has a practically proven analgesic and anti-inflammatory effect [6,7]. Gas chromatographic analysis and mass spectrometry showed that OVEO contains more than a hundred different low-molecular substances (flavonoids, sesquiterpenes, and others) [7,30].

Upon IP injection, BCP, a bioactive sesquiterpene, is absorbed through the peritoneal cavity’s capillary network into the bloodstream. Its lipophilic nature facilitates rapid absorption into lipid-rich tissues and blood plasma. BCP is transported via the bloodstream and widely distributed due to its high lipophilicity. Even inhaled, BCP preferentially accumulates in lipid-rich tissues such as the liver, adipose tissue, and brain [31]. It crosses biological membranes, including the blood-brain barrier, potentially interacting with the endocannabinoid system by binding to CB2 receptors. BCP binds to plasma proteins, which modulates its bioavailability and distribution. The metabolism produces oxidized derivatives, such as epoxides (e.g., caryophyllene oxide, a major metabolite) and hydroxylated products that enhance water solubility. BCP activates CB2 receptors, reducing inflammation and oxidative stress. This action might influence its metabolism by reducing inflammatory-mediated metabolic shifts. BCP and its metabolites can reduce oxidative stress in tissues, potentially modifying local metabolic pathways. In some tissues (e.g., brain), BCP may undergo local enzymatic transformations contributing to neuroprotective effects.

In the case of the ointment, BCP acts as an analgesic agent, which is an agonist of cannabinoid CB2 receptors, the activation of which leads to the effect on keratinocytes and the subsequent synthesis and release of endorphins, which reduces local acute and inflammatory pain [6]. Undoubtedly, depending on the dose, the effect of essential oil can be irritating (high dose), or soothing (low dose). Unlike venom, which includes high molecular weight components, such as polypeptides with a molecular weight of 3–4 kDa and proteins with a molecular weight from 13 to 70 or more kDa, essential oil components are low molecular weight substances with an order of 120–250 Da. Moreover, all components of the venom are hydrophilic substances, very soluble in aqueous solutions, while the components of the essential oil have exclusively lipophilic properties. Therefore, oregano essential oil applied to the skin is absorbed more quickly and reaches its targets. For example, BCP, after absorption, is almost completely distributed in all organs, passing all histo-hematic barriers, including the blood-brain barrier [32]. In other words, the two active components in the combined preparation have fundamentally different methods of action, depending on their absorption and distribution paths in the body.

The intensity and duration of the analgesic and anti-inflammatory effect of the preparation may exceed the same abilities of individual raw materials. That is, after spreading the combined ointment on the skin, the essential oil components are absorbed first, and the venom components are deposited in the subcutaneous tissue. Of particular interest is the MLO venom, which demonstrate significant anti-inflammatory effect in the carrageenan test when used alone. However, its antinociceptive action in the FT test is considerably delayed due to its poor permeability across the skin (Figs. 6, 7). The combination of MLO venom with OVEO, which is a proven penetration enhancer (Fig. 11), assumes an increase of permeability of such hydrophilic macromolecules, such as enzymes and polypeptides of venom. So, the OVEO plays a dual role in the ointment, exerting antinociceptive properties and helping the venom to more easily penetrate the multiple layers of the skin.

The β-caryophyllene of the OVEO binds to CB2 receptors, leading to the activation of keratinocytes and the release of endorphins, leading to the activation of ORs and an analgesic effect [6]. The comparative investigation of Ointment + Naloxone and Ointment + SR144528 shows that the CB2R participation in the analgesic effect is more accented than that of the ORs. The blockade of the CB2Rs by SR144528 showed that their participation in the ointment's analgesic mechanism was much more significant than that of ORs blocked by naloxone.

Along with the activation of the ‘exoCBs - CB2R - keratinocytes - opioid release – OR’ cascade, the components of the venom manage to leave the epidermal strata, reaching the afferent nerve endings and causing their significant desensitization and blocking of nociceptive signal transduction. In addition to β-caryophyllene (8.2%), OVEO contains β-caryophyllene oxide (13.4%) [7]. There is some evidence that the β-caryophyllene oxide may contribute its anti-inflammatory and related analgesic action in tested ointment effect [33,34]. So, the OVEO action is combined, and its components may exert an “entourage” effect. The special interest may concern other components of essential oil and venom, which can provoke some hypersensitization in cases of experimental blockade of the CB2Rs, as can be seen in Fig. 8 and Table 2. The most detailed molecular mechanisms of the effect of the ointment have not yet been revealed, but it is a fact that the ointment reduces inflammatory pain by more than 68% and reduces the volume of inflammatory edema up to 36%. At the same time, according to the research data, it does not irritate the skin or have any allergic effect.

Also, there is not any data suggesting that viper venom has any addictive properties, and for OVEO, moreover, there is some evidence that it inhibits methamphetamine-taking and methamphetamine-seeking behaviors in rats [35].

So, the authors tested the recipe for a new analgesic and anti-inflammatory preparation that does not contain any compounds that can cause addiction. Due to the content of its two main components, tested in optimal proportions, this preparation has a dual action: desensitization of nociceptive nerve fibers with a decrease of nociceptive impulses transduction (venom), and activation of peripheral opioid and cannabinoid anti-nociceptive system with a decrease of nociperception in central structures of the brain (essential oil). So, the tested combined preparation may be a candidate for the development of prospective anti-nociceptive and anti-inflammatory drug, but there is a need for further investigation of this preparation’s dermatokinetics and the exact mechanisms of its action. Despite the experimental data having shown that OVEO facilitates MLO venom toxicity upon IP injection, the mechanisms of the mutual action of the venom and essential oil has not been investigated yet.