big in vegas
MuscleChemistry Registered Member
[h=3][/h][h=2]MC Receptor Agonists[/h]A variety of research modalities have been used to elucidate the action of MC compounds on penile erection . MC compound affinity and activity properties are determined by cell culture and membrane receptor assays. In general, MC agonists bind strongly to subsets of the five G-protein coupled MC receptors and cause increased intracellular production of cAMP while MC antagonists bind strongly but do not stimulate cAMP production. Notably MCRs 1, 3, 4 and 5 have high constitutive (ligand-independent) activity enabling antagonists to decrease basal levels of cAMP production.
[h=3]Table 1[/h]Models for study of MC compounds on Penile Erection
<colgroup><col style="width: 115px"><col width="502"></colgroup><tbody>
</tbody>
[h=4]Endogenous MC Receptor Agonists[/h]ACTH, α-MSH, β-MSH and γ-MSH are the known endogenous agonists of the MC system. Each hormone is a product of posttranslational modification of the POMC gene transcript and contains the sequence of His-Phe-Arg-Trp, considered to be the “core” of agonist activity []. Only ACTH and α-MSH have shown the ability to generate sexual stimulation and penile erection in various animal species including rats, rabbits, cats, dogs and monkeys [. These pro-erectile effects appear to be androgen-dependent as castration abolishes the aforementioned response []. Notably, many of the synthetic MC agonists contain the “core” sequence present in ACTH and α-MSH, particularly the agents MT-II and PT-141.
[h=4]MT-II (Melanotan-II)[/h][h=5]Pharmacology[/h]MT-II is a synthetic cyclic heptapeptide that was initially designed as an artificial tanning agent. Its structure is based on an earlier linear peptide, Melanotan-I, however cyclization was introduced to prevent degradation and allow both N and C terminal truncation of the peptide [. The pro-erectile activity of MT-II was reported as a significant unexpected reaction during a phase-I human trial of human tanning [. MT-II contains a seven amino acid sequence with homology to receptor binding portions of α-MSH and ACTH. The compound is thought to cross the blood brain barrier and has high affinity for the MC1R, MC3R and MC4R. MT-II has a similar affinity for MC4R compared with MC3R and may be considered “superpotent” because of its relatively high affinity for MC4R compared with the endogenous peptides α-MSH and ACTH (10-100 fold difference).
[h=5]In-vitro and animal studies[/h]The pro-erectile activity of MT-II appears to be both forebrain and spinally mediated, with little, if any, peripheral effect. Dose dependent increases in spontaneous erections in awake Long-Evans rats were noted with administration of MT-II intracerebrally, intrathecally and intravenously Increases in yawning and grooming behaviors paralleled erectile activity with intracerebral administration but not spinal administration. As discussed previously, when the non-selective MCR antagonist SHU-9119 was given spinally, it blocked spinal MT-II induced erections, however intrathecal SHU-9119 failed to block intracerebral MT-II induced erections. This indicates potentially independent sites of melanocortin action along the CNS axis with intracerebral sites activating multiple downstream pathways including those independent of melanocortinergic activation.
In parallel with the above observations, Vemulapalli et al found that isolated corpus cavernosal strips from rabbits had no relaxation in response to electrical field stimulation in the presence or absence of MT-II. [ As well, MT-II associated elevations in intracavernosal pressure were abolished when anesthetized rabbits underwent bilateral pudendal nerve ablation. Inhibition of NO-synthase enzymes with L-NAME also blocked the erectile effect of systemic MT-II. These pieces of data indicate that MT-II exerts direct actions through CNS brain and spinal activation, which is then mediated peripherally through established NO vasodilatory pathways.
[h=5]Human studies[/h]A double blind placebo-controlled crossover study by Wessells et al. demonstrated the safety and pro-erectile activity of subcutaneous MT-II in humans [ In the absence of erotic stimulation, 10 men with psychogenic (non-organic) erectile dysfunction received subcutaneous doses ranging from 0.025 to 0.157 mg/kg, while erections were monitored by RigiScan over a 6-hour period. Eight of the 10 men developed clinically apparent erections with greater than 80% rigidity of an average duration of 38 minutes compared with 3 minutes for placebo controls. The time to onset ranged from 15 to 270 minutes. Side effects were dose dependent included nausea, stretching, yawning and decreased appetite. At the preferred dose of 0.025 mg/kg side effects were mild.
The above study documented erectogenic effects of MT-II in men with presumed normal underlying physiology. A subsequent study of MT-II was carried out on men with organic ED . In a similar double blind, placebo-controlled crossover study, 10 men received 2 subcutaneous doses of 0.025 mg/kg MT-II and 2 doses of vehicle. MT-II initiated subjectively reported erections following 63% of the drug injection verses 5% of the placebo injections. The mean rigidity score of the responders was 6.9 on a scale of 0 to 10. Mean duration of tip rigidity greater than 80% was 45 minutes with Melanotan II compared to two minutes for placebo. There was increased subjective reporting of sexual desire after MT-II administration compared with placebo, although the question used to assess desire was not designed specifically to measure desire in men not engaging in sexual intercourse.
[h=4]PT 141 (Bremelanotide®)[/h]
[h=5]Pharmacology[/h]PT-141 (Bremelanotide®) is currently the most studied melanocortinergic compound with regard to therapeutic potential for treatment of erectile dysfunction. PT-141 is a synthetic heptapeptide. It is a deaminated derivative and likely metabolite of MT-II. This compound has strong binding to MC receptors 1, 3 and 4, with a higher affinity for MC4R over MC3R. Application of PT-141 to HEK-293 cells expressing MC4R increases cAMP production, indicating that this compound, like MT-II, acts as an agonist
[h=5]
Animal studies[/h]Studies with adult male Sprague-Dawley rats indicate pro-erectile responses through multiple modes of delivery [42]. Intranasal injection of 50μg/kg PT-141 produced a significant increase in spontaneous erections compared with saline controls in rats observed over a 30-minute period. As well, 100% of the drug treated rats had at least one erection. In this study the pro-erectile effect of PT-141 was attributed to hypothalamic stimulation of MC3R and/or MC4R. Two hours after PT-141 (50μg/kg IN) administration, immunostaining for FOS, a measure of neural activation, showed increased expression in the paraventricular nucleus compared with rats administered saline.
[h=5]
Human studies[/h]Preliminary trials in humans have established both safety and efficacy of PT-141. A phase 1 randomized double-blind placebo controlled trial involved 24 healthy male subjects without erectile dysfunction [42, 43]. Intranasal doses of 4 to 20mg were delivered to patients in the absence of visual sexual stimulation (VSS). Safety and tolerability were monitored revealing no significant hemodynamic changes or side effects, including priapism. Serum concentration of drug was dose dependent and peaked at 30 minutes in the maximum dose group. Serum half-life was measured at 120 minutes. Rigi-Scan monitoring of erectile response revealed a significantly increased duration of rigid erections of 140 minutes compared to 22 minutes in the placebo group. Time to onset of erection ranged from 34 to 63 minutes. Erections were considered rigid if they were more than 60% of base rigidity.
Based on the above results, phase II studies were initiated in patients with mild to moderate ED who showed positive erectile response to PDE-5 inhibitors [44]. RigiScan monitoring in the presence of VSS detected a 3-fold increase in erectile activity with PT-141 (20mg intranasal) administration. The duration of base rigidity was significantly increased utilizing both a 60% and 80% cut-off versus placebo [43]. Timing of erections corresponded well to visual stimulation indicating a potential facilitator mechanism of drug action.
In a first Phase IIB at home study, PT-141 induced dose dependent improvements in erectile function as assessed by the International Index of Erectile Function (IIEF) [45]. Of the patients who completed at least 3 at home attempts (n = 203), the mean IIEF erectile function domain score increased in a dose dependent fashion (p < 0.05 for 10, 15, and 20 mg). Normal erectile function (EF>26) was achieved by 10, 30, 36, 53, and 50% of patients in the placebo, 5, 10, 15, and 20 mg groups respectively. Improved erections as defined by a global assessment question were reported by 17, 49, 67, 66, and 66% of patients in the placebo, 5, 10, 15, and 20 mg groups respectively. There were no episodes of syncope or hypotension. The only serious adverse event reported in this study occurred in one patient who reported a prolonged erection that was painless and required no treatment.
Gastrointestinal side-effects were the primary reasons for discontinuation in the higher two higher dose groups.
In summary, PT-141 is a potent initiator of erection with minimal side effects, a rapid onset of action and a sufficiently long duration of action. Notably, the recent Phase II studies confirm that the erectile responses are augmented by sexual stimulation. With its central mechanism of action, PT-141 may act independent or synergistically with PDE-5 inhibitors and provide a useful alternative therapy for erectile dysfunction, both from organic and psychogenic origin.
A randomized prospective placebocontrolled study compared treatement of ED with sildenafil alone verses sildenafil with 7.5mg intranasal PT-141 [46]. Co-administration of the two agents resulted in significantly prolonged time of increased base rigidity (>60%) compared with sildenafil alone during a 2.5 hour monitoring session. The combination of drugs was well tolerated with no significantly increased side-effects over either sildenafil or PT-141 alone. The ability of the peptide to safely and effectively induce high quality erections allowed Palatin Technologies to initiate a second Phase IIB study in 2006 and propose Phase III studies in 2007 [47].
[h=4]
THIQ[/h]THIQ is a synthetic small molecule hMC4R agonist with moderate bioavailability (14%), rapid absorption (Tmax = 1hr) and a short T1/2 (0.5hrs) [
]. THIQ has a >600 selectivity for the MC4R compared with MC3R
. Studies by Martin and Van der Ploeg evaluated the effects of THIQ delivered both systemically and intracerebrally in rodents. Systemic administration (1mg/kg) potentiated electrically stimulated erections as well as decreasing mounting and intromission latency mating behaviors in wild type mice [30]. In an ex copula model using male rats, systemic THIQ dose dependently increased total numbers of erections
. This effect was blocked by central administration of the non-specific antagonist, AgRP, as well as the MC4R specific antagonist, MBP-10. ICV delivery of 20μg of THIQ increased reflexive penile erections. There are no studies of THIQ in humans to date. Interestingly, and in contradistinction to MT-II, THIQ has not been reported to initiate spontaneous erections in rodents.
[h=3]
MC Receptor Antagonists[/h]Endogenous and synthetic antagonists have been used to explore melanocortin signaling. When MCR antagonists bind to the MC receptors they either decrease constitutive levels of cAMP production or prevent agonist induced increases in cAMP production. In studies of penile erection, MCR antagonists have been primarily utilized to identify the mechanisms and location of action of MCR agonists as well as parcel out specific receptor subtype activity. These compounds have not been utilized as therapeutic agents.
Endogenous melanocortin receptor inhibitors include agouti or agouti-related peptide (AgRP). AgRP is a 132 amino acid peptide which competitively antagonizes both MC3R and MC4R [51]. While AgRP has primarily been studied for its role in energy homeostasis, this peptide is principally expressed in the arcuate nucleus of the hypothalamus, a potential site for regulation of melanocortin mediated erection [14]. As mentioned, intracerebral delivery of AgRP (5.5μg) was shown to block erections in rats induced by the MC4R agonist, THIQ [ There have been no studies in humans with regard to erection.
SHU-9119 is a classical inhibitor of both the MC3R and the MC4R. This synthetic cyclic lactam α-MSH analogue is closely related in structure to MT-II [52]. SHU-9119 actually has agonist properties at MC1R and MC5R, but for the purposes of discussing erection, this compound is considered primarily an antagonist because of the lack of these receptors in the CNS. In rabbits this highly potent compound readily blocked MT-II induced erections when administered systemically [34]. In rats, SHU-9119 blocked erections and grooming/yawning behaviors stimulated by MT-II both at supraspinal and spinal locations [31]. This compound has not been studied in humans.
Two other synthetic MC receptor antagonists that have been utilized in studies of erectogenisis include MPB-10 and HS014. Both of these compounds preferentially block the MC4 receptor. Their use in animal studies has primarily been related to determination of receptor specification as described in the following section. These compounds have not been studied in humans.
[h=3]MC3R vs. MC4R[/h]Of the 5 melanocortin receptor subtypes, only the MC3R and MC4R have been identified in CNS regions associated with activation of penile erection [1], particularly the PVN of the hypothalamus. Many of the more commonly studied compounds, such as MT-II and αMSH, activate both MC3 and MC4 receptor subtypes to some degree. This lack of receptor specificity has limited our understanding of each receptor’s contribution toward erectile behaviors and has prompted studies utilizing receptor specific agonists and antagonists as well as receptor knock out mouse models. Contrary evidence has pointed to each receptor as the principle subtype mediating erection. Although the weight of evidence leans towards MC4R activation being responsible for activation of erection, the debate remains unresolved.
Evidence of MC3Rs participation in sexual stimulation and erection comes from a series of studies in the late 1990s utilizing an MC4R specific antagonist, HS014 [53]. Vergoni et al. administered ACTH and α-MSH into the lateral ventricle of adult male Sprague-Dawley rats and showed predictable responses with grooming, stretching, yawning and erections [2]. Co-administration of these compounds with HS014 completely blocked grooming, stretching and yawning behaviors, but only partially reduced erections. Argiolas et al. studied this effect further with ACTH, α-MSH and HS014 microinjections into regions surrounding the 3rd ventricle of adult rats [54]. The effect was a dose dependent elicitation of yawns, grooms and erections when only ACTH and α-MSH were administered. Co-administration of these compounds with HS014 significantly blocked yawns and grooms but erections were unaffected. This evidence suggested that the MC4R was not involved in the sexual response to ACTH and α-MSH. As the only other MC receptor in the region, the MC3R was attributed partial credit for the erectile response.
However, HS014 does have MC3R antagonist activity and the relatively small difference in affinity for MC4 vs. MC3 receptors makes interpretation difficult. If MC3R were the primary mediator of erection, one would have expected some diminution of erections with this compound. Another possible consideration in the interpretation of these studies is that a different level of MC4R occupancy may stimulate yawning/ grooming behaviors and erection. Thus a higher dose of antagonist may be required to block penile erection. Finally, the proerectile effects of MSH are not as potent as synthetic analogs such as MT-II, raising the possibility that an inadequate stimulatory dose of the agonist prevented a measurable effect of the antagonist (floor effect).
Evidence in support of MC4R as the principle receptor mediating erections is provided by multiple converging lines of evidence using MC4R knock out mice as well as rat studies with MC4R specific agonists and antagonists. Van der Ploeg et al stimulated the cavernous nerves of wild type and MC4R knockout mice in the presence or absence of systemic THIQ (10mg/kg IV), an agonist with >1000-fold selectivity for MC4R compared with MC3R [30]. Wild-type mice had significant augmentation of erectile activity while knockout mice did not show any changes. Copulatory behavior in these mice was evaluated as well. MC4R wild type mice received THIQ (2.5mg/kg IP) or vehicle and were paired with estrous females. THIQ treatment decreased mounting and intromission latencies in WT mice. Interestingly, MC4R knock out mice had impaired copulatory behavior compared with littermate wild type controls, showing baseline dysfunctional mating characteristics with loss of the MC4R. The systemic delivery of THIQ in these studies led the authors to question whether the effect could be related to direct activation of cavernosal tissue. Although they demonstrated MC4R mRNA in rat cavernosum, application of THIQ to cavernosal strips did not result in tissue relaxation.
Martin et al. utilized a slightly different approach to investigate MC3/4R question, by administering selective and non-selective antagonists to MC4R in combination with the MC4R agonist THIQ [48]. MBP10 is a synthetic MCR antagonist with at least a 125-fold selectivity for MC4R over MC3R [55] while AgRP is an endogenous antagonist with comparable inhibition of both MC3R and MC4R. In this study, an ex copula model of monitoring penile reflexes was measured after drug delivery. Consistent with the work of Van der Ploeg et al., systemic THIQ increased intracavernosal pressures and dose-dependently increased reflex erectile activity in restrained rats. Central administration of THIQ into the 3rd ventricle of rats increased reflexive penile erections. This centrally mediated effect was blocked by pretreatment with both AgRP as well as MPB10. Unfortunately, an MC3R selective antagonist was not employed in this study. The conclusion of this study was that MC4R activation was sufficient for penile erectile activity, but did not exclude a possible role for MC3R.
Although the pro-erectile effects of MC4R activation appear well established, the contribution of MC3R towards erection is incompletely understood. An alternative hypothesis to the above studies is that stimulation of the MC3R may actually be inhibitory toward erectile activity. In support of this hypothesis are neuroanatomical pathways involving AgRP (endogenous melanocortin antagonist) and POMC neurons, which travel in parallel throughout much of the central nervous system. MC3R mRNA has been co-localized to both AgRP and POMC neurons in a rostrocaudal gradient in the arcuate nucleus [56]. In contrast, MC4R mRNA was not found in AgRP or POMC expressing cells. This implies a potential negative feedback loop whereby “expression of MC3R by POMC neurons provides a potential circuit for amplification of AgRP-mediated signals, because AgRP-induced inhibition of POMC neurons via the MC3R would reinforce the postsynaptic effects of AGRP. Furthermore, the expression of the MC3R by AGRP neurons provides a potential circuit for negative autoregulation of POMC-mediated signals, because POMC-induced activation of AGRP neurons via the MC3R would terminate the postsynaptic effects of POMC”.
Preliminary studies in our laboratory using MC3R specific agonists and antagonists may have unmasked a proposed autoinhibitory pathway of the MC3 receptor. AVM-127 is a cyclic γ-MSH analog with selective MC3R/ MC5R Antagonist activities in vitro; it is a synthetic compound with antagonist activity and 100 fold selectivity for MC3R compared to MC4 [57]. A study of intracerebroventricular administration of AVM-127 (750ng) prior to MT-II (1μg) in adult male rats was performed with the initial hypothesis that antagonism of the MC3 receptor would either reduce or have no effect on MT-II stimulated erections. This hypothesis was based on previous studies indicating that MC3R activation was “pro-erectile”. Paradoxically, rats administered AVM-127 in combination with MT-II had significantly more erections over a 90 minute observation period when compared with rats given MT-II alone (see Fig. 1). Rats given AVM-127 alone had no significant erectile activity compared with controls. AVM-127 had no effect on MT-II induced yawns or genital grooming behaviors. These results were highly surprising and unexpected. An inverse approach to this study involved specific stimulation of central MC3 receptors with a novel specific MC3R agonist, the γ-MSH analog PBIII-93 [58]. When administered ICV to male rats, this compound failed to produce erections despite dose-dependently stimulating yawns. These results taken together suggest that MC3 activation does not stimulate erections but rather inhibits erections. As well, MC3 receptor antagonism (inhibition of inhibition) may not be sufficient to induce erections, but may facilitate erections initiated by MC4R activation. A proposed model for this can be seen in Fig. (2). An alternative interpretation of the data is simply that by occupying the MC3R, the antagonist allows greater amounts of MT-II to bind to MC4R.
[h=2][/h]
[h=3]Table 1[/h]Models for study of MC compounds on Penile Erection
Model | Comment |
| In vitro | |
| Cell culture |
|
| Organ bath | • Measure corpus cavernosal (end organ) relaxation in response to direct drug application |
| In vivo | |
| Animal studies | |
| Spontaneous erections | • Site specific drug delivery: intracerebral, intrathecal, intracavernosal or systemically followed by observation period. May or may not mimic behaviorally stimulated erection. Observer dependent results |
| Mating behaviors | • Site-specific delivery as above. Place trained male in contact with trained receptive female in artificial estrous or separated by barrier (non-contact erections). Measure specific aspects of erection: mounts, intromissions, ejaculations, etc |
| Reflexogenic erections | • Site-specific delivery in a restrained or anesthetized animal. Monitor specific penile responses (cups, flares, flips) or intracorporal pressure respectively |
| Nerve stimulation studies | • Site-specific drug delivery with nerve stimulation (usually cavernous nerve) in an anesthetized animal. Evaluation for facilitation or inhibition of erection by drug |
| Human studies | |
| Laboratory based | • Drug delivery IV, SQ or intranasal. Monitor penile tumescence w/ Rigi-scan with or without visual sexual stimulation. (VSS) |
| At home trials | • Phase II and III trials to determine safety, pharmacokinetics and efficacy using validated self report questionnaires and event diaries |
<colgroup><col style="width: 115px"><col width="502"></colgroup><tbody>
</tbody>
[h=4]Endogenous MC Receptor Agonists[/h]ACTH, α-MSH, β-MSH and γ-MSH are the known endogenous agonists of the MC system. Each hormone is a product of posttranslational modification of the POMC gene transcript and contains the sequence of His-Phe-Arg-Trp, considered to be the “core” of agonist activity []. Only ACTH and α-MSH have shown the ability to generate sexual stimulation and penile erection in various animal species including rats, rabbits, cats, dogs and monkeys [. These pro-erectile effects appear to be androgen-dependent as castration abolishes the aforementioned response []. Notably, many of the synthetic MC agonists contain the “core” sequence present in ACTH and α-MSH, particularly the agents MT-II and PT-141.
[h=4]MT-II (Melanotan-II)[/h][h=5]Pharmacology[/h]MT-II is a synthetic cyclic heptapeptide that was initially designed as an artificial tanning agent. Its structure is based on an earlier linear peptide, Melanotan-I, however cyclization was introduced to prevent degradation and allow both N and C terminal truncation of the peptide [. The pro-erectile activity of MT-II was reported as a significant unexpected reaction during a phase-I human trial of human tanning [. MT-II contains a seven amino acid sequence with homology to receptor binding portions of α-MSH and ACTH. The compound is thought to cross the blood brain barrier and has high affinity for the MC1R, MC3R and MC4R. MT-II has a similar affinity for MC4R compared with MC3R and may be considered “superpotent” because of its relatively high affinity for MC4R compared with the endogenous peptides α-MSH and ACTH (10-100 fold difference).
[h=5]In-vitro and animal studies[/h]The pro-erectile activity of MT-II appears to be both forebrain and spinally mediated, with little, if any, peripheral effect. Dose dependent increases in spontaneous erections in awake Long-Evans rats were noted with administration of MT-II intracerebrally, intrathecally and intravenously Increases in yawning and grooming behaviors paralleled erectile activity with intracerebral administration but not spinal administration. As discussed previously, when the non-selective MCR antagonist SHU-9119 was given spinally, it blocked spinal MT-II induced erections, however intrathecal SHU-9119 failed to block intracerebral MT-II induced erections. This indicates potentially independent sites of melanocortin action along the CNS axis with intracerebral sites activating multiple downstream pathways including those independent of melanocortinergic activation.
In parallel with the above observations, Vemulapalli et al found that isolated corpus cavernosal strips from rabbits had no relaxation in response to electrical field stimulation in the presence or absence of MT-II. [ As well, MT-II associated elevations in intracavernosal pressure were abolished when anesthetized rabbits underwent bilateral pudendal nerve ablation. Inhibition of NO-synthase enzymes with L-NAME also blocked the erectile effect of systemic MT-II. These pieces of data indicate that MT-II exerts direct actions through CNS brain and spinal activation, which is then mediated peripherally through established NO vasodilatory pathways.
[h=5]Human studies[/h]A double blind placebo-controlled crossover study by Wessells et al. demonstrated the safety and pro-erectile activity of subcutaneous MT-II in humans [ In the absence of erotic stimulation, 10 men with psychogenic (non-organic) erectile dysfunction received subcutaneous doses ranging from 0.025 to 0.157 mg/kg, while erections were monitored by RigiScan over a 6-hour period. Eight of the 10 men developed clinically apparent erections with greater than 80% rigidity of an average duration of 38 minutes compared with 3 minutes for placebo controls. The time to onset ranged from 15 to 270 minutes. Side effects were dose dependent included nausea, stretching, yawning and decreased appetite. At the preferred dose of 0.025 mg/kg side effects were mild.
The above study documented erectogenic effects of MT-II in men with presumed normal underlying physiology. A subsequent study of MT-II was carried out on men with organic ED . In a similar double blind, placebo-controlled crossover study, 10 men received 2 subcutaneous doses of 0.025 mg/kg MT-II and 2 doses of vehicle. MT-II initiated subjectively reported erections following 63% of the drug injection verses 5% of the placebo injections. The mean rigidity score of the responders was 6.9 on a scale of 0 to 10. Mean duration of tip rigidity greater than 80% was 45 minutes with Melanotan II compared to two minutes for placebo. There was increased subjective reporting of sexual desire after MT-II administration compared with placebo, although the question used to assess desire was not designed specifically to measure desire in men not engaging in sexual intercourse.
[h=4]PT 141 (Bremelanotide®)[/h]
[h=5]Pharmacology[/h]PT-141 (Bremelanotide®) is currently the most studied melanocortinergic compound with regard to therapeutic potential for treatment of erectile dysfunction. PT-141 is a synthetic heptapeptide. It is a deaminated derivative and likely metabolite of MT-II. This compound has strong binding to MC receptors 1, 3 and 4, with a higher affinity for MC4R over MC3R. Application of PT-141 to HEK-293 cells expressing MC4R increases cAMP production, indicating that this compound, like MT-II, acts as an agonist
[h=5]
Animal studies[/h]Studies with adult male Sprague-Dawley rats indicate pro-erectile responses through multiple modes of delivery [42]. Intranasal injection of 50μg/kg PT-141 produced a significant increase in spontaneous erections compared with saline controls in rats observed over a 30-minute period. As well, 100% of the drug treated rats had at least one erection. In this study the pro-erectile effect of PT-141 was attributed to hypothalamic stimulation of MC3R and/or MC4R. Two hours after PT-141 (50μg/kg IN) administration, immunostaining for FOS, a measure of neural activation, showed increased expression in the paraventricular nucleus compared with rats administered saline.
[h=5]
Human studies[/h]Preliminary trials in humans have established both safety and efficacy of PT-141. A phase 1 randomized double-blind placebo controlled trial involved 24 healthy male subjects without erectile dysfunction [42, 43]. Intranasal doses of 4 to 20mg were delivered to patients in the absence of visual sexual stimulation (VSS). Safety and tolerability were monitored revealing no significant hemodynamic changes or side effects, including priapism. Serum concentration of drug was dose dependent and peaked at 30 minutes in the maximum dose group. Serum half-life was measured at 120 minutes. Rigi-Scan monitoring of erectile response revealed a significantly increased duration of rigid erections of 140 minutes compared to 22 minutes in the placebo group. Time to onset of erection ranged from 34 to 63 minutes. Erections were considered rigid if they were more than 60% of base rigidity.
Based on the above results, phase II studies were initiated in patients with mild to moderate ED who showed positive erectile response to PDE-5 inhibitors [44]. RigiScan monitoring in the presence of VSS detected a 3-fold increase in erectile activity with PT-141 (20mg intranasal) administration. The duration of base rigidity was significantly increased utilizing both a 60% and 80% cut-off versus placebo [43]. Timing of erections corresponded well to visual stimulation indicating a potential facilitator mechanism of drug action.
In a first Phase IIB at home study, PT-141 induced dose dependent improvements in erectile function as assessed by the International Index of Erectile Function (IIEF) [45]. Of the patients who completed at least 3 at home attempts (n = 203), the mean IIEF erectile function domain score increased in a dose dependent fashion (p < 0.05 for 10, 15, and 20 mg). Normal erectile function (EF>26) was achieved by 10, 30, 36, 53, and 50% of patients in the placebo, 5, 10, 15, and 20 mg groups respectively. Improved erections as defined by a global assessment question were reported by 17, 49, 67, 66, and 66% of patients in the placebo, 5, 10, 15, and 20 mg groups respectively. There were no episodes of syncope or hypotension. The only serious adverse event reported in this study occurred in one patient who reported a prolonged erection that was painless and required no treatment.
Gastrointestinal side-effects were the primary reasons for discontinuation in the higher two higher dose groups.
In summary, PT-141 is a potent initiator of erection with minimal side effects, a rapid onset of action and a sufficiently long duration of action. Notably, the recent Phase II studies confirm that the erectile responses are augmented by sexual stimulation. With its central mechanism of action, PT-141 may act independent or synergistically with PDE-5 inhibitors and provide a useful alternative therapy for erectile dysfunction, both from organic and psychogenic origin.
A randomized prospective placebocontrolled study compared treatement of ED with sildenafil alone verses sildenafil with 7.5mg intranasal PT-141 [46]. Co-administration of the two agents resulted in significantly prolonged time of increased base rigidity (>60%) compared with sildenafil alone during a 2.5 hour monitoring session. The combination of drugs was well tolerated with no significantly increased side-effects over either sildenafil or PT-141 alone. The ability of the peptide to safely and effectively induce high quality erections allowed Palatin Technologies to initiate a second Phase IIB study in 2006 and propose Phase III studies in 2007 [47].
[h=4]
THIQ[/h]THIQ is a synthetic small molecule hMC4R agonist with moderate bioavailability (14%), rapid absorption (Tmax = 1hr) and a short T1/2 (0.5hrs) [
]. THIQ has a >600 selectivity for the MC4R compared with MC3R
. Studies by Martin and Van der Ploeg evaluated the effects of THIQ delivered both systemically and intracerebrally in rodents. Systemic administration (1mg/kg) potentiated electrically stimulated erections as well as decreasing mounting and intromission latency mating behaviors in wild type mice [30]. In an ex copula model using male rats, systemic THIQ dose dependently increased total numbers of erections
. This effect was blocked by central administration of the non-specific antagonist, AgRP, as well as the MC4R specific antagonist, MBP-10. ICV delivery of 20μg of THIQ increased reflexive penile erections. There are no studies of THIQ in humans to date. Interestingly, and in contradistinction to MT-II, THIQ has not been reported to initiate spontaneous erections in rodents.
[h=3]
MC Receptor Antagonists[/h]Endogenous and synthetic antagonists have been used to explore melanocortin signaling. When MCR antagonists bind to the MC receptors they either decrease constitutive levels of cAMP production or prevent agonist induced increases in cAMP production. In studies of penile erection, MCR antagonists have been primarily utilized to identify the mechanisms and location of action of MCR agonists as well as parcel out specific receptor subtype activity. These compounds have not been utilized as therapeutic agents.
Endogenous melanocortin receptor inhibitors include agouti or agouti-related peptide (AgRP). AgRP is a 132 amino acid peptide which competitively antagonizes both MC3R and MC4R [51]. While AgRP has primarily been studied for its role in energy homeostasis, this peptide is principally expressed in the arcuate nucleus of the hypothalamus, a potential site for regulation of melanocortin mediated erection [14]. As mentioned, intracerebral delivery of AgRP (5.5μg) was shown to block erections in rats induced by the MC4R agonist, THIQ [ There have been no studies in humans with regard to erection.
SHU-9119 is a classical inhibitor of both the MC3R and the MC4R. This synthetic cyclic lactam α-MSH analogue is closely related in structure to MT-II [52]. SHU-9119 actually has agonist properties at MC1R and MC5R, but for the purposes of discussing erection, this compound is considered primarily an antagonist because of the lack of these receptors in the CNS. In rabbits this highly potent compound readily blocked MT-II induced erections when administered systemically [34]. In rats, SHU-9119 blocked erections and grooming/yawning behaviors stimulated by MT-II both at supraspinal and spinal locations [31]. This compound has not been studied in humans.
Two other synthetic MC receptor antagonists that have been utilized in studies of erectogenisis include MPB-10 and HS014. Both of these compounds preferentially block the MC4 receptor. Their use in animal studies has primarily been related to determination of receptor specification as described in the following section. These compounds have not been studied in humans.
[h=3]MC3R vs. MC4R[/h]Of the 5 melanocortin receptor subtypes, only the MC3R and MC4R have been identified in CNS regions associated with activation of penile erection [1], particularly the PVN of the hypothalamus. Many of the more commonly studied compounds, such as MT-II and αMSH, activate both MC3 and MC4 receptor subtypes to some degree. This lack of receptor specificity has limited our understanding of each receptor’s contribution toward erectile behaviors and has prompted studies utilizing receptor specific agonists and antagonists as well as receptor knock out mouse models. Contrary evidence has pointed to each receptor as the principle subtype mediating erection. Although the weight of evidence leans towards MC4R activation being responsible for activation of erection, the debate remains unresolved.
Evidence of MC3Rs participation in sexual stimulation and erection comes from a series of studies in the late 1990s utilizing an MC4R specific antagonist, HS014 [53]. Vergoni et al. administered ACTH and α-MSH into the lateral ventricle of adult male Sprague-Dawley rats and showed predictable responses with grooming, stretching, yawning and erections [2]. Co-administration of these compounds with HS014 completely blocked grooming, stretching and yawning behaviors, but only partially reduced erections. Argiolas et al. studied this effect further with ACTH, α-MSH and HS014 microinjections into regions surrounding the 3rd ventricle of adult rats [54]. The effect was a dose dependent elicitation of yawns, grooms and erections when only ACTH and α-MSH were administered. Co-administration of these compounds with HS014 significantly blocked yawns and grooms but erections were unaffected. This evidence suggested that the MC4R was not involved in the sexual response to ACTH and α-MSH. As the only other MC receptor in the region, the MC3R was attributed partial credit for the erectile response.
However, HS014 does have MC3R antagonist activity and the relatively small difference in affinity for MC4 vs. MC3 receptors makes interpretation difficult. If MC3R were the primary mediator of erection, one would have expected some diminution of erections with this compound. Another possible consideration in the interpretation of these studies is that a different level of MC4R occupancy may stimulate yawning/ grooming behaviors and erection. Thus a higher dose of antagonist may be required to block penile erection. Finally, the proerectile effects of MSH are not as potent as synthetic analogs such as MT-II, raising the possibility that an inadequate stimulatory dose of the agonist prevented a measurable effect of the antagonist (floor effect).
Evidence in support of MC4R as the principle receptor mediating erections is provided by multiple converging lines of evidence using MC4R knock out mice as well as rat studies with MC4R specific agonists and antagonists. Van der Ploeg et al stimulated the cavernous nerves of wild type and MC4R knockout mice in the presence or absence of systemic THIQ (10mg/kg IV), an agonist with >1000-fold selectivity for MC4R compared with MC3R [30]. Wild-type mice had significant augmentation of erectile activity while knockout mice did not show any changes. Copulatory behavior in these mice was evaluated as well. MC4R wild type mice received THIQ (2.5mg/kg IP) or vehicle and were paired with estrous females. THIQ treatment decreased mounting and intromission latencies in WT mice. Interestingly, MC4R knock out mice had impaired copulatory behavior compared with littermate wild type controls, showing baseline dysfunctional mating characteristics with loss of the MC4R. The systemic delivery of THIQ in these studies led the authors to question whether the effect could be related to direct activation of cavernosal tissue. Although they demonstrated MC4R mRNA in rat cavernosum, application of THIQ to cavernosal strips did not result in tissue relaxation.
Martin et al. utilized a slightly different approach to investigate MC3/4R question, by administering selective and non-selective antagonists to MC4R in combination with the MC4R agonist THIQ [48]. MBP10 is a synthetic MCR antagonist with at least a 125-fold selectivity for MC4R over MC3R [55] while AgRP is an endogenous antagonist with comparable inhibition of both MC3R and MC4R. In this study, an ex copula model of monitoring penile reflexes was measured after drug delivery. Consistent with the work of Van der Ploeg et al., systemic THIQ increased intracavernosal pressures and dose-dependently increased reflex erectile activity in restrained rats. Central administration of THIQ into the 3rd ventricle of rats increased reflexive penile erections. This centrally mediated effect was blocked by pretreatment with both AgRP as well as MPB10. Unfortunately, an MC3R selective antagonist was not employed in this study. The conclusion of this study was that MC4R activation was sufficient for penile erectile activity, but did not exclude a possible role for MC3R.
Although the pro-erectile effects of MC4R activation appear well established, the contribution of MC3R towards erection is incompletely understood. An alternative hypothesis to the above studies is that stimulation of the MC3R may actually be inhibitory toward erectile activity. In support of this hypothesis are neuroanatomical pathways involving AgRP (endogenous melanocortin antagonist) and POMC neurons, which travel in parallel throughout much of the central nervous system. MC3R mRNA has been co-localized to both AgRP and POMC neurons in a rostrocaudal gradient in the arcuate nucleus [56]. In contrast, MC4R mRNA was not found in AgRP or POMC expressing cells. This implies a potential negative feedback loop whereby “expression of MC3R by POMC neurons provides a potential circuit for amplification of AgRP-mediated signals, because AgRP-induced inhibition of POMC neurons via the MC3R would reinforce the postsynaptic effects of AGRP. Furthermore, the expression of the MC3R by AGRP neurons provides a potential circuit for negative autoregulation of POMC-mediated signals, because POMC-induced activation of AGRP neurons via the MC3R would terminate the postsynaptic effects of POMC”.
Preliminary studies in our laboratory using MC3R specific agonists and antagonists may have unmasked a proposed autoinhibitory pathway of the MC3 receptor. AVM-127 is a cyclic γ-MSH analog with selective MC3R/ MC5R Antagonist activities in vitro; it is a synthetic compound with antagonist activity and 100 fold selectivity for MC3R compared to MC4 [57]. A study of intracerebroventricular administration of AVM-127 (750ng) prior to MT-II (1μg) in adult male rats was performed with the initial hypothesis that antagonism of the MC3 receptor would either reduce or have no effect on MT-II stimulated erections. This hypothesis was based on previous studies indicating that MC3R activation was “pro-erectile”. Paradoxically, rats administered AVM-127 in combination with MT-II had significantly more erections over a 90 minute observation period when compared with rats given MT-II alone (see Fig. 1). Rats given AVM-127 alone had no significant erectile activity compared with controls. AVM-127 had no effect on MT-II induced yawns or genital grooming behaviors. These results were highly surprising and unexpected. An inverse approach to this study involved specific stimulation of central MC3 receptors with a novel specific MC3R agonist, the γ-MSH analog PBIII-93 [58]. When administered ICV to male rats, this compound failed to produce erections despite dose-dependently stimulating yawns. These results taken together suggest that MC3 activation does not stimulate erections but rather inhibits erections. As well, MC3 receptor antagonism (inhibition of inhibition) may not be sufficient to induce erections, but may facilitate erections initiated by MC4R activation. A proposed model for this can be seen in Fig. (2). An alternative interpretation of the data is simply that by occupying the MC3R, the antagonist allows greater amounts of MT-II to bind to MC4R.
[h=2][/h]
