Synthetic psychoactive cathinones: hypothermia and reduced lethality\ncompared to methamphetamine and methylenedioxymethamphetamine
Rationale: Synthetic psychoactive cathinones (SPCs) are drugs with psychostimulant and entactogenic properties like methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). Despite clinical reports of human overdose, it remains to be determined if SPCs have greater propensity for adverse effects than MA or MDMA. Objectives: To determine whether the SPCs cathinone (CAT), methcathinone (MCAT), mephedrone (MMC), and methylenedioxypyrovalerone (MDPV) have lower LD50 values than MA or MDMA. Methods: Male and female C57Bl/6J mice received single injections of one of 6 doses of a test drug (0\xe2\x80\x93160 mg/kg IP). Temperature and behavioral observations were taken every 20 min for 2 hr followed by euthanasia of surviving mice. Organs were weighed and evaluated for histopathological changes. Results: LD50 values for MA and MDMA, 84.5 and 100.9 mg/kg respectively, were similar to previous observations. The LD50 for MMC was 118.8 mg/kg, but limited lethality was observed for other SPCs (CAT, MCAT, MDPV), so LD50 values could not be calculated. For all drugs, death was associated with seizure, when it was observed. Rather than hyperthermia, dose-dependent hypothermia was observed for MMC, MDPV, CAT, and MCAT. Contrary to initial expectations, none of the SPCs studied here had LD50 values lower than MA or MDMA. Conclusions: These data indicate that, under the conditions studied here: (1) SPCs exhibit less lethality than MA and MDMA; (2) SPCs impair thermoregulation; (3) effects of SPCs on temperature appear to be independent of effects on lethality.
Introduction<!>Subjects<!>Drug Treatments:<!>Statistical Analysis:<!>Lethality<!>Drug-Induced Seizures<!>MA<!>MDMA<!>MMC<!>MCAT<!>CAT<!>MDPV<!>Organ Weights<!>DISCUSSION<!>Temperature<!>Hepatotoxicity, ammonia, and brain glutamate function
<p>Synthetic psychoactive cathinones (SPCs) are drugs with psychostimulant
properties similar to those of methamphetamine (MA) and
3,4-methylenedioxymethamphetamine (MDMA), derived from the naturally occurring
molecule cathinone (CAT), found in the khat plant (Catha edulis)
(Prosser and Nelson, 2012). Similar to
those more well-known stimulants, SPCs show clear potential for abuse ((Javadi-Paydar et al., 2018; Schindler et al., 2016; Vandewater et al., 2015; Watterson et
al., 2012a) for review see (Negus and
Banks, 2017; Riley et al., In
Press; Watterson and Olive, 2017))
and pose a threat to public health (Madras,
2017; Zaami et al., 2018). Their
use has been associated with side effects that include neurotoxicity, hyperthermia
and lethality (Marusich et al., 2014).
However, it is difficult to determine the dangers presented by this new class of
drugs from clinical reports alone. Clinical reports are usually single case reports
or small studies that may lack confirmatory toxicology. Moreover, clinical
circumstances most often involve the ingestion of multiple drugs, making it
difficult to evaluate the contribution of particular drugs to these adverse
outcomes, and consequently the dangers that individual drugs may pose. Furthermore,
illicit drugs have an inconsistent composition that often include drug mixtures, of
variable doses, that can create a mismatch between what the patients believe they
have consumed and the actual drugs consumed (Araújo et al., 2015). Indeed, hair samples from self-reported
MDMA users have been found to contain a wide variety of both SPCs and other
amphetamine derivatives (Caudevilla-Gálligo
et al., 2013; Palamar et al.,
2016). In many cases from those studies samples tested negatively for MDMA
but were positive for one or more SPCs, such as methylone or butylone. The
possibility of unknowing consumption of SPCs by illicit drug users additionally
suggests that the use of these drugs may be more widespread than is currently
thought.</p><p>SPCs are commonly known as "bath salts", because they were
originally marketed with such labels to circumvent in initial legal restrictions
(Spiller et al., 2011), prior to the
majority of these drugs being made illegal in the US and Europe. At that time, these
drugs were labelled "not-for human consumption" to hide their intended
use as recreational drugs and, before 2011, they could be easily purchased in smoke
shops and gas stations in the US. Thus, they were also known as "legal
highs" and were even presumed by some users to be safe alternatives to
illicit drugs such as MA, amphetamine and cocaine (Jerry et al., 2012). Currently, the majority of SPCs are illegal, but
analogues are continually emerging as illicit manufacturers attempt to circumvent
legal restrictions or to appeal to illicit markets, as is happening for other
classes of drugs, including opioids and cannabinoids. These emerging drugs may
differ in a number of properties, one of which is potency, which may increase the
risk for adverse effects, including death, as it has for the emergence of potent
fentanyl analogues as part of the current opioid crisis in the US and elsewhere
(Misailidi et al., 2018).</p><p>The abuse and addiction potential of SPCs is now well recognized ((Watterson et al., 2014), and see review by
Watterson and Olive (2017)), and may be
greater than that of MA and MDMA for some SPCs, although many remain to be
characterized. Overall, users seek out these drugs because of their similar
subjective effects to other drugs of abuse, including MA, MDMA and cocaine (Ashrafioun et al., 2016; Johnson and Johnson, 2014), that include euphoria,
alertness, wakefulness, an increased sense of empathy and/or connection with others
and orgasm intensification (Rosenbaum et al.,
2012). The reported adverse effects of these drugs are similar to other
stimulants, and wide in scope, including agitation, tachycardia, palpitations, chest
pain, hypertension, violent behavior, hallucinations, paranoia, confusion,
mydriasis, vomiting, myoclonus, seizures, organ toxicity, hyperthermia, and
lethality (Spiller et al., 2011).</p><p>The similarities between the effects of SPCs and other stimulant drugs are
not surprising given the strong similarities in their chemical structures and
pharmacological activity. SPCs share a phenethylamine pharmacophore with amphetamine
(Smith et al., 2017), and really only
differ based on the presence of a β-ketone moiety. Like amphetamines,
variations in the length of carbon substitutions at the α-carbon and
expression of the nitrogen terminus generate analogues that vary in pharmacological
effects (Banks et al., 2014), as well as
pharmacokinetic properties ((Grecco et al.,
2017), for review see Calinski et al.
(2019)). The length of the alkyl chain and the presence or absence of a
pyrrolidine moiety, along with the β-ketone addition (Namera et al., 2015), have been reported to affect the
mechanism of action of CAT analogues and, in particular, for their selectivity to
particular monoamine transporters (Simmler et al.,
2013). To one degree or another, all of these stimulant drugs increase
extracellular norepinephrine, serotonin, or dopamine levels by acting as monoamine
transporter inhibitors or monoamine releasers (Baumann et al., 2012). Shorter alkyl chains and modification of the
pyrrolidine moiety reduce dopamine transporter activity, for example (Kolanos et al., 2013). The pharmacokinetic
properties of cathinones were also altered by extension of the α-alkyl chain,
but these effects were different centrally and peripherally (Grecco et al., 2017), increasing Cmax, AUC and
t1/2 peripherally, while reducing Cmax and AUC centrally.
Although not widely studies yet, it is clear that effects of structural changes on
pharmacokinetic properties are likely to contribute to their addictive and toxic
potential. However, it remains unclear how particular modifications to the core
cathinone structure may alter toxicity or lethality.</p><p>At least some SPCs appear to have less neurotoxic potential than MA and MDMA
(Angoa-Pérez et al., 2014; Angoa-Pérez et al., 2012), including
methylenedioxypyrovalerone (MDPV) and mephedrone (MMC; 4-methyl-methcathinone).
Methcathinone (MCAT) produces dopaminergic neurotoxicity, although this is reduced
compared to MA. MMC does enhance damage to dopaminergic nerve endings when
administered with sub-optimally neurotoxic doses of MA (Angoa-Pérez et al., 2013). In the case of MMC, it
has been hypothesized that reduced neurotoxicity is attributable to the 4-methyl
group, as the equivalent substitution in MA yields a psychoactive drug devoid of
neurotoxic potential. Much still remains to be done to fully characterize the
structure-activity relationships underlying the neurotoxic effects of SPCs, and what
other actions of these drugs may contribute to their toxicity. As is the case for
amphetamines, the effects of these drugs on temperature may be a critical factor
influencing their adverse effects. Both MMC and methylone damage serotonergic
systems at elevated temperatures (Pantano et al.,
2017; Wright Jr et al., 2012), as
is the case for amphetamines (Miller and
O'Callaghan, 2003; O'Shea
et al., 2006). In any case, it remains to be seen if the mechanisms
underlying the neurotoxic potential of SPCs is related to the mechanisms underlying
the acute neurological and cardiac effects associated with overdose and death.</p><p>The psychiatric consequences of SPC overdose may be consistent with the
known neural mechanisms of these drugs, but because case reports also describe
cardiac, renal, and hepatic injury following SPC overdose, peripheral toxicity has
been suggested to be a primary cause of lethality in many cases of acute overdose.
Clinical reports that point to cardiac injury as cause of death include reports
sinus tachycardia, reduced cardiac output and heart failure (Beck et al., 2018; Marinetti and Antonides, 2013; Spiller
et al., 2011). These consequences may be caused by effects of these drugs
on the heart itself, myocardial damage due to rhabdomyolysis, or perhaps indirectly
through overstimulation of adrenoreceptors in the central nervous system. Reported
renal symptoms include high blood urea nitrogen levels, hyperkalemia, dehydration,
hyponatremia and rhabdomyolysis resulting in acute renal injury (Benzer et al., 2013; Borek and Holstege, 2012; Eiden et al.,
2013). Some overdose cases also present with hepatotoxicity, including
post mortem signs of hepatic inflammation (Marinetti and Antonides, 2013). SPCs may act similarly to
MA, resulting in elevated hepatic levels of reactive metabolites, mitochondrial
impairment, hyperthermia and apoptosis (for review see (Carvalho, M. et al., 2012)). In vitro
studies have assessed some potential aspects of SPC toxicity, and shown that a
number of SPCs are hepatotoxic (Araújo et
al., 2015). MDPV shows a temperature-dependent hepatotoxicity (Valente et al., 2016), and MMC is more
cytotoxic in SH-SY5Y cells than MA (Valente et al.,
2017). MMC toxicity was partially prevented by dopamine transporter (DAT;
Slc6a3) blockade or overexpression of Bcl2.
MMC also depletes ATP stores in hepatocytes by disruption of the electron transport
chain (Luethi et al., 2017). Although clearly
demonstrating several types of toxicity, it remains to be seen which of these
effects underlies the acute lethality of SPCs, and whether SPCs have more, or less,
potential for lethality compared to MA and MDMA.</p><p>To date, only one non-clinical study (Piao
et al., 2015) has explicitly examined the lethality of any SPCs. This
study found that the LD50 for methylone is slightly lower than that of MA or MDMA.
Moreover, methylone lethality was reduced in DAT knockout mice. Moreover, several
approaches in that report showed that the mechanisms underlying methylone lethality
could be dissociated from methylone effects on temperature, similar to a study of
METH-induced lethality (Numachi et al.,
2007). This is not to say that temperature does not affect the lethality of
these drugs, but rather that the lethal effects of these drugs occur even in the
absence of hyperthermia. The experiments described in this report, begin to
elucidate the potential mechanisms underlying the lethal effects of SPCs: LD50
studies were conducted with several SPCs, and for comparison, MA and MDMA. Effects
on temperature and peripheral organs were also examined.</p><!><p>Adult male and female C57Bl/6J mice were obtained from the Jackson
Laboratory (Bar Harbor, ME, USA) at 7 weeks of age. Subjects were housed in
groups of 4–5 mice for one week in standard plastic cages (7.5 ×
11.75 × 5″) prior to testing for habituation to animal facility
conditions. Food (Teklad rodent diet 2916; 4% fat) and water were available
ad libitum. Temperature was maintained at 22–23
°C and humidity was maintained at 40–45%. Subjects were tested
between 8 and 12 weeks of age (average weight: males, 27.1 ± 0.9;
females, 20.5 ± 0.7).</p><!><p>On test day, mice were taken from the colony and transported to the
experimental room where they were allowed to acclimate for 2 hours prior to the
first temperature readings. During testing, temperature and humidity remained at
the same values as the housing conditions. Water was not available during this
time. During this time, they were housed singly in cages identical to their home
cages, but with bench paper placed on the bottom of the cages. Rectal
temperature was measured twice before injection at twenty-minute intervals, to
establish a baseline, and then for two hours after the injections. Subjects (N=5
males, and N=5 females, for each experimental condition) were weighed prior to
testing, and injected with one of 6 different drugs (MA, MDMA, MCAT, CAT, MDPV,
or MMC) at one of six different doses (20–160 mg/kg IP, calculated as
salt, and dissolved in sterile saline). Drugs were kindly provided by the NIDA
Drug Supply Program, except for MA, which was purchased from Sigma (St. Louis).
These drugs were chosen for study primarily because of their abuse potential,
and common use for that purpose, not for structure-activity considerations,
something that will need to be addressed in future studies. After each
temperature reading, behavioral observations were recorded: general level of
activity, presence of stereotypical behavior, aspects of 5-HT behavioral
syndrome (e.g. Straub tail, head shakes, ptosis), seizure, tremors, grooming,
sniffing, vacuous chewing, and righting. Upon death, either by lethal
administration of drug or euthanasia, the following organs were collected:
brain, heart, lungs, kidney, intestines, and stomach. Organs were weighed and
then immediately immersed in 10% formaldehyde. All experiments were conducted in
accordance with all applicable NIH guidelines, and in accordance with
The Guide for the Care and Use of Laboratory Animals
(National Academies of Sciences Press), under animal protocols approved by the
University of Toledo Institutional Animal Care and Use Committee.</p><!><p>χ2 tests were initially used to evaluate lethality,
comparing sex and dose. As no significant effects of sex were observed, LD50
values were calculated for each drug for the pooled subjects. The same analysis
was used to examine the relationship of dose to the incidence of seizure.</p><p>Analysis of temperature data was performed using repeated measures ANOVA
with TIME as a repeated measure with DOSE and SEX as a between-subjects factor.
Temperature data was only analyzed for subjects for which there was complete
temperature data, and groups were dropped from the analysis if all measurements
were available for less than 4 subjects. Thus, for some drugs (as noted below)
only some doses were used in the temperature analysis. Temperature data from
male and female subjects were analyzed separately after the initial omnibus
ANOVA. Over all effects of drugs on organ weights were performed using two-way
ANOVA. Post hoc analyses were performed using Bonferroni
comparisons. ANOVA were performed using SPSS 23.0 (IBM). LD50 and ED50
calculations were made using Prism 5.04 (GraphPad Corporation). Temperature and
organ weight data are presented as mean ± standard error of the mean
(SEM). The α level was 0.05.</p><!><p>MA and MDMA both induced lethality. Lethal effects were confirmed as
significant effects of dose in χ2 tests for MA
(χ2[5, 55] = 34.0, p<0.0001) and MDMA
(χ2[6, 69] = 49.4, p<0.0001). Sex was not
significant in either analysis. The LD50 for MA was estimated to be
105.2 mg/kg calculated as the salt or 84.5 mg/kg as base and MDMA was estimated
to be 119.2 mg/kg (salt) or 100.92 mg/kg (base) (Fig. 1). LD50 values could only be calculated for one of the four
SPCs tested, MMC, for which there was a significant effect of dose in the
X2 test (χ2[5, 60] = 34.0, p<0.0001).
Sex was not significant. The LD50 for MMC was estimated to be 143.2
mg/kg (salt) or 118.8 mg/kg (base) (Fig.
1). The only other SPC to induce a lethal effect was MCAT
(χ2[5, 60] = 5.1, p=NS), however, this occurred at the
highest dose and only in one subject. CAT and MDPV produced no lethal effects at
any dose.</p><p>Behavioral observations demonstrated a high incidence of hyperlocomotion
and stereotypical behavior for all drugs, even from the lowest doses. These
behaviors were not always observed for higher doses as these animals lost
righting ability and quickly developed seizures. Symptoms of 5-HT behavioral
syndrome were sometimes observed, including Straub tail, but were not observed
consistently in all subjects for any drug. When lethality did occur, it
typically occurred within 20–60 mins of drug administration, generally
associated with seizure activity beginning within 5 or 10 minutes after drug
administration. This was observed for all cases of lethality for those drugs
that did induce lethality at the doses tested.</p><!><p>MA, MDMA, and MMC induced seizures in a dose dependent manner. Seizure
effects were confirmed as significant effects of dose in χ2
tests for MA (χ2[5, 55] = 21.1, p<0.0001), MDMA
(χ2[6, 69] = 34.3, p<0.0001), and MMC
(χ2[5, 60] = 30.9, p<0.0001). Sex was not
significant in either analysis. The ED50 for MA was estimated to be
97.1 mg/kg calculated as the salt or 78 mg/kg as base while MDMA was estimated
to be 137.6 mg/kg (salt) or 116.5 mg/kg (base) (Fig. 2). MMC had the second highest incidence of seizure with an
estimated ED50 of 124 mg/kg as the salt or 102.9 mg/kg as base,
slightly lower than the observed value for MDMA. Sex was not significant.</p><!><p>The effects of MA on temperature were only examined for doses of 20,
40, and 80 mg/kg as higher doses caused lethal effects in most subjects
within 2 hrs (Fig. 3A/B). MA administration in male but not female mice
increased body temperature. An omnibus repeated measures ANOVA for
temperature revealed a significant effect of TIME (F[8,168] = 5.0,
p<0.0001) and a significant interaction of TIME × SEX
(F[24,200] = 2.8, p<0.0001). In subsequent 1-way ANOVA examining each
dose separately, only males at the 40 mg/kg dose showed a significant
overall effect of TIME (F[8,80] = 5.38, p<0.0001). Temperature
increased after injection by about 1.5 °C, reaching a maximum at the
60 min timepoint (p<0.04 vs. baseline). Temperature subsequently
returned to baseline by the 100 min timepoint. Neither the 20 mg/kg dose,
nor the 80 mg/kg dose produced significant changes in temperature in males.
When compared to baseline, females showed no significant differences in
temperature post-injection.</p><!><p>The effects of MDMA on temperature were only compared for the 20,
40, and 80 mg/kg doses due to lethal effects of MDMA at higher doses. MDMA
was found to increase temperature in males while causing hypothermic effects
in females (Figs. 3C/D). An omnibus
two-way ANOVA found a significant effect of SEX (F[1,26] = 9.0,
p<0.006) while a repeated measures ANOVA indicated a significant
effect of TIME (F[8,208) = 6.2, p<0.0001), as well as significant
interactions of TIME × SEX (F[8,208) = 4.6, p<0.0001) and TIME
× DOSE (F[16,208] = 1.7, p<0.05). Post hoc
analysis revealed, however, there were no significant differences between
doses. Subsequent 1-way ANOVA examining the effect of sex revealed that only
females had a significant effect of TIME (F[8,96] = 10.9, p<0.001).
When compared to baseline temperatures, a treatment with 20 mg/kg was found
to induce a hypothermic response from 20 (p<0.0001) to 80
minutes(p<0.004)) minutes after injection. This effect was not
significant at any dose in males.</p><!><p>Treatment with MMC produced profound hypothermic effects in females
as well as producing the greatest temperature decreases in males compared to
the five other drugs (Fig. 3 E/F; compare to Figures 3 and 4). An overall ANOVA indicated a significant main effect of DOSE
(F[4,39] = 3.6, p<0.01), but there was no main effect of SEX. A
repeated measures ANOVA indicated a significant main effect of TIME
(F[8,312] = 158.2, p<0.0001) as well as interactions of TIME ×
SEX (F[8,312] = 3.9, p<0.0001) and TIME × DOSE (F[32, 312] =
4.8, p<0.00001). The data were further examined in separate ANOVA
split by sex, which indicated a significant effect of TIME in both males
(F[8,160] = 58.1, p<0.0001) and females (F[8,152] = 107.4,
p<0.0001). An interaction of TIME × DOSE was also seen for
both males (F[32,160] = 2.9, p<0.0001) and females (F[32,152] = 3.3,
p<0.0001). Subsequent one-way ANOVA examining each dose separately
indicated significant effects of TIME at 20 mg/kg (F[8, 32] = 53.9,
p<0.0001), 40 mg/kg (F[8, 32] = 24.3, p<0.0001), 80 mg/kg
(F[8, 32] = 17.4, p<0.0001), 100 mg/kg (F[8, 32] = 16.2,
p<0.0001), and 120 mg/kg (F[8, 24] = 30.9, p<0.0001) for
females and 20 mg/kg (F[8, 32] = 16.0, p<0.0001), 40 mg/kg (F[8, 32]
= 106.8, p<0.0001), 80 mg/kg (F[8, 32] = 11.9, p<0.0001), 100
mg/kg (F[8, 32] = 12.5, p<0.0001), and 120 mg/kg (F[8, 32] = 5.2,
p<0.0004) for males. Post hoc analysis indicated
substantial decreases in temperature in both males and females. At 20 mg/kg,
females had hypothermic responses lasting from 20 minutes (p<0.0001)
to 40 minutes (p<0.0001) post injection, while males experienced less
prolonged effects lasting only 20 minutes (p<0.0001). Administration
of 40 mg/kg of MMC produced hypothermic changes extending from 20 minutes
(p<0.0001) to 60 minutes (p<0.05) post injection in females
and in males (p<0.0001). In females, 80 mg/kg doses produced similar
responses with effects peaking at 20 minutes (p<0.0001) post
injection and lasting 60 minutes. (p<0.02). A similar, but slightly
more prolonged effect was observed in males given this dose, peaking at 20
minutes (p<0.0001) and lasting up until 80 minutes (p<0.03)
after administration. When injected with 100 mg/kg MMC, slightly more
prolonged hypothermic effects were observed in females which lasted from 20
minutes (p<0.0001) to 60 minutes (p<0.0003). Moreover, males
showed similar effects with hypothermic responses lasting from 20 minutes
(p<0.0001) to 80 minutes (p<0.03) after injection. Finally,
administration of 120mg/kg of MMC produced the most significant and
prolonged decreases in temperature in females lasting from 20 minutes
(p<0.0001) to 60 minutes (p<0.0001), but this dose only
elicited significant hypothermic effects at 20 minutes (p<0.04) post
injection in males.</p><!><p>Administration of MCAT also induced pronounced hypothermic effects,
the severity of which was dependent on both sex and dose (Fig. 4A/B).
An overall two-way ANOVA for temperature indicated significant effects of
SEX (F[1,47] = 14.2, p<0.0001) and DOSE (F[5,47] = 6.570,
p<0.0001), as well as an interaction of SEX × DOSE (F[5,47] =
2.5, p<0.047). A repeated measures ANOVA indicated a significant
effect of TIME (F[8,376] = 94.6, p<0.0001) as well as interactions of
TIME × SEX (F[8,376] = 13.8, p<0.0001), TIME × DOSE
(F[40, 376] = 7.2, p<0.0001), and TIME × SEX × DOSE
(F[40, 376] = 1.6, p<0.01).</p><p>Subsequent ANOVA examining males and females individually indicated
a significant main effect of DOSE (F[5,24] = 9.8, p<0.0001) in
females but not males. Additionally, a significant main effect of TIME was
found in both males (F[8, 184] = 19.3, p<0.0001) and females
(F[8,192] = 98.0, p<0.0001), however, an interaction of TIME ×
DOSE (F[40,192] = 8.7, P<0.0001) was found in females only.
Subsequent ANOVA examining DOSE indicated a significant main effect of TIME
for 20 mg/kg (F[8, 32] = 18.0, p<0.0001), 40 mg/kg (F[8, 32] = 8.014,
p<0.0001), 80 mg/kg (F[8, 32] = 22.8, p<0.0001), 100 mg/kg
(F[8, 32] = 18.3, p<0.0001), 120 mg/kg (F[8, 32] = 28.1,
p<0.0001), and 160 mg/kg (F[8, 32] = 32.6, p<0.0001), in
females, and 20 mg/kg (F[8, 32] = 7.12, p<0.0001), 40 mg/kg (F[8, 32]
= 6.1, p<0.0001), 80 mg/kg (F[8, 32] = 4.6, p<0.001), 100
mg/kg (F[8, 32] = 8.9, p<0.0001), 120 mg/kg (F[8, 32] = 2.3,
p<0.05), and 160 mg/kg (F[8, 24] = 2.9, p<0.03) in males.
Post hoc analysis of all timepoints compared to
baseline indicated that the hypothermic effects MCAT were more prolonged at
higher doses in both males and females. Females treated with 20 mg/kg of
MCAT experienced hypothermic effects from 20 minutes (p<0.0001) to 40
minutes (p<0.005) post injection (vs baseline) while the duration of
this effect was only observed at 20 minutes (p<0.001) in males.
Treatment with 40 mg/kg of MCAT again produced pronounced hypothermic
effects which lasted longer than the effects of 20 mg/kg MCAT. Females
treated with this dose experienced decreases in temperature from 20 minutes
(p<0.0001) to 60 minutes (p<0.03) post injection. Similarly,
males showed prolonged temperature decreases as well, lasting from 20
minutes (p<0.0001) to 40 minutes (p<0.002) post injection. At
80 mg/kg of MCAT females showed hypothermic effects between 20 minutes
(p<0.0001) and 60 minutes (p<0.0001) post injection, while
males failed to show significance. Treatment with 100 mg/kg elicited even
more prolonged effects in females lasting from 20 minutes post injection
(p<0.0001) to 80 minutes post injection. Males at this same dose also
experienced prolonged hypothermia lasting from 20 minutes (p<0.002)
to 60 minutes (p<0.002) post injection. The hypothermic effects of
MCAT were further prolonged at 120 mg/kg in females, starting 20 minutes
(p<0.0001) and never fully returning to baseline temperatures even
120 minutes (p<0.02) after injection. Post hoc
comparisons did not identify an significant effects in males given 120 mg/kg
MCAT. Finally, treatment with 160 mg/kg of MCAT produced the most pronounced
prolonged hypothermic effect in females starting at 20 minutes
(p<0.0001) post injection and never fully returning to basal
temperatures 120 minutes later (p<0.0001). Males experienced some
hypothermic response at this dose, peaking at 60 minutes (p<0.02)
post injection.</p><p>The peak hypothermic effect occurred 60 min after injection in both
males and females, however, there was a more pronounced and prolonged effect
in females as opposed to males. The most significant difference in
temperature among females at this point was observed between the lowest dose
of 20mg and the two highest doses of 120mg (p<0.000) and 160mg
(p<0.000). When compared to baseline temperatures, all doses induced
hypothermic responses 20 minutes post injection (p<0.000) in females.
This response was the most prolonged at the 160mg/kg, preventing the return
to normothermic temperatures throughout the experiment (p<0.000). In
males, no significant differences in dose were observed at any of the time
points.</p><!><p>Treatment with CAT produced a similar hypothermic state to that
observed with MCAT, an effect again dependent on both sex and dose (Fig. 4C/D). An overall two way ANOVA indicated significant main effects
of both SEX (F[1,45] = 8.5, p<0.006) and DOSE (F[5,45] = 8.2,
p<0.0001). A repeated measures ANOVA examining the effect of TIME,
showed a significant main effect of TIME (F[8,360] = 168.1, p<0.0001)
as well as significant interaction of TIME × SEX (F[8,360] = 12.9,
p<0.0001), TIME × DOSE (F[40,360] = 12.9, p<0.0001),
and TIME × SEX × DOSE (F[40,360] = 1.5, p<0.03).
Post hoc analysis showed that the largest hypothermic
effect occurred at a dose of 160 mg/kg when compared to doses of 20
(p<0.0001), 40 (p<0.0001), and 100 mg/kg (p<0.005), but
not 120 mg/kg. Additional ANOVA analyzing the data separately for each sex
showed significant effects of TIME for females (F[8,192] = 113.4,
p<0.0001) and males (F[8,168] = 67.0, p<0.0001) as well as
interactions of TIME × DOSE (Females: F[40,192] = 8.4,
p<0.0001; Males: F[40, 168] = 5.9, p<0.0001). Subsequent ANOVA
examining DOSE separately revealed a significant effect of TIME for females
at doses of 20 mg/kg (F[8, 32] = 16.6, p<0.0001), 40 mg/kg (F[8, 32]
= 39.9, p<0.0001), 80 mg/kg (F[8, 32] = 16.05, p<0.0001), 100
mg/kg (F[8, 32] = 16.6, p<0.0001), 120 mg/kg (F[8, 32] = 17.5,
p<0.0001), and 160 mg/kg (F[8, 32] = 40.5, p<0.0001) and males
at doses of 20 mg/kg (F[8, 24] = 4.6, p<0.002), 40mg/kg (F[8, 24] =
3.1, p<0.0001), 80 mg/kg (F[8, 24] = 5.0, p<0.002), 100 mg/kg
(F[8, 32] = 22.9, p<0.0001), 120 mg/kg (F[8, 32] = 16.2,
p<0.0001), and 160 mg/kg (F[8, 32] = 48.7, p<0.0001).
Post hoc comparisons of all timepoints compared to
baseline indicated that females showed hypothermic responses at all doses,
differing only in the duration of hypothermia, which lasted longer at higher
doses. Males showed hypothermic responses, but these responses were not as
prolonged as females. Administration of 20 mg/kg CAT in females lead to
decreases in temperature 20 minutes (p<0.0001 vs baseline) post
injection, which lasted until approximately 60 minutes (p<0.01).
Males also had decreased temperatures at this dose, but only at 20 minutes
(p<0.005) after administration. In females 40 mg/kg CAT elicited
hypothermic responses from 20 minutes (p<0.0001) post injection which
lasted the entire duration of the experiment (p<0.01). Conversely,
males only showed decreases in temperature at 20 minutes (p<0.007)
post injection. Doses of 80 mg/kg produced hyperthermic effects in females,
lasting from 20 minutes (p<0.0001) to 60 minutes post injection
(p<0.05), but in males hypothermia was only observed at 20 minutes
(p<0.004) post injection. Administration of 100 mg/kg of CAT lead to
decreased temperatures from 20 minutes (p<0.0001) to 40 minutes
(p<0.0002) post injection in males and females. 160 mg/kg injections
of CAT induced hypothermic changes in females from 20 minutes
(p<0.0001) to 80 minutes (0.0001) after injection. Males showed
similar but less prolonged effects at this same dose lasting from 20 minutes
(p<0.0001) to 60 minutes (p<0.0001) post injection.</p><!><p>Administration of MDPV produced significant hypothermic effects in
both males and females, which were most pronounced at the highest doses,
primarily at the 20 min time-point, and then returning to basal levels
(Fig. 4E/F). The exception here was the highest dose in
females, which did not return to baseline levels for nearly the entire
assessment period. An omnibus ANOVA for temperature revealed a significant
effects of DOSE (F[5,48] = 2.6, p<0.04) and TIME (F[8,384] = 21.2,
p<0.0001), and significant interactions with TIME: TIME × SEX
(F[8,384] = 3.3, p<0.001) and TIME × DOSE (F[40,384] = 2.1,
p<0.0001). The data were then split by sex to further analyze these
results in separate ANOVA. Both males (F[8,192] = 5.1, p<0.0001) and
females (F[8,192] = 21.7, p<0.0001) showed a significant effect of
TIME, however, only females had a significant of TIME × DOSE
interaction (F[40,192] = 2.3, p<0.0001). Subsequent ANOVA examining
individual doses indicated significant effects of TIME for 40 mg/kg (F[8,
32] = 6.5, p<0.0001), 80 mg/kg (F[8, 32] = 6.2, p<0.0001), 100
mg/kg (F[8, 32] = 3.2, p=0.01), 120 mg/kg (F[8, 32] = 12.35,
p<0.0001), and 140 mg/kg (F[8, 32] = 5.0, p<0.001) in females.
Males, however, only showed effects of TIME at the 80 mg/kg (F[8, 32] = 2.4,
p<0.04) and 140 mg/kg (F[8, 32] = 3.2, p=0.01) doses. Post
hoc comparisons of all time-points compared to baseline
confirmed that profound hypothermic responses were observed. Females treated
with 80 mg/kg (p<0.0003), 100mg/kg (p<0.002), and 120 mg/kg
(p<0.0001) of MDPV displayed substantial decreases in temperature 20
minutes after injection compared to baseline. Prolonged hypothermia was
observed for doses of 140 mg/kg from 20 (p<0.007) to 40 minutes
(p<0.005) post-injection in females. Only a dose of 140 mg/kg,
however, was able to elicit hypothermia in males at 20 minutes after
injection (p<0.003) compared to baseline. Post hoc
means comparisons did not identify any significant differences for the 80
mg/kg dose in males.</p><!><p>Organ weight data was normalized according to body weight (Fig. 5). Differences were seen in only the
heart and liver and only for MA, MDMA and MMC. MA, MDMA and MMC increased
normalized heart weight in a dose dependent manner. MA and MMC, but not MDMA,
significantly increased normalized liver weight. A two-way ANOVA for normalized
heart weight after treatment with MA indicated a significant effect of DOSE
(Fig. 5A; F[5,46] = 2.5, p<0.05)
for normalized heart weight. Heart weight increased at intermediate doses but
then returned to levels observed at the lower concentrations. The only
significant difference in the means comparisons was between the 40 mg/kg MA dose
and the 100 and 120 mg/kg MA doses (p<0.05). A two-way ANOVA examining
normalized liver weights after MA treatment revealed a significant main effect
of DOSE (Fig. 5B; F[5,46] = 16.7,
p<0.001). Post hoc means comparisons revealed a
significant increase in liver weight at both the 160 mg/kg (p<0.001) and
120 mg/kg (p<0.001) doses when compared to the lowest MA dose. No effect
of SEX was observed. MDMA also increased normalized heart rates, as shown by a
significant effect of dose (Fig. 5C;
F[5,42] = 3.7, p<0.01)). Post hoc means comparisons
found that there were significant differences for the 80 and 100 mg/kg IP doses
versus the 20 mg/kg dose. MDMA did not significantly affect liver weight (Fig. 5D; F[5,42] = 1.5, NS). MMC
administration increased heart weight at the 100 mg/kg dose (Fig. 5E) and liver weight at the 160 mg/kg dose (Fig. 5F). A two-way ANOVA examining
normalized heart indicated a significant effect of DOSE (F[5,51] = 5.6,
p<0.006). Post hoc means comparisons showed that the
largest increase occurred at the 100 mg/kg dose (p<0.02), after which
weight again returned to levels observed at lower, non-lethal, doses. A two-way
ANOVA examining normalized liver weight showed a significant effect of DOSE
(F[5,51] = 4.2, p<0.003). Post hoc means comparisons
indicated that the largest differences in liver weight occurred between doses of
160 mg/kg and 20 mg/kg MMC (p<0.05). No differences of SEX were
observed.</p><p>Histological analysis, by hematoxylin-eosin staining, did not show any
changes for most organs examined, but did shown minor changes in cell morphology
in the liver (data not presented), similar to those observed by Halpin et al. (2013) for MA at similar timepoints
post-injection. Such changes were only observed for MA, MDMA and MMC at lethal
doses.</p><!><p>Contrary to initial predictions based upon a previous study that examined
the lethal effects of methylone (Piao et al.,
2015), none of the SPCs studied here (MMC, CAT, MCAT, and MDPV) were
found to induce greater lethality than MA or MDMA (e.g. to have lower
LD50 values). Indeed, for several of these drugs (CAT, MCAT, and
MDPV) it was not possible to calculate an LD50 value at all over the
doses studied. This would seem to suggest, in contrast to expectations based upon
clinical reports (for examples see (Fudin et al.,
2018)), that these drugs pose less risk of overdose than the more
commonly abused amphetamine drugs MA and MDMA. However, as will be discussed here,
it is too early to make a broad determination regarding the risks of these drugs and
several factors may influence lethality that were not considered in the current
experiments.</p><!><p>In a similar manner to the failure of these drugs to induce substantial
lethality, these cathinones did not induce hyperthermia, which is one of the
primary adverse outcomes of MA and MDMA overdose. MA induced hyperthermia in
male, but not female, mice, and MDMA produced only a marginal hyperthermia at
one dose in males. In part, this may reflect the conditions used to examine
lethality in the present studies – overdose resulting from exposure to a
single drug dose. This was an intentional choice for these initial studies, to
examine the lethal and toxic effects of these drugs after a single exposure
before moving on to studies examining the effects of chronic or repeated
exposures in subsequent studies. In particular, hyperthermia is more typically
observed using "binge-dosing" regimens (repeated moderate dosing
cumulatively equivalent to about an LD50 dose over a period of a few
hours). Indeed, most of the studies demonstrating hyperthermia and other adverse
effects resulting from MA and MDMA have used these binge-dosing or chronic
escalating regimens of drug exposure (for review see Cadet et al. (2007) and Krasnova and Cadet (2009)). It is important to note
that even within a single MA binge, the initial dose does not necessarily induce
hyperthermia, and indeed can produce hypothermia, while subsequent doses do
induce hyperthermia (Granado et al.,
2010), suggestive of acute adaptations underlying hyperthermia. A
virtually identical pattern was observed recently for both MDMA and methylone
(Miner et al., 2017), both drugs
exhibiting robust decreases in temperature after the first injection, of 3
°C and 4 °C respectively, but increases in temperature after the
fourth injection. The doses of MDMA and methylone tested in that study were in
the range of the lowest doses studied in the present experiments. Although those
authors did not find effects of MDPV on temperature, they only used a dose of 1
mg/kg IP, well below the range of doses assessed here. MMC has also been found
to produce hypothermia acutely, but hyperthermia when administered in a
binge-paradigm (Shortall, 2015). Caffeine
can also convert MMC-induced hypothermia into hyperthermia (Shortall et al., 2016).</p><p>In the present study, MDPV produced pronounced decreases in temperature
after single injections of large doses (≈ 2 °C in males and
females). These effects were mostly transient, except in females at the highest
dose tested. The other cathinones produced even more robust and lasting
hypothermia: MCAT (maximal hyperthermia ≈ 3 °C in males and
≈ 7 °C females), CAT (maximal hyperthermia ≈ 5 °C in
males and ≈ 7 °C females), and MMC (maximal hyperthermia ≈
4 °C in males and ≈ 6 °C females). Hypothermic effects were
greater in females for all of these cathinones. Susceptibility to changes in
body temperature in males and females have been tied to differences in body mass
and susceptibility to cutaneous vasodilation in males and females (Ford and Klugman, 1980). Hypothermia is
observed after administration of many cathinones under standard temperature
conditions in rodents, including MMC (Aarde et
al., 2013a; Anneken et al.,
2017a; Shortall et al., 2013),
MCAT (Anneken et al., 2017a; Anneken et al., 2017b), and MDPV (Aarde et al., 2015). An important factor
here may be the ambient temperature. Single injections of MMC (Aarde et al., 2013a; Anneken et al., 2017a; Shortall et
al., 2013), MCAT (Anneken et al.,
2017a), MDPV (Aarde et al.,
2015), and Î±-pyrrolidinopentiophenone (Î±PVP; (Aarde et al., 2015; Aarde et al., 2013b)) have been shown to induce
hypothermia rather than hyperthermia in rodents at normal room temperature. A
direct comparison of MA and MDPV found that MA induced hyperthermia but MDPV did
not affect temperature substantially (Aarde et
al., 2013b). Lower doses (<10 mg/kg IP) of CAT and MCAT, below
those doses examined in the present study, produce a slight (≈ 1
°C) hyperthermia. An important factor here in the observation of
hypothermia versus hyperthermia may be the ambient temperature.
The current experiments were conducted at "room temperature" (e.g.
22–23 °C) and singly. Ambient temperature and the presence of
other animals (also affecting heat dissipation) is known to affect the effects
of MDMA and MA on temperature (Fantegrossi et
al., 2003; Huether et al.,
1997). Similarly, MDPV has been shown to produce hyperthermia at
elevated temperatures but not at normal ambient temperature (Gannon et al., 2017), although the robustness of
hyperthermia with increased temperature is certainly less that MA (Aarde et al., 2013b). By contrast, MMC
potentiated the hyperthermic effects of MA, as well as MA-induced neurotoxicity
(Angoa-Pérez et al., 2013).
This occurs despite MMC not producing monoaminergic toxicity on its own.
Repeated MMC binge injections (at 2 hr intervals), while continuing to produce
transient hypothermia, produced an opponent hyperthermia in recovery (Angoa-Pérez et al., 2012). The
hypothermia observed here might certainly have affected lethality, indeed it
might have exerted a protective effect, although hyperthermia is certainly not
necessary for lethality, and vice versa (Numachi
et al., 2007; Piao et al.,
2015). It remains to be seen whether the effects of the SPCs studied
here might produce hyperthermia at higher ambient temperatures, and what effect
this might have on their lethality.</p><!><p>There was evidence for acute hepatotoxicity in response to MA in the
present studies based upon increased liver weight and post
mortem histology. The histological results are similar to what was
observed for MA at a similar time-point by Halpin et al. (2013). Similar results were observed for MMC, but not
any of the other SPCs tested. Doses of MA and SPCs that induced evidence of
liver toxicity largely corresponded with lethal doses. No evidence of liver
toxicity was observed for SPCs that did not induce toxicity. Although liver
toxicity was not observed in vivo here for many SPCs, toxicity
in primary hepatocytes has been demonstrated for many SPCs, including
pentedrone, MDPV, methylone, and 4-methylethcathinone (4-MEC) (Araújo et al., 2015). Numerous factors could
influence the observation of liver toxicity, including the route of
administration, dosing regimen, ambient temperature, and the presence of
hyperthermia/hypothermia. Some of these factors may also differentially affect
the observation of hepatotoxicity between in vitro and
in vivo studies.</p><p>In any case, the observation of hepatotoxicity is consistent with
previous observations for MA that also found elevations in plasma concentrations
of ammonia (Halpin et al., 2014). Those
authors further demonstrated that increased plasma levels of ammonia were
associated with increased brain glutamate levels, and that these mechanisms were
important mediators of MA neurotoxicity. Neurotoxicity was assessed in terms of
the well-known ability of MA to reduce brain tissue concentrations of dopamine
and serotonin. This study differed from the present studies in terms of
time-course about outcome measures, focusing on sub-lethal binge-dosing and
neurotoxicity. However, with regards the connection between hepatotoxicity,
plasma ammonia and elevated brain glutamate levels, it is interesting to note,
in addition to the present observations of hepatotoxicity at lethal drug doses,
that lethality was almost universally associated with seizure in the present
experiments, a likely indicator of elevated glutamate function, although this
hypothesis will certainly need to be confirmed directly.</p><p>Regarding the potential for cathinones to induce neurotoxicity, many
studies have failed to show that many cathinones induce neurotoxicity using
standard binge-regimen approaches (Angoa-Pérez et al., 2012; Anneken et al., 2015; Lopez-Arnau et
al., 2015; Martinez-Clemente et al.,
2014; Thomas et al., 2004).
These studies examined a wide range of markers for neurotoxic effects, including
tissue monoamine levels, markers for monoamine terminals, and markers for
reactive gliosis. Given the important role of glutamate in neurotoxicity, this
might indicate that these cathinones have less ability to elevate glutamate
levels than MA. However, it should be noted that despite a lack of neurotoxicity
(Anneken et al., 2015), methylone does
induce lethality at doses slightly lower than MA or MDMA (Piao et al., 2015). This might suggest that there may
be a divergence between the factors influencing neurotoxicity and acute
lethality. Alternatively, some other factor that differed between those studies,
such as ambient temperature or species/strain, may influence the observation of
lethality or neurotoxicity. These differences might occur in both directions.
MCAT, which showed no signs of lethality in the present studies, did produce
some neurotoxicity (Anneken et al., 2017b;
Sparago et al., 1996), although this
was much reduced in comparison to MA.</p><p>It has been suggested that cathinone use is associated with a greater
incidence of emergency room admissions than other abused drugs (American Association of Poison Control Centers, 2011,
Wood et al., 2015, 2013). Among the symptoms noted in reports of
cathinone overdoses are hepatic impairments or hepatic failure (Fröhlich et al., 2011; Riyaz et al., 2014; Smith et al., 2013; Somi et al.,
2014). Elevated levels of aspartate and alanine aminotransferase are
reported in many of these cases (Borek and
Holstege, 2012; Kramer et al.,
2016; Murray et al., 2012;
Thirakul et al., 2017), which are
common indicators of liver damage. Post mortem histological
analysis of methylone overdose patients has also found evidence of hepatic
portal and lobular inflammation (Benzer et al.,
2013; Carbone et al., 2013).
Even when many of the acute physiological and neurological symptoms associated
with cathinone overdose have abated, within about 24 hours of the initial
overdose, hepatic impairments can persist, sometimes resulting in hepatic
failure in the days following initial hospitalization. One case report of a
cathinone overdose involved mildly elevated aspartate and alanine transaminase
levels initially, but these levels increased dramatically over the next two
days, even as other symptoms abated (Kramer et
al., 2016). It is not surprising that cathinones would be associated
with hepatic damage since MDMA and MA also produce hepatic impairments (for a
review, see Carvalho et al., 2012). Very
few studies have examined cathinone toxicity or lethality in
vivo. Most of what is known about the hepatotoxic effects of
cathinones comes from a few in vitro studies using either
primary hepatocytes or hepatic cell lines. These studies have shown that
methylone (Nakagawa et al., 2009; Valente et al., 2017), MDPV, pentedrone,
and 4-MEC (Valente et al., 2017) are
hepatotoxic. Methylone was less toxic than MDMA, while MDPV, pentedrone and
4-MEC were more toxic.</p><p>Although much remains to be done to study the cellular toxicity of
cathinones, hepatic or otherwise, it certainly appears that cathinones, like MA
and MDMA, can damage the liver. It remains to be directly observed whether this
is associated with elevated plasma ammonia and brain glutamate function, and
whether these effects contribute to lethality. The present study clearly
demonstrates that several cathinones have less potential for inducing lethal
effects than MA or MDMA. Some evidence here suggests that this may be due, in
part, to their propensity to induce liver toxicity, although much remains to be
done in this regard, particularly with respect to structural features of
cathinones that may contribute to greater liver toxicity and other effects.
Reduced lethality was observed for all of the cathinones studied here. Indeed,
lethality was so much reduced for CAT, MCAT and MDPV that the LD50 values could
not even be calculated. This does not mean that differences in lethality will
not be observed for other cathinones, and it will be essential to study a
sufficient number of these analogues to identify structure-activity
relationships. Moreover, the reduced lethality observed here may not carry over
to other situations, relevant to the ways that human consume these drugs,
including under conditions of higher ambient temperatures, in the presence of
other factors contributing to liver toxicity, and under binge or chronic
administration conditions.</p>