- Email: [email protected]
Therapeutic Effects of Ether Lipids
CHAPTER «f
f-
Therapeutic Effects of Ether Lipids Bo Hallgren
I. Introduction II. Alkylglycerols and Their Esters A. Protection against Radiation-Induced Leukopenia and Thrombocytopenia B. Protection against Local Irradiation Injuries C. Shift in Tumor Stages When Alkylglycerols Are Given Prophylactically before Radiation D. Effect of Alkylglycerols on Survival Rates III. Methoxy-Substituted Alkylglycerols A. Antibacterial and Antifungal Effects B. Experimental Antitumor Activity C. Influence on Immune Reactivity IV. Acetal Analogs of Phosphatidic Acids V. Alkyl Ethers of Halogenopropanediols References
261 262 262 263 265 267 268 270 270 271 273 274 274
I. Introduction
Numerous biological effects have been attributed to alkylglycerols. Supposedly, these compounds exhibit bacteriostatic properties, hemopoietic effects, and neuromuscular activities; they have been reported to protect against radiation damage, stimulate the formation of red blood cells, inhibit neoplastic growth, accelerate wound healing, and be of use in the treatment of bracken poisoning (Boeryd et aL, 1971; Man« gold, 1972). Not all these alleged beneficial effects have been verified in different laboratories; in fact, the validity of some reports has been questioned (Mangold, 1972). 261 Ether Lipids: Biochemical and Biomédical Aspects
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-468780-6
262
Bo
HALLGREN
This chapter places emphasis on the description of findings that have been published during the past decade. IL Alkylglycerols and Their Esters
In most biological studies, shark liver oils or l-0-alkyl-2,3-diacylglycerols isolated therefrom or 1-0-alkylglycerols prepared from their nonsaponifiable fraction have been used. Synthetic preparations were tested after improved methods of synthesis became available (see Chapters 3 and 20). A. Protection against Radiation-Induced Leukopenia and Thrombocytopenia In a study reported by Brohult (1963) patients suffering from cancer of the uterine cervix were given radiotherapy: One group of 122 patients received a daily dose (0.3-2.6 g) of alkyldiacylglycerols from the Greenland shark {Somniosus microcephalus), whereas another group of 132 patients served as controls. The average initial leukocyte count for both the treated and the nontreated patients was 5900/mm3. External treatment with x rays commenced 3 weeks after intracavitary application of radium. At that time, the patients who had received alkyldiacylglycerols had an average leukocyte count of 4600/mm3, whereas the patients in the control group had a mean count of 4000/mm3. At the end of the treatment, the mean value for the white cell count was 3900/mm3 for patients in the group that had received alkyldiacylglycerols as compared to 3200/mm3 for patients in the control group; the difference was highly significant (p < 0.001). A significant difference between the treated group and the controls was also observed with respect to the number of thrombocytes: 43% of the treated patients had values below 150,000 mm3, compared to 60% of the controls. An additional study on the effect of alkylglycerols on radiation damage to the bone marrow was performed by Nyström (1973). The subjects were 150 consecutive patients with cancer of the uterine cervix referred to the hospital for radiation therapy. This study was performed as a double-blind trial, and alkylglycerols derived from Greenland shark liver oil were used instead of the esters. The patients were selected at random; half of them were given a placebo and the other half 600 mg of alkylglycerols per day for 10 days before the start of the radiation treatment and throughout. After 10 days' prophylactic treatment, the average number of leuko-
15.
THERAPEUTIC E F F E C T S OF E T H E R L I P I D S
263
cytes was 5700/mm3 in the treated group and 5200/mm3 in the control group. The difference was not significant. The mean of the lowest values during and after radiation treatment was 3040/mm3 for the treated group and 3050/mm3 for the controls. After dividing the patients into classes (<2000/mm3, 2000-3000/mm3, and >3000/mm3) a significant difference between treated patients and controls became obvious. It was found that only 10% of the treated patients had values below 2000/mm3, compared to 23% of the controls. The two independent studies referred to (one double-blind) indicate that alkylglycerols from Greenland shark liver oil as well as the alkylglycerols derived therefrom have a clinically important effect on leukopenia caused by irradiation. B. Protection against Local Irradiation Injuries Studies of patients treated in 1963-1966 by Brohult et al. (1977) were aimed at evaluating further the protective effects of ether lipids against irradiation injuries in patients with cancer of the cervix of the uterus. Alkylglycerols derived from Greenland shark liver oil were administered to one group of patients at a level of 600 mg/day during the radiation treatment and 300 mg/day for 1-3 months after treatment. Another group of patients was treated the same way during and after radiation but was also treated prophylactically 8 days before the radiation with 600 mg of alkylglycerols per day. The patients receiving alkylglycerols during and after radiation treatment are referred to as the nonprophylactic group, and the patients also given alkylglycerols before the radiation as the prophylactic group. The results obtained are summarized in Table 1. The system proposed by Kottmeier and Gray (1961) was used for evaluation of the radiation injuries that had occurred in bladder, rectum, uterus, and intestine. Radiation injuries of grade I, with minimal objective changes in the mucosa, were excluded. Patients with radiation injuries of grades II-IV were treated as a single group, "patients with radiation injuries." Grade II was characterized by moderate to severe changes, such as necroses, ulcérations, moderate stenoses, and/or reactions with lengthy bleeding. Radiation complications of grade III included injuries to the bladder, radiation fistulas from the ureters, and rectal and intestinal stenoses of such severity that colostomy or resection was needed. Grade IV was characterized by rectal and intestinal fistulas. Complications included patients with clinical features of radiation injury in whom the symptoms were found to be caused by tumor growth or a combination of tumor growth and radiation injury. These
264
Bo HALLGREN
TABLE I Injuries Following Radiation Therapy—All Stages Included0*
Group 1963-1966 P IP IIP 1970-1972 P IP
Number I of patients No.
R
C
M
Nc
Nj
%
No.
%
No.
%
No.
%
No.
%
No.
%
458 381 657
83 18.1 93 24.4 244 37.1
59 34 150
12.9 8.9 22.8
24 59 94
5.2 15.5 14.3
78 69 189
17.0 18.1 28.8
22 41 76
4.8 10.8 11.6
5 23 43
1.1 6.0 6.5
137 142
36 26.3 74 52.1
28 40
20.4 28.2
8 34
5.8 23.9
32 52
23.4 36.6
8 24
5.8 16.9
4 16
2.9 11.3
α From Brohult^i al. (1977). * I, Total injuries; R, injuries due to radiation treatment; C, complex injuries due to tumour growth or to a combination of tumor growth and radiation treatment; N t , number of patients with injuries; N c , number of patients with complex injuries; M, more than one injury per patient—multiple injuries. c Administration of alkylglycerols prophylactically and during radiation treatment. d Administration of alkylglycerols only during radiation treatment. e Radiation treatment only.
complications were termed complex injuries and represented a very serious situation. All patients with complex injuries were dead after 5 years. The incidence of radiation injuries varies with the spread of the cancer and the radiation technique. The incidence is higher in the more advanced tumor stages than in the lower ones. It is also higher after 60 Co three-beam treatment of combined high-voltage and x-ray treatment than when conventional x rays or only radium is used. When comparing the groups statistically, standardized proportions have been used in order to cancel out differences with regard to stage distribution and radiation technique. The total incidence of injuries was lower in the groups that had received alkylglycerols (18.1% in the prophylactic group and 24.4% in the nonprophylactic group) than in the controls (37.1%) (Table I). The prophylactic group had a considerably lower incidence of complex injuries and multiple injuries than both the controls and the nonprophylactic groups. The differences were highly significant (p < 0.001). Analysis of the patients divided into groups according to tumor stage or radiation technique showed that the incidence of complex injuries was lower in all subgroups of prophylactically treated patients than in the corresponding control groups. A double-blind study in 1970-1972 showed a pronounced protective effect of prophylactic treatment
15.
THERAPEUTIC E F F E C T S OF E T H E R LIPIDS
265
against injuries after radiation therapy (Table I). The use of increased doses of radium in intracavitary irradiation was followed by a high incidence of radiation injuries, which was considerably reduced by treatment with alkylglycerols, especially when these compounds were administered prophylactically (Brohult et al., 1977). In the author's opinion it is unquestionable that prophylactic treatment with alkylglycerols has a protective effect against radiation injuries irrespective of the radiation technique used and the stage of the tumor. This is valid for all radiation injuries, including those where tumor growth is involved. C. Shift in Tumor Stages When Alkylglycerols Are Given Prophylactically before Radiation Cases of cancer of the uterine cervix are classified into six different clinical stages according to the severity of the disease, IA being the mildest form. Patients treated prophylactically with alkylglycerols showed a statistically significant shift toward lower stages compared to the controls (p < 0.01) (Brohult, 1963). If the stage distribution of a group prophylactically treated in 1965 (period I in Table II) is compared to that of a control group consisting of patients treated in the periods 1960-1963 and 1966-1969, the prophylactic group shows a shift to lower stages, the difference being highly significant (p < 0.001). When the prophylactic group is compared with the 1964 nonprophylactic group, the difference is also significant (p < 0.01) (Brohult et al., 1978). In another study, patients treated with alkylglycerols also had a more favorable stage distribution than the controls, but the difference was not statistically significant (Nyström, 1973). In the double-blind study performed in 1970-1972, there was the same tendency toward lower stages in patients treated with alkylglycerols in comparison to the controls (Brohult et al., 1978). In Table II the stage distribution for the years from 1958 through 1975 has been summarized. The ratios between the lower stages, IA + IB + IIA, and the more advanced stages, IIB + III + IV, have been calculated. Calculations have also been performed with stage IA cases, those not clearly defined during the 1950s, excluded. As may be seen from Table II, the ratios were higher when alkylglycerols were given than in the years before and after. A study carried out in the years 1973-1975, not mentioned before, also showed a very high ratio for patients treated with alkylglycerols (Table II).
•ri c«
^
^ ^ Η ^ Η ^ ^ Η ^ Η ^ Η ^
^H
^
^
CN
1s
^
Ä *
^Η
^ »Λ) »O
N rt
»Λ VO r f Tf
T-H r>-
rr> r -
N O > n O Tt Tt ^ m
Tf
3s
*-* i o
■a-S iä *° *>> ^ o
o
*-■ *r!
oooNON^^ONmr^mvOi-HOs^oor^fN
3
· g
O
I l
SÎ.
w
CÖ
cd ö Tfc400^Hf«">r^t^SO
-* x: — wΟ (u -^ αχί cd ο Ο
S
a
X> .Si 3 ex,
CA
c
w
•S .2 »
S
m so NO ^ vo Tt rq ^ r i ^
^ ^ Η ^ ^ Η ^ Η ^ ^ , ^ Η
4>
χ : cd ^ o,
'S m ^f <+-. t^ t i 0 os w
T j - T T ^ ^ h ^ ^ f ^ ^ ^ O O ^ X O ^
*C O cd cd ^ * ο S *3
vO vo io ON m
c .3 ω
a
·- Ê ° 1 c2 ft - S "o "-> & h
vot^ioasraoNrsjOvo,^t
*a l
w
115 3 Si" o a δ
M ( S ( S M N ( S ( S n H ( S H H
JD
^ r- .> 33 2 5P c§
7-S s
U ^
■
^
η
η
^
η
^
Ν
Ν
Η
Η
Ν
Ν
x> B ·<π S o g
o
^
s-f ^
0 \ Ö\ ON
7 7 7 7 5 + <« 3 o« t^ oo os O O m m NO NO vp r- r- r^ r* II ON ON Q\ ON G \ 0 \ 5a + ^ S > ON
PQ
•g
tas
S : 04
^^ . . ON
15.
THERAPEUTIC E F F E C T S OF E T H E R L I P I D S
267
The shift of the stage distribution to lower stages indicates that prophylactic treatment with alkylglycerols might have a regressive effect on tumors. However, this could also be an effect on the tissues around the tumor, leading to a change in the clinician's impression when palpating the tumor. In this context, a report by Kato et al. (1971) on the cytostatic effects of alkylglycerols is of interest. D. Effect of Alkylglycerols on Survival Rates The aim of Brohult's early investigations was to study the effect of alkyldiacylglycerols on granulocytopenia after radiation. When the results were analyzed later, it was found that patients given alkyldiacylglycerols had a higher survival rate than the controls. When comparing the groups, standardized proportions of stages were used. The radiation treatment was the same for the two groups. Patients treated with ether lipids showed a somewhat more favorable stage distribution. After 3 years, the group treated with alkyldiacylglycerols showed a higher survival rate than the controls, but this difference was not significant. After 5 years the survival rate was still higher, and the difference was significant (p < 0.05). It is interesting to note that the survival rate was higher for all tumor stages in patients treated with ether lipids than in the corresponding control groups. In the 1960s, several new methods of high-voltage treatment were introduced in cancer therapy. When groups of patients are compared statistically, it is therefore necessary to use standardized proportions with regard to radiation technique. Even if it is assumed that the more favorable stage distribution of a prophylactically treated group in 1965 (Table II) is due to chance (standardized proportions with regard to stage distribution being used in the statistical comparison), the survival rate after 3 years is higher for the prophylactic group than for the controls (p < 0.05). There is still a difference after 5 years, but it is not statistically significant. When the groups are compared statistically using standarized proportions with regard to radiation technique instead of stage, the difference in survival rate in favor of the prophylactic group is even more evident. More patients in the 1964 nonprophylactic group had received highvoltage treatment. Brohult et al. (1981) have therefore compared the survival rates of patients treated with high-voltage irradiation in the periods 1960-1963 and 1966-1969. The survival rate was lower in the first period than in the second, the 1964 nonprophy lactic group being
268
Bo
HALLGREN
an intermediate group. A positive effect on the survival rate of patients given alkylglycerols only during and after radiation therapy could thus not be demonstrated in this study. It is important to give alkylglycerols prophylactically before radiation treatment in order to improve the survival rate. The survival rates of the patients in the study performed by Nyström (1973) have also been analyzed. The groups were small, and no significant differences in survival rates between patients treated with alkylglycerols and controls were found. The tumor recurrence rate and incidence of metastasis were in favor of the group treated with alkylglycerols, but the differences were not significant. In the double-blind study performed in 1970-1972 on 120 treated patients and 125 controls, the survival rate after 3 years was 78% in the group treated with alkylglycerols and 70% in the controls. The difference is not statistically significant, but such a high survival rate was achieved only once in the years 1950-1969, and that was in the 1965 prophylactically treated group. In summary, clinical studies on the effects of alkylglycerols from Greenland shark liver oil given orally to patients undergoing radiation treatment for cancer of the uterine cervix have produced the following findings: First, prophylactic treatment with alkylglycerols or their esters has a protective effect against radiation injuries. This effect is reflected both in the white blood cell count and in the local radiation reactions within the irradiated area. Second, tumor growth within the irradiated area is also influenced by prophylactic treatment with alkylglycerols—this is the only explanation that can be given for the observation that the incidence of complex injuries associated with tumor growth, hence the mortality, is lower in patients treated prophylactically with alkylglycerols than in controls. Moreover, all clinical studies on prophylactic treatment with alkylglycerols show a shift to a more favorable stage distribution in treated patients than in the controls. Finally, an increased survival rate is observed after prophylactic treatment with alkylglycerols. This may partly be explained by the more favorable stage distribution, but it is partly independent of this. UK Methoxy-Substituted Alkylglycerols
During work on the nonsaponifiable fraction of Greenland shark liver oil, 3-4% alkylglycerols more polar than the common ones were dis-
15.
THERAPEUTIC EFFECTS OF ETHER LIPIDS
269
OCH* I H2C-0-CH2-CH-CH2-CH=:CH-(CH2)n-CH3 HO-CH H2C —OH Fig. 1. Methoxy-substituted alkylglycerols. n = 10, 12.
covered; they were shown to be methoxy-substituted alkylglycerols (Hallgren and Ställberg, 1967) (Fig. 1). The quantitative distribution within the mixture of methoxy-substituted alkylglycerols in Greenland shark liver oil was found to be about 60% l-0-(2'-methoxyhexadecenyl)glycerol, 15% l-0-(2'-methoxyhexadecyl)glycerol, and 20% 1-0(2'-methoxyoctadecenyl)glycerol. A polyunsaturated methoxy-substituted alkylglycerol was also isolated from Greenland shark liver oil (Hallgren et al., 1971). Various biological materials, principally from humans and from marine animals, have been analyzed for their content of methoxy-substituted alkylglycerols. The following mammalian materials were investigated: human milk from three different periods of lactation, cow's milk, sheep's milk, human red bone marrow, red blood cells, blood plasma, and a uterine carcinoma (Hallgren et al., 1974a). The samples of marine origin included fillets of Atlantic and Baltic herring as well as mackerel, the edible parts of marine and freshwater crayfish, shrimp and sea mussels, and cod liver oil (Hallgren et al., 1974b). 2-Methoxy-substituted alkyl moieties were found together with unsubstituted alkyl moieties in the neutral lipids as well as in the phospholipids in all the tissues investigated. In the various samples from humans, cows, and sheep only trace quantities of methoxyalkylglycerols were detected. As a rule, the phospholipids contained larger proportions than the neutral lipids. In the samples from marine animals the percentages of methoxy-substituted alkylglycerols were higher than in mammals, especially in the respective phospholipids. The highest content was found in sea mussels and marine crayfish, where 0.47 and 0.35% of the phospholipids contained methoxy-substituted alkyl moieties, respectively. The principal components of the methoxy-substituted alkylglycerols from the mammals studied are the same as those found in the ether lipids of Greenland shark liver, namely, 2-methoxyhexadecyl-, 2-methoxyhexadecenyl-, and 2-methoxyoctadecenylglycerols. Compounds having C16 chains account for 50-90% of the total methoxyalkylglycerols; particularly large proportions are found in the milk of humans,
270
Bo
HALLGREN
and sheep. The methoxy-substituted docosahexaenylglycerol first demonstrated in Greenland shark liver oil is also present in some of the other materials investigated, namely, human red blood cells, shrimp, mackerel, and cod liver oil (Hallgren et al., 1974a). The synthesis of methoxy-substituted alkylglycerols and methods for the characterization of these compounds have been described (Ställberg, 1975) (see Chapter 3). COWS,
A. Antibacterial and Antifungal Effects The mixture of methoxy-substituted alkylglycerols isolated from Greenland shark liver oil, as well as synthetic 2-methoxyhexadecylglycerol (a mixture of stereoisomers), showed an antibiotic effect in vitro against several types of bacteria, especially Corynebacterium Hofmannii, Diplococcus pneumoniae, Staphylococcus pyogenes A, and Staphylococcus pyogenes H. Oxford, Streptococcus pyogenes, and Streptococcus viridans (Boeryd et al., 1971). The antibiotic activity was about equal to that of nitrofurantoin. Fungistatic and fungicidal activity has also been demonstrated in vitro for the methoxy-substituted alkylglycerols (Hallgren et al., 1978). Monosporically selected strains of the dermatophytes Epidermophyton floccosum, Microsporum canis, Trichophyton mentagrophytes, and Trichophyton rubrum were used in these studies. Fungistatic tests showed the mixture of methoxy-substituted alkylglycerols from Greenland shark liver oil to be a more potent inhibitor of dermatophyte growth than the synthetic compounds, 2-methoxyhexadecylglycerol and 2-methoxyhexadecenylglycerol, which produced inhibition in the range 5-15% at a concentration of 100 /xg/ml. In fungicidal tests, the methoxy-substituted alkylglycerols in high concentrations (1000 ^g/ml) inhibited the growth of T. rubrum and E. floccosum. B. Experimental Antitumor Activity The methoxy-substituted alkylglycerols were found to inhibit tumor growth in cultured cells. Two cell lines were used, a methylcholanthrene-induced murine sarcoma (MCG1-SS) and a juvenile osteogenic sarcoma (2T). Marked growth inhibition was noted for the mixture of 2-methoxyalkylglycerols from Greenland shark liver oil, different single components derived from this oil (2-methoxyhexadecylglycerol, 2-methoxyhexadecenylglycerol , and 2-methoxy octadeceny lgly cerol), and various synthetic compounds including, for example, 2-ethoxyhexa-
15.
THERAPEUTIC E F F E C T S OF E T H E R L I P I D S
271
decylglycerol, 2-methoxyhexadecenylglycerol, and 3-methoxyhexadecylglycerol (Hallgren et al., 1978). In several tumor-host systems, including solid tumors, leukemias, and lymphomas, the methoxy-substituted alkylglycerols were incorporated into the feed in different concentrations (0.1-2%, w/w). Growth inhibition was noted for melanoma B16, for a methylcholanthrene-induced sarcoma (MCG101) and Lewis lung tumor (LLT) in C57BL/6J mice, for lymphoma LAA in A/Sn mice with synthetic 2-methoxyhexadecylglycerol, and for a spontaneous mammary carcinoma in C3H mice with methoxy-substituted alkylglycerols from Greenland shark liver oil. The survival time of DBA/2J mice transplanted with lymphatic leukemia PI534 was increased by synthetic 2-methoxyhexadecylglycerol (Hallgren et al., 1978; Boeryd and Hallgren, 1980a). Métastases induced by the injection of MCGl-SS cells into a tail vein or into the portal vein were inhibited in the liver by methoxy-substituted alkylglycerols from Greenland shark liver oil (Boeryd et al., 1971). Spontaneous metastasis formation from a methylcholanthrene-induced sarcoma (MCGl-SS) in lymph nodes and lungs of CBA mice was inhibited by methoxy-substituted alkylglycerols derived from Greenland shark liver oil as well as by synthetic 2-methoxyhexadecylglycerol (Boeryd et al., 1971). Spontaneous metastasis formation from melanoma B16 was inhibited by synthetic 2-methoxyhexadecylglycerol (Boeryd and Hallgren, 1980a). C. Influence on Immune Reactivity Methoxy-substituted alkylglycerols in the feed stimulated the immune reactivity in mice against sheep red blood cells (SRBCs) (Boeryd et al., 1978) as determined by the number of plaque-forming cells (PFC) (Cunningham and Szenberg, 1968). Further, they stimulated cellular immunoreactivity as demonstrated by the increased ability of parenteral spleen cells to induce a graft-versus-host reaction (GVHR) in DBA/1 J x C57BL/6J Ft (H-2bQ) hybrid mice (Boeryd et al., 1978) tested according to Michie (1967). The effect on humoral immunoreactivity was studied mainly in CBA mice but to some extent also in C57BL/6J and DBA/2J mice. The mixture of methoxy-substituted alkylglycerols from Greenland shark liver oil and synthetic 2-methoxyhexadecylglycerol, given in concentrations of 0.1, 0.25, and 0.5% of the feed for 4 or 14 days before SRBC injection, significantly increased the number of PFCs. To achieve immunostimulation by the mixture of alkylglycerols (unsubstituted and methoxy-substituted) from Greenland
272
Bo
HALLGREN
shark liver oil, it was necessary to increase the content of these compounds in the diet to 3%. This concentration corresponds to about 0,1% methoxy-substituted alkylglycerols, as 3-4% of the alkylglycerols in Greenland shark liver oil are methoxy-substituted. It is probable that the immunostimulation caused by the complete mixture was due to its content of methoxy-substituted alkylglycerols. The cellular immunoreactivity tested by the GVHR was stimulated by synthetic 2-methoxyhexadecylglycerol given in a concentration of 0.1% of the feed for 44 days to the spleen cell donors. In this connection, a study by Brohult et al. (1972) is of great interest. These authors observed that patients vaccinated against typhoidparatyphoid 1 day before and 1 day after implantation of radium for uterine cancer and given alkylglycerols and methoxy-substituted alkylglycerols from Greenland shark liver oil produced antibodies to a larger extent than a group receiving radiation treatment only. As methoxy-substituted alkylglycerols occur in the milk of different species, immunoreactivity was tested in adult mice whose mothers had been deprived of these substances during lactation. Exclusion of animal fat from the diets of lactating mothers profoundly decreased the immunoreactivity of the adult offspring. This reactivity was partly restored in male offspring when synthetic 2-methoxyalkylglycerols were added to the feed of the mothers. In mice deprived of animal fat at weaning and for the following 21 days immunoreactivity to SRBCs, tested about 3 month after stopping the diet, was not influenced. However, resistance to a transplanted tumor in similarly fed mice was increased, and this resistance was brought approximately to the control level by methoxy-substituted alkylglycerols (Boeryd and Hallgren, 1980b). In summary, the mechanisms behind the clinical and experimental effects of methoxy-substituted and unsubstituted alkylglycerols are still unknown. Methoxy-substituted alkylglycerols stimulate immunoreactivity, and it is probable that these substances are the immunostimulating components of the nonsaponifiable fraction from Greenland shark liver oil. It has not been possible, however, to evaluate the importance of immunostimulation for the antitumor activity demonstrated on experimental tumors, either for tumor growth or for metastasis formation. Growth retardation of tumor cells has been demonstrated in vitro. It therefore seems probable that the substituted alkylglycerols to some extent exert direct effects on the tumor cells. They might be incorporated into membrane lipids, changing their structure and thereby influencing the growth and spread of the tumor cells. The unique combination of a stimulatory effect on host defense
15.
THERAPEUTIC E F F E C T S OF E T H E R L I P I D S
273
mechanisms and an antitumor effect in the same molecule has also been demonstrated for a synthetic ether phospholipid with a methoxy group in sn-2 position of glycerol (Munder et al., 1979). In patients treated with radiation for cancer of the uterine cervix, the protective effect of the mixture of methoxy-substituted and unsubstituted alkylglycerols from Greenland shark liver oil against leukopenia and thrombocytopenia might be due to the latter compounds, as a stimulatory effect on bone marrow has been demonstrated for both chimyl alcohol and batyl alcohol (natural l-O-hexadecyl-s/i -glycerol and 1-Öoctadecyl-sfl-glycerol, respectively). One can only speculate about the mechanisms behind the other effects observed in patients treated with radiation. The protective effect against local radiation injuries with tumor growth involved in the clinical condition, that is, complex injuries, according to Brohult et al. (1977), might be explained by a membrane effect of the alkylglycerols, leading to an increase in the radiation sensitivity of the tumor in relation to the normal tissue. The effect on the stage distribution might be explained by some direct effect on the tumor but also by a decreased reaction to, for example, necrotic tumor cells in the tissues surrounding the tumor. The relative importance for the clinical effects of methoxy-substituted alkylglycerols and the more common alkylglycerols of Greenland shark liver oil has not yet been determined. IV. Acetal Analogs of Phosphatidic Acids Long-chain cyclic acetals of glycerophosphoric acid (I) Darmstoff (Vogt, 1963), are potent stimulators of gastrointestinal smooth muscle (Wiley et al.y 1970; Vogt, 1963) and inhibitors of renin (Bunag and Walaszek, 1973). It has been suspected that the activity of the natural product may in fact be due to the presence of prostaglandins (Andersen, 1974), but studies with synthetic preparations have demonstrated similar activities (Wiley et al., 1970). Long-chain cyclic acetals of glycerol that could serve as intermediates in the synthesis of the corresponding phosphorylated compounds have been prepared (Baumann, 1971; Wedmid and Baumann, 1977). The biologically active acetal analogs of phosphatidic acid (I) have also been synthesized (Wiley et aL, 1970). As these compounds are unstable in neutral aqueous solution, Milbert and Wiley (1978) have prepared the tetrahydrofuran and cyclopentyl derivatives of Darms toff II and III, respectively. A comparison of compounds I, II, and III has shown that they ex-
274 H
Bo
HALLGREN
0-
FT V I
^
ΟΡΟοΗΝα
R
λ
Λ./ΟΡΟοΗΝα
0 ^ ^ II
1
λ
.ΟΡΟοΗΝα
~ / \ / \ / III
Fig. 2. Darmstoff (I) and analogous compounds (II, III). R = C 15 H 31 .
hibit similar effects on the blood pressure and heart rate of rats (Milbe rt and Wiley, 1978). In contrast to aqueous solutions of compound I, which loses all biological activity within 1 h, solutions of compounds II and of III remain active for days (Milbert and Wiley, 1978). The compounds described may find therapeutic use as hypotensive agents. V. Alkyl Ethers of Halogenopropanediols
Langen et al. (1979) have recently demonstrated that long-chain alkyl ethers of halogenopropanediols exhibit a strong inhibitory effect on the proliferation of Ehrlich ascites carcinoma cells in suspension cultures. The same compounds have been found to have a cytostatic activity in ascites cells in vivo. Most of the substances investigated had no effect on the survival time or cell number after subcutaneous administration, and there was no prolongation of the survival time of leukemia LI210- or L184-bearing mice. Nevertheless, some halo analogs of alkyl lysophospholipids appear to be promising as cytostatic agents (Brachwitz et al., 1982). References Andersen, N. H. (1974). Arch. Intern. Med. 133, 30. Baumann, W. J. (1971). J. Org. Chem. 36, 2743. Boeryd, B., and Hallgren, B. (1980a). Acta PathoL Microbol. Scand.y Sect. A 88A, 11. Boeryd, B., and Hallgren, B. (1980b). Int. J. Cancer 26, 241. Boeryd, B., Hallgren, B., and Ställberg, G. (1971). Br. J. Exp. PathoL 52, 20. Boeryd, B., Nilsson, T., Lindholm, L., Lange, S., Hallgren, B., and Ställberg, G. (1978). Eur. J. Immunol. 8, 678. Brachwitz, H., Langen, P., Hintsche, R., and Schildt, J. (1982). Chem. Phys. Lipids 31, 33. Brohult, A. (1963). Acta Radiol., Suppl. No. 233. Brohult, A., Brohult, J., and Brohult, S. (1972). Experientia 28, 954. Brohult, A., Brohult, J., and Brohult, S. (1977). Acta Obstet. Gynecol. Scand. 56, 441. Brohult, A., Brohult, J., and Brohult, S. (1978). Acta Obstet. Gynecol. Scand. 57, 79. Brohult, A., Brohult, J., and Brohult, S. (1981). Unpublished observations. Bunag, R. D., and Walaszek, E. J. (1973). Eur. J. Pharmacol. 23, 191. Cunningham, A. J., and Szenberg, A. (1968). Immunology 14, 599.
15.
THERAPEUTIC EFFECTS OF ETHER LIPIDS
275
Hallgren, B., and Ställberg, G. (1967). Acta Chem. Scand. 21, 1519. Hallgren, B., Ställberg, G., and Thorin, H. (1971). Acta Chem. Scand. 25, 3781. Hallgren, B., Niklasson, A., Ställberg, G., and Thorin, H. (1974a). Acta Chem. Scand., Ser. B 28B, 1029. Hallgren, B., Niklasson, A., Ställberg, G., and Thorin, B. (1974b). Acta Chem. Scand., Ser. B 28B, 1035. Hallgren, B., Ställberg, G., and Boeryd, B. (1978). Prog. Chem. Fats Other Lipids 16, 45. Kato, A., Ando, K., Tamura, G., and Arima, K. (1971). Cancer Res. 31, 501. Kottmeier, H. L., and Gray, M. J. (1961). Am. J. Obstet. Gynecol. 82, 74. Langen, P., Brachwitz, H., and Schildt, J. (1979). Acta Biol. Med. Ger. 38, 965. Mangold, H. K. (1972). In "Ether Lipids: Chemistry and Biology" (F. L. Snyder, ed.), pp. 157-176. Academic Press, New York. Michie, D. (1967). In " H a n d b o o k s Experimental Immunology" (D. M. Weir, ed.), pp. 969-987. Blackwell, Oxford. Milbert, A. N., and Wiley, R. O. (1978). J. Med. Chem. 21, 245. Münder, P. G., Modolell, M., Andreesen, R., Weltzien, H. V., and Westphal, O. (1979). Springer Sem. Immuno. 2, 187. Nyström, C. (1973). Unpublished observations. Ställberg, G. (1975). Chem. Ser. 7, 31. Vogt, W. (1963). Biochem. Pharmacol. 12, 415. Wedmid, Y., and Baumann, W. J. (1977). J. Org. Chem. 42, 3624. Wiley, R. O., Sumner, D. D., and Walaszek, E. J. (1970). Lipids 5, 803.
f-
Therapeutic Effects of Ether Lipids Bo Hallgren
I. Introduction II. Alkylglycerols and Their Esters A. Protection against Radiation-Induced Leukopenia and Thrombocytopenia B. Protection against Local Irradiation Injuries C. Shift in Tumor Stages When Alkylglycerols Are Given Prophylactically before Radiation D. Effect of Alkylglycerols on Survival Rates III. Methoxy-Substituted Alkylglycerols A. Antibacterial and Antifungal Effects B. Experimental Antitumor Activity C. Influence on Immune Reactivity IV. Acetal Analogs of Phosphatidic Acids V. Alkyl Ethers of Halogenopropanediols References
261 262 262 263 265 267 268 270 270 271 273 274 274
I. Introduction
Numerous biological effects have been attributed to alkylglycerols. Supposedly, these compounds exhibit bacteriostatic properties, hemopoietic effects, and neuromuscular activities; they have been reported to protect against radiation damage, stimulate the formation of red blood cells, inhibit neoplastic growth, accelerate wound healing, and be of use in the treatment of bracken poisoning (Boeryd et aL, 1971; Man« gold, 1972). Not all these alleged beneficial effects have been verified in different laboratories; in fact, the validity of some reports has been questioned (Mangold, 1972). 261 Ether Lipids: Biochemical and Biomédical Aspects
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-468780-6
262
Bo
HALLGREN
This chapter places emphasis on the description of findings that have been published during the past decade. IL Alkylglycerols and Their Esters
In most biological studies, shark liver oils or l-0-alkyl-2,3-diacylglycerols isolated therefrom or 1-0-alkylglycerols prepared from their nonsaponifiable fraction have been used. Synthetic preparations were tested after improved methods of synthesis became available (see Chapters 3 and 20). A. Protection against Radiation-Induced Leukopenia and Thrombocytopenia In a study reported by Brohult (1963) patients suffering from cancer of the uterine cervix were given radiotherapy: One group of 122 patients received a daily dose (0.3-2.6 g) of alkyldiacylglycerols from the Greenland shark {Somniosus microcephalus), whereas another group of 132 patients served as controls. The average initial leukocyte count for both the treated and the nontreated patients was 5900/mm3. External treatment with x rays commenced 3 weeks after intracavitary application of radium. At that time, the patients who had received alkyldiacylglycerols had an average leukocyte count of 4600/mm3, whereas the patients in the control group had a mean count of 4000/mm3. At the end of the treatment, the mean value for the white cell count was 3900/mm3 for patients in the group that had received alkyldiacylglycerols as compared to 3200/mm3 for patients in the control group; the difference was highly significant (p < 0.001). A significant difference between the treated group and the controls was also observed with respect to the number of thrombocytes: 43% of the treated patients had values below 150,000 mm3, compared to 60% of the controls. An additional study on the effect of alkylglycerols on radiation damage to the bone marrow was performed by Nyström (1973). The subjects were 150 consecutive patients with cancer of the uterine cervix referred to the hospital for radiation therapy. This study was performed as a double-blind trial, and alkylglycerols derived from Greenland shark liver oil were used instead of the esters. The patients were selected at random; half of them were given a placebo and the other half 600 mg of alkylglycerols per day for 10 days before the start of the radiation treatment and throughout. After 10 days' prophylactic treatment, the average number of leuko-
15.
THERAPEUTIC E F F E C T S OF E T H E R L I P I D S
263
cytes was 5700/mm3 in the treated group and 5200/mm3 in the control group. The difference was not significant. The mean of the lowest values during and after radiation treatment was 3040/mm3 for the treated group and 3050/mm3 for the controls. After dividing the patients into classes (<2000/mm3, 2000-3000/mm3, and >3000/mm3) a significant difference between treated patients and controls became obvious. It was found that only 10% of the treated patients had values below 2000/mm3, compared to 23% of the controls. The two independent studies referred to (one double-blind) indicate that alkylglycerols from Greenland shark liver oil as well as the alkylglycerols derived therefrom have a clinically important effect on leukopenia caused by irradiation. B. Protection against Local Irradiation Injuries Studies of patients treated in 1963-1966 by Brohult et al. (1977) were aimed at evaluating further the protective effects of ether lipids against irradiation injuries in patients with cancer of the cervix of the uterus. Alkylglycerols derived from Greenland shark liver oil were administered to one group of patients at a level of 600 mg/day during the radiation treatment and 300 mg/day for 1-3 months after treatment. Another group of patients was treated the same way during and after radiation but was also treated prophylactically 8 days before the radiation with 600 mg of alkylglycerols per day. The patients receiving alkylglycerols during and after radiation treatment are referred to as the nonprophylactic group, and the patients also given alkylglycerols before the radiation as the prophylactic group. The results obtained are summarized in Table 1. The system proposed by Kottmeier and Gray (1961) was used for evaluation of the radiation injuries that had occurred in bladder, rectum, uterus, and intestine. Radiation injuries of grade I, with minimal objective changes in the mucosa, were excluded. Patients with radiation injuries of grades II-IV were treated as a single group, "patients with radiation injuries." Grade II was characterized by moderate to severe changes, such as necroses, ulcérations, moderate stenoses, and/or reactions with lengthy bleeding. Radiation complications of grade III included injuries to the bladder, radiation fistulas from the ureters, and rectal and intestinal stenoses of such severity that colostomy or resection was needed. Grade IV was characterized by rectal and intestinal fistulas. Complications included patients with clinical features of radiation injury in whom the symptoms were found to be caused by tumor growth or a combination of tumor growth and radiation injury. These
264
Bo HALLGREN
TABLE I Injuries Following Radiation Therapy—All Stages Included0*
Group 1963-1966 P IP IIP 1970-1972 P IP
Number I of patients No.
R
C
M
Nc
Nj
%
No.
%
No.
%
No.
%
No.
%
No.
%
458 381 657
83 18.1 93 24.4 244 37.1
59 34 150
12.9 8.9 22.8
24 59 94
5.2 15.5 14.3
78 69 189
17.0 18.1 28.8
22 41 76
4.8 10.8 11.6
5 23 43
1.1 6.0 6.5
137 142
36 26.3 74 52.1
28 40
20.4 28.2
8 34
5.8 23.9
32 52
23.4 36.6
8 24
5.8 16.9
4 16
2.9 11.3
α From Brohult^i al. (1977). * I, Total injuries; R, injuries due to radiation treatment; C, complex injuries due to tumour growth or to a combination of tumor growth and radiation treatment; N t , number of patients with injuries; N c , number of patients with complex injuries; M, more than one injury per patient—multiple injuries. c Administration of alkylglycerols prophylactically and during radiation treatment. d Administration of alkylglycerols only during radiation treatment. e Radiation treatment only.
complications were termed complex injuries and represented a very serious situation. All patients with complex injuries were dead after 5 years. The incidence of radiation injuries varies with the spread of the cancer and the radiation technique. The incidence is higher in the more advanced tumor stages than in the lower ones. It is also higher after 60 Co three-beam treatment of combined high-voltage and x-ray treatment than when conventional x rays or only radium is used. When comparing the groups statistically, standardized proportions have been used in order to cancel out differences with regard to stage distribution and radiation technique. The total incidence of injuries was lower in the groups that had received alkylglycerols (18.1% in the prophylactic group and 24.4% in the nonprophylactic group) than in the controls (37.1%) (Table I). The prophylactic group had a considerably lower incidence of complex injuries and multiple injuries than both the controls and the nonprophylactic groups. The differences were highly significant (p < 0.001). Analysis of the patients divided into groups according to tumor stage or radiation technique showed that the incidence of complex injuries was lower in all subgroups of prophylactically treated patients than in the corresponding control groups. A double-blind study in 1970-1972 showed a pronounced protective effect of prophylactic treatment
15.
THERAPEUTIC E F F E C T S OF E T H E R LIPIDS
265
against injuries after radiation therapy (Table I). The use of increased doses of radium in intracavitary irradiation was followed by a high incidence of radiation injuries, which was considerably reduced by treatment with alkylglycerols, especially when these compounds were administered prophylactically (Brohult et al., 1977). In the author's opinion it is unquestionable that prophylactic treatment with alkylglycerols has a protective effect against radiation injuries irrespective of the radiation technique used and the stage of the tumor. This is valid for all radiation injuries, including those where tumor growth is involved. C. Shift in Tumor Stages When Alkylglycerols Are Given Prophylactically before Radiation Cases of cancer of the uterine cervix are classified into six different clinical stages according to the severity of the disease, IA being the mildest form. Patients treated prophylactically with alkylglycerols showed a statistically significant shift toward lower stages compared to the controls (p < 0.01) (Brohult, 1963). If the stage distribution of a group prophylactically treated in 1965 (period I in Table II) is compared to that of a control group consisting of patients treated in the periods 1960-1963 and 1966-1969, the prophylactic group shows a shift to lower stages, the difference being highly significant (p < 0.001). When the prophylactic group is compared with the 1964 nonprophylactic group, the difference is also significant (p < 0.01) (Brohult et al., 1978). In another study, patients treated with alkylglycerols also had a more favorable stage distribution than the controls, but the difference was not statistically significant (Nyström, 1973). In the double-blind study performed in 1970-1972, there was the same tendency toward lower stages in patients treated with alkylglycerols in comparison to the controls (Brohult et al., 1978). In Table II the stage distribution for the years from 1958 through 1975 has been summarized. The ratios between the lower stages, IA + IB + IIA, and the more advanced stages, IIB + III + IV, have been calculated. Calculations have also been performed with stage IA cases, those not clearly defined during the 1950s, excluded. As may be seen from Table II, the ratios were higher when alkylglycerols were given than in the years before and after. A study carried out in the years 1973-1975, not mentioned before, also showed a very high ratio for patients treated with alkylglycerols (Table II).
•ri c«
^
^ ^ Η ^ Η ^ ^ Η ^ Η ^ Η ^
^H
^
^
CN
1s
^
Ä *
^Η
^ »Λ) »O
N rt
»Λ VO r f Tf
T-H r>-
rr> r -
N O > n O Tt Tt ^ m
Tf
3s
*-* i o
■a-S iä *° *>> ^ o
o
*-■ *r!
oooNON^^ONmr^mvOi-HOs^oor^fN
3
· g
O
I l
SÎ.
w
CÖ
cd ö Tfc400^H
-* x: — wΟ (u -^ αχί cd ο Ο
S
a
X> .Si 3 ex,
CA
c
w
•S .2 »
S
m so NO ^ vo Tt rq ^ r i ^
^ ^ Η ^ ^ Η ^ Η ^ ^ , ^ Η
4>
χ : cd ^ o,
'S m ^f <+-. t^ t i 0 os w
T j - T T ^ ^ h ^ ^ f ^ ^ ^ O O ^ X O ^
*C O cd cd ^ * ο S *3
vO vo io ON m
c .3 ω
a
·- Ê ° 1 c2 ft - S "o "-> & h
vot^ioasraoNrsjOvo,^t
*a l
w
115 3 Si" o a δ
M ( S ( S M N ( S ( S n H ( S H H
JD
^ r- .> 33 2 5P c§
7-S s
U ^
■
^
η
η
^
η
^
Ν
Ν
Η
Η
Ν
Ν
x> B ·<π S o g
o
^
s-f ^
0 \ Ö\ ON
7 7 7 7 5 + <« 3 o« t^ oo os O O m m NO NO vp r- r- r^ r* II ON ON Q\ ON G \ 0 \ 5a + ^ S > ON
PQ
•g
tas
S : 04
^^ . . ON
15.
THERAPEUTIC E F F E C T S OF E T H E R L I P I D S
267
The shift of the stage distribution to lower stages indicates that prophylactic treatment with alkylglycerols might have a regressive effect on tumors. However, this could also be an effect on the tissues around the tumor, leading to a change in the clinician's impression when palpating the tumor. In this context, a report by Kato et al. (1971) on the cytostatic effects of alkylglycerols is of interest. D. Effect of Alkylglycerols on Survival Rates The aim of Brohult's early investigations was to study the effect of alkyldiacylglycerols on granulocytopenia after radiation. When the results were analyzed later, it was found that patients given alkyldiacylglycerols had a higher survival rate than the controls. When comparing the groups, standardized proportions of stages were used. The radiation treatment was the same for the two groups. Patients treated with ether lipids showed a somewhat more favorable stage distribution. After 3 years, the group treated with alkyldiacylglycerols showed a higher survival rate than the controls, but this difference was not significant. After 5 years the survival rate was still higher, and the difference was significant (p < 0.05). It is interesting to note that the survival rate was higher for all tumor stages in patients treated with ether lipids than in the corresponding control groups. In the 1960s, several new methods of high-voltage treatment were introduced in cancer therapy. When groups of patients are compared statistically, it is therefore necessary to use standardized proportions with regard to radiation technique. Even if it is assumed that the more favorable stage distribution of a prophylactically treated group in 1965 (Table II) is due to chance (standardized proportions with regard to stage distribution being used in the statistical comparison), the survival rate after 3 years is higher for the prophylactic group than for the controls (p < 0.05). There is still a difference after 5 years, but it is not statistically significant. When the groups are compared statistically using standarized proportions with regard to radiation technique instead of stage, the difference in survival rate in favor of the prophylactic group is even more evident. More patients in the 1964 nonprophylactic group had received highvoltage treatment. Brohult et al. (1981) have therefore compared the survival rates of patients treated with high-voltage irradiation in the periods 1960-1963 and 1966-1969. The survival rate was lower in the first period than in the second, the 1964 nonprophy lactic group being
268
Bo
HALLGREN
an intermediate group. A positive effect on the survival rate of patients given alkylglycerols only during and after radiation therapy could thus not be demonstrated in this study. It is important to give alkylglycerols prophylactically before radiation treatment in order to improve the survival rate. The survival rates of the patients in the study performed by Nyström (1973) have also been analyzed. The groups were small, and no significant differences in survival rates between patients treated with alkylglycerols and controls were found. The tumor recurrence rate and incidence of metastasis were in favor of the group treated with alkylglycerols, but the differences were not significant. In the double-blind study performed in 1970-1972 on 120 treated patients and 125 controls, the survival rate after 3 years was 78% in the group treated with alkylglycerols and 70% in the controls. The difference is not statistically significant, but such a high survival rate was achieved only once in the years 1950-1969, and that was in the 1965 prophylactically treated group. In summary, clinical studies on the effects of alkylglycerols from Greenland shark liver oil given orally to patients undergoing radiation treatment for cancer of the uterine cervix have produced the following findings: First, prophylactic treatment with alkylglycerols or their esters has a protective effect against radiation injuries. This effect is reflected both in the white blood cell count and in the local radiation reactions within the irradiated area. Second, tumor growth within the irradiated area is also influenced by prophylactic treatment with alkylglycerols—this is the only explanation that can be given for the observation that the incidence of complex injuries associated with tumor growth, hence the mortality, is lower in patients treated prophylactically with alkylglycerols than in controls. Moreover, all clinical studies on prophylactic treatment with alkylglycerols show a shift to a more favorable stage distribution in treated patients than in the controls. Finally, an increased survival rate is observed after prophylactic treatment with alkylglycerols. This may partly be explained by the more favorable stage distribution, but it is partly independent of this. UK Methoxy-Substituted Alkylglycerols
During work on the nonsaponifiable fraction of Greenland shark liver oil, 3-4% alkylglycerols more polar than the common ones were dis-
15.
THERAPEUTIC EFFECTS OF ETHER LIPIDS
269
OCH* I H2C-0-CH2-CH-CH2-CH=:CH-(CH2)n-CH3 HO-CH H2C —OH Fig. 1. Methoxy-substituted alkylglycerols. n = 10, 12.
covered; they were shown to be methoxy-substituted alkylglycerols (Hallgren and Ställberg, 1967) (Fig. 1). The quantitative distribution within the mixture of methoxy-substituted alkylglycerols in Greenland shark liver oil was found to be about 60% l-0-(2'-methoxyhexadecenyl)glycerol, 15% l-0-(2'-methoxyhexadecyl)glycerol, and 20% 1-0(2'-methoxyoctadecenyl)glycerol. A polyunsaturated methoxy-substituted alkylglycerol was also isolated from Greenland shark liver oil (Hallgren et al., 1971). Various biological materials, principally from humans and from marine animals, have been analyzed for their content of methoxy-substituted alkylglycerols. The following mammalian materials were investigated: human milk from three different periods of lactation, cow's milk, sheep's milk, human red bone marrow, red blood cells, blood plasma, and a uterine carcinoma (Hallgren et al., 1974a). The samples of marine origin included fillets of Atlantic and Baltic herring as well as mackerel, the edible parts of marine and freshwater crayfish, shrimp and sea mussels, and cod liver oil (Hallgren et al., 1974b). 2-Methoxy-substituted alkyl moieties were found together with unsubstituted alkyl moieties in the neutral lipids as well as in the phospholipids in all the tissues investigated. In the various samples from humans, cows, and sheep only trace quantities of methoxyalkylglycerols were detected. As a rule, the phospholipids contained larger proportions than the neutral lipids. In the samples from marine animals the percentages of methoxy-substituted alkylglycerols were higher than in mammals, especially in the respective phospholipids. The highest content was found in sea mussels and marine crayfish, where 0.47 and 0.35% of the phospholipids contained methoxy-substituted alkyl moieties, respectively. The principal components of the methoxy-substituted alkylglycerols from the mammals studied are the same as those found in the ether lipids of Greenland shark liver, namely, 2-methoxyhexadecyl-, 2-methoxyhexadecenyl-, and 2-methoxyoctadecenylglycerols. Compounds having C16 chains account for 50-90% of the total methoxyalkylglycerols; particularly large proportions are found in the milk of humans,
270
Bo
HALLGREN
and sheep. The methoxy-substituted docosahexaenylglycerol first demonstrated in Greenland shark liver oil is also present in some of the other materials investigated, namely, human red blood cells, shrimp, mackerel, and cod liver oil (Hallgren et al., 1974a). The synthesis of methoxy-substituted alkylglycerols and methods for the characterization of these compounds have been described (Ställberg, 1975) (see Chapter 3). COWS,
A. Antibacterial and Antifungal Effects The mixture of methoxy-substituted alkylglycerols isolated from Greenland shark liver oil, as well as synthetic 2-methoxyhexadecylglycerol (a mixture of stereoisomers), showed an antibiotic effect in vitro against several types of bacteria, especially Corynebacterium Hofmannii, Diplococcus pneumoniae, Staphylococcus pyogenes A, and Staphylococcus pyogenes H. Oxford, Streptococcus pyogenes, and Streptococcus viridans (Boeryd et al., 1971). The antibiotic activity was about equal to that of nitrofurantoin. Fungistatic and fungicidal activity has also been demonstrated in vitro for the methoxy-substituted alkylglycerols (Hallgren et al., 1978). Monosporically selected strains of the dermatophytes Epidermophyton floccosum, Microsporum canis, Trichophyton mentagrophytes, and Trichophyton rubrum were used in these studies. Fungistatic tests showed the mixture of methoxy-substituted alkylglycerols from Greenland shark liver oil to be a more potent inhibitor of dermatophyte growth than the synthetic compounds, 2-methoxyhexadecylglycerol and 2-methoxyhexadecenylglycerol, which produced inhibition in the range 5-15% at a concentration of 100 /xg/ml. In fungicidal tests, the methoxy-substituted alkylglycerols in high concentrations (1000 ^g/ml) inhibited the growth of T. rubrum and E. floccosum. B. Experimental Antitumor Activity The methoxy-substituted alkylglycerols were found to inhibit tumor growth in cultured cells. Two cell lines were used, a methylcholanthrene-induced murine sarcoma (MCG1-SS) and a juvenile osteogenic sarcoma (2T). Marked growth inhibition was noted for the mixture of 2-methoxyalkylglycerols from Greenland shark liver oil, different single components derived from this oil (2-methoxyhexadecylglycerol, 2-methoxyhexadecenylglycerol , and 2-methoxy octadeceny lgly cerol), and various synthetic compounds including, for example, 2-ethoxyhexa-
15.
THERAPEUTIC E F F E C T S OF E T H E R L I P I D S
271
decylglycerol, 2-methoxyhexadecenylglycerol, and 3-methoxyhexadecylglycerol (Hallgren et al., 1978). In several tumor-host systems, including solid tumors, leukemias, and lymphomas, the methoxy-substituted alkylglycerols were incorporated into the feed in different concentrations (0.1-2%, w/w). Growth inhibition was noted for melanoma B16, for a methylcholanthrene-induced sarcoma (MCG101) and Lewis lung tumor (LLT) in C57BL/6J mice, for lymphoma LAA in A/Sn mice with synthetic 2-methoxyhexadecylglycerol, and for a spontaneous mammary carcinoma in C3H mice with methoxy-substituted alkylglycerols from Greenland shark liver oil. The survival time of DBA/2J mice transplanted with lymphatic leukemia PI534 was increased by synthetic 2-methoxyhexadecylglycerol (Hallgren et al., 1978; Boeryd and Hallgren, 1980a). Métastases induced by the injection of MCGl-SS cells into a tail vein or into the portal vein were inhibited in the liver by methoxy-substituted alkylglycerols from Greenland shark liver oil (Boeryd et al., 1971). Spontaneous metastasis formation from a methylcholanthrene-induced sarcoma (MCGl-SS) in lymph nodes and lungs of CBA mice was inhibited by methoxy-substituted alkylglycerols derived from Greenland shark liver oil as well as by synthetic 2-methoxyhexadecylglycerol (Boeryd et al., 1971). Spontaneous metastasis formation from melanoma B16 was inhibited by synthetic 2-methoxyhexadecylglycerol (Boeryd and Hallgren, 1980a). C. Influence on Immune Reactivity Methoxy-substituted alkylglycerols in the feed stimulated the immune reactivity in mice against sheep red blood cells (SRBCs) (Boeryd et al., 1978) as determined by the number of plaque-forming cells (PFC) (Cunningham and Szenberg, 1968). Further, they stimulated cellular immunoreactivity as demonstrated by the increased ability of parenteral spleen cells to induce a graft-versus-host reaction (GVHR) in DBA/1 J x C57BL/6J Ft (H-2bQ) hybrid mice (Boeryd et al., 1978) tested according to Michie (1967). The effect on humoral immunoreactivity was studied mainly in CBA mice but to some extent also in C57BL/6J and DBA/2J mice. The mixture of methoxy-substituted alkylglycerols from Greenland shark liver oil and synthetic 2-methoxyhexadecylglycerol, given in concentrations of 0.1, 0.25, and 0.5% of the feed for 4 or 14 days before SRBC injection, significantly increased the number of PFCs. To achieve immunostimulation by the mixture of alkylglycerols (unsubstituted and methoxy-substituted) from Greenland
272
Bo
HALLGREN
shark liver oil, it was necessary to increase the content of these compounds in the diet to 3%. This concentration corresponds to about 0,1% methoxy-substituted alkylglycerols, as 3-4% of the alkylglycerols in Greenland shark liver oil are methoxy-substituted. It is probable that the immunostimulation caused by the complete mixture was due to its content of methoxy-substituted alkylglycerols. The cellular immunoreactivity tested by the GVHR was stimulated by synthetic 2-methoxyhexadecylglycerol given in a concentration of 0.1% of the feed for 44 days to the spleen cell donors. In this connection, a study by Brohult et al. (1972) is of great interest. These authors observed that patients vaccinated against typhoidparatyphoid 1 day before and 1 day after implantation of radium for uterine cancer and given alkylglycerols and methoxy-substituted alkylglycerols from Greenland shark liver oil produced antibodies to a larger extent than a group receiving radiation treatment only. As methoxy-substituted alkylglycerols occur in the milk of different species, immunoreactivity was tested in adult mice whose mothers had been deprived of these substances during lactation. Exclusion of animal fat from the diets of lactating mothers profoundly decreased the immunoreactivity of the adult offspring. This reactivity was partly restored in male offspring when synthetic 2-methoxyalkylglycerols were added to the feed of the mothers. In mice deprived of animal fat at weaning and for the following 21 days immunoreactivity to SRBCs, tested about 3 month after stopping the diet, was not influenced. However, resistance to a transplanted tumor in similarly fed mice was increased, and this resistance was brought approximately to the control level by methoxy-substituted alkylglycerols (Boeryd and Hallgren, 1980b). In summary, the mechanisms behind the clinical and experimental effects of methoxy-substituted and unsubstituted alkylglycerols are still unknown. Methoxy-substituted alkylglycerols stimulate immunoreactivity, and it is probable that these substances are the immunostimulating components of the nonsaponifiable fraction from Greenland shark liver oil. It has not been possible, however, to evaluate the importance of immunostimulation for the antitumor activity demonstrated on experimental tumors, either for tumor growth or for metastasis formation. Growth retardation of tumor cells has been demonstrated in vitro. It therefore seems probable that the substituted alkylglycerols to some extent exert direct effects on the tumor cells. They might be incorporated into membrane lipids, changing their structure and thereby influencing the growth and spread of the tumor cells. The unique combination of a stimulatory effect on host defense
15.
THERAPEUTIC E F F E C T S OF E T H E R L I P I D S
273
mechanisms and an antitumor effect in the same molecule has also been demonstrated for a synthetic ether phospholipid with a methoxy group in sn-2 position of glycerol (Munder et al., 1979). In patients treated with radiation for cancer of the uterine cervix, the protective effect of the mixture of methoxy-substituted and unsubstituted alkylglycerols from Greenland shark liver oil against leukopenia and thrombocytopenia might be due to the latter compounds, as a stimulatory effect on bone marrow has been demonstrated for both chimyl alcohol and batyl alcohol (natural l-O-hexadecyl-s/i -glycerol and 1-Öoctadecyl-sfl-glycerol, respectively). One can only speculate about the mechanisms behind the other effects observed in patients treated with radiation. The protective effect against local radiation injuries with tumor growth involved in the clinical condition, that is, complex injuries, according to Brohult et al. (1977), might be explained by a membrane effect of the alkylglycerols, leading to an increase in the radiation sensitivity of the tumor in relation to the normal tissue. The effect on the stage distribution might be explained by some direct effect on the tumor but also by a decreased reaction to, for example, necrotic tumor cells in the tissues surrounding the tumor. The relative importance for the clinical effects of methoxy-substituted alkylglycerols and the more common alkylglycerols of Greenland shark liver oil has not yet been determined. IV. Acetal Analogs of Phosphatidic Acids Long-chain cyclic acetals of glycerophosphoric acid (I) Darmstoff (Vogt, 1963), are potent stimulators of gastrointestinal smooth muscle (Wiley et al.y 1970; Vogt, 1963) and inhibitors of renin (Bunag and Walaszek, 1973). It has been suspected that the activity of the natural product may in fact be due to the presence of prostaglandins (Andersen, 1974), but studies with synthetic preparations have demonstrated similar activities (Wiley et al., 1970). Long-chain cyclic acetals of glycerol that could serve as intermediates in the synthesis of the corresponding phosphorylated compounds have been prepared (Baumann, 1971; Wedmid and Baumann, 1977). The biologically active acetal analogs of phosphatidic acid (I) have also been synthesized (Wiley et aL, 1970). As these compounds are unstable in neutral aqueous solution, Milbert and Wiley (1978) have prepared the tetrahydrofuran and cyclopentyl derivatives of Darms toff II and III, respectively. A comparison of compounds I, II, and III has shown that they ex-
274 H
Bo
HALLGREN
0-
FT V I
^
ΟΡΟοΗΝα
R
λ
Λ./ΟΡΟοΗΝα
0 ^ ^ II
1
λ
.ΟΡΟοΗΝα
~ / \ / \ / III
Fig. 2. Darmstoff (I) and analogous compounds (II, III). R = C 15 H 31 .
hibit similar effects on the blood pressure and heart rate of rats (Milbe rt and Wiley, 1978). In contrast to aqueous solutions of compound I, which loses all biological activity within 1 h, solutions of compounds II and of III remain active for days (Milbert and Wiley, 1978). The compounds described may find therapeutic use as hypotensive agents. V. Alkyl Ethers of Halogenopropanediols
Langen et al. (1979) have recently demonstrated that long-chain alkyl ethers of halogenopropanediols exhibit a strong inhibitory effect on the proliferation of Ehrlich ascites carcinoma cells in suspension cultures. The same compounds have been found to have a cytostatic activity in ascites cells in vivo. Most of the substances investigated had no effect on the survival time or cell number after subcutaneous administration, and there was no prolongation of the survival time of leukemia LI210- or L184-bearing mice. Nevertheless, some halo analogs of alkyl lysophospholipids appear to be promising as cytostatic agents (Brachwitz et al., 1982). References Andersen, N. H. (1974). Arch. Intern. Med. 133, 30. Baumann, W. J. (1971). J. Org. Chem. 36, 2743. Boeryd, B., and Hallgren, B. (1980a). Acta PathoL Microbol. Scand.y Sect. A 88A, 11. Boeryd, B., and Hallgren, B. (1980b). Int. J. Cancer 26, 241. Boeryd, B., Hallgren, B., and Ställberg, G. (1971). Br. J. Exp. PathoL 52, 20. Boeryd, B., Nilsson, T., Lindholm, L., Lange, S., Hallgren, B., and Ställberg, G. (1978). Eur. J. Immunol. 8, 678. Brachwitz, H., Langen, P., Hintsche, R., and Schildt, J. (1982). Chem. Phys. Lipids 31, 33. Brohult, A. (1963). Acta Radiol., Suppl. No. 233. Brohult, A., Brohult, J., and Brohult, S. (1972). Experientia 28, 954. Brohult, A., Brohult, J., and Brohult, S. (1977). Acta Obstet. Gynecol. Scand. 56, 441. Brohult, A., Brohult, J., and Brohult, S. (1978). Acta Obstet. Gynecol. Scand. 57, 79. Brohult, A., Brohult, J., and Brohult, S. (1981). Unpublished observations. Bunag, R. D., and Walaszek, E. J. (1973). Eur. J. Pharmacol. 23, 191. Cunningham, A. J., and Szenberg, A. (1968). Immunology 14, 599.
15.
THERAPEUTIC EFFECTS OF ETHER LIPIDS
275
Hallgren, B., and Ställberg, G. (1967). Acta Chem. Scand. 21, 1519. Hallgren, B., Ställberg, G., and Thorin, H. (1971). Acta Chem. Scand. 25, 3781. Hallgren, B., Niklasson, A., Ställberg, G., and Thorin, H. (1974a). Acta Chem. Scand., Ser. B 28B, 1029. Hallgren, B., Niklasson, A., Ställberg, G., and Thorin, B. (1974b). Acta Chem. Scand., Ser. B 28B, 1035. Hallgren, B., Ställberg, G., and Boeryd, B. (1978). Prog. Chem. Fats Other Lipids 16, 45. Kato, A., Ando, K., Tamura, G., and Arima, K. (1971). Cancer Res. 31, 501. Kottmeier, H. L., and Gray, M. J. (1961). Am. J. Obstet. Gynecol. 82, 74. Langen, P., Brachwitz, H., and Schildt, J. (1979). Acta Biol. Med. Ger. 38, 965. Mangold, H. K. (1972). In "Ether Lipids: Chemistry and Biology" (F. L. Snyder, ed.), pp. 157-176. Academic Press, New York. Michie, D. (1967). In " H a n d b o o k s Experimental Immunology" (D. M. Weir, ed.), pp. 969-987. Blackwell, Oxford. Milbert, A. N., and Wiley, R. O. (1978). J. Med. Chem. 21, 245. Münder, P. G., Modolell, M., Andreesen, R., Weltzien, H. V., and Westphal, O. (1979). Springer Sem. Immuno. 2, 187. Nyström, C. (1973). Unpublished observations. Ställberg, G. (1975). Chem. Ser. 7, 31. Vogt, W. (1963). Biochem. Pharmacol. 12, 415. Wedmid, Y., and Baumann, W. J. (1977). J. Org. Chem. 42, 3624. Wiley, R. O., Sumner, D. D., and Walaszek, E. J. (1970). Lipids 5, 803.